NanoMed 2009 - Berlin

Study of the micro- and nanostructured silicon

for biosensing and medical applications

Irina Kleps (irinaklepsimtro) Mihaela Miu Monica Simion Teodora Ignat Adina Bragaru

Florea Craciunoiu Mihai Danila National Institute for Research and Development in Microtechnology (IMT-

Bucharest) Erou Iancu Nicolae 126 A 72996 Bucharest Romania

OUTLINE

bull Motivationbull Micro- and nanostructured silicon preparationbull PS as sensing element

- Photoluminescence sensors- Microarray substrates for protein detection- DNA detection by Impedance spectroscopy- SERS sensors

bullNanostructured Si as carriers for controlled drug deliverybullConclusions

Motivation

- porous silicon (PS) is a low cost material- PS has controllable pore size- it has high surface area within a small volume (internal surface of 600 m2cm3)- convenient surface chemistry- compatibility with conventional silicon microfabrication technologies - PS is biocompatible [1 2] and bioresorbable (nanometric size Si) with silicic acid releasing without toxic effects for the body [3]

[1] S C Bayliss R Heald D I Fletcher L D Buckberry Adv Mater 11 318 (1999)[2] A Angelescu I Kleps M Miu M Simion T Neghina A Bragaru S Petrescu C Paduraru A Raducanu N Moldovan Rev Adv Mater Sci 5 34-40 (2003)[3] Uracha Rungsardthong Susan I Anderson and Leigh T Canham Chiang Mai J Sci 2005 32(3) 487-494

Micro- and nanostructured silicon preparation

Porous silicon ndash PS - is obtained by electrochemical dissolution of silicon in HF-based

solution

Etching requires holes (electron injection) to break bonds

Resistivity ~ 106 Ωcm similar to intrinsic Si

Schematic view of anodization cell

Si + 4HF + (4-n)h+ rarr SiF4 + 4H+ + ne-

Dissolution chemistries

AMMT etching system for 4rsquorsquo Si wafers with programmable power supply and dedicated software for time-based current profiles

PS morphology ndash SEM characterisation

nano-PS (lt 15 nm)

meso-PS (50- 100 nm)macro-PS (ca 1microm)

Pore size wire size

Electrolyte type HF concentration Doping type and level

Illumination

Experimental ndash PS fabricated by electrochemical etching

p-type silicon (100) with 6 - 10 middotcm resistivity

HF - C2H5OH electrolyte with 15 18 25

concentration

current densities have been 15 mAcm2 and 25 mAcm2

NoConcetration

HF()

Current density

(mAcm2)

Time(min)

Porosity

()

1 25 15 6 58

2 25 20 6 60

3 25 25 6 62

4 18 15 6 65

5 18 20 6 70

6 15 15 6 80

7 15 25 6 88

The orange-red photoluminescence from the porous

silicon is clearly visible when the wafer illuminated by UV

light

550 600 650 700 750 800 850 00

02

04

06

08

10 88 82 70 65 62 60 58

PL

Inte

nsity (n

orm

ate

va

lue

s)

Wavelength (nm)

Photoluminescence spectra of PS Si-p samples with different porosities (58 - 88)

PL

peak

pos

ition

(nm

)

Porosity ()

PL maximum dependence with porosities

The PL emission from PS is observable at

wavelengths ranging from the ultraviolet to

the infrared the normalized spectra recorded

for different experimental samples

demonstrate the dependence on porosity

PL emission from PS

bull the PL peaks for high porosity PS samples are centred around 650-720 nm in visible range due to quantuum confinement and surface states effectsbull a shift of the PL peak position towards high photon energies with the increase of the PS porosity is observed

thermal treatment at different temperatures between

300degC and 750degC

550 600 650 700 750 800 85000

20x103

40x103

60x103

80x103

10x104

12x104

14x104

16x104

18x104

20x104

22x104

24x104

62 PS 62 PS + TT 315oC 62 PS + TT 750oC

Inte

nsita

te P

L (u

a)

Lungime de unda (nm)Wavelength (nm)

PL

inte

nsi

ty (

au

)

550 600 650 700 750 800 8500

10000

20000

30000

40000

50000

676

667

70 PS 70 PS + oxidare anodica

Inte

nsita

te P

L (u

a)

Lungime de unda (nm)

PL

inte

nsi

ty (

au

)

Wavelength (nm)

The PL peaks for high porosity PS samples are centred around 650-720 nm in visible range due to quantuum confinement and surface states effects

bull a shift of the PL peak position towards high photon energies with the increase of the PS porosity is

observed

bull the stabilisation methods lead to a redshift of PL peak when thermal treatments were used and a

blueshift of PL peak when anodic oxidation was used supplementary an improvement of PL intensity was

observed

Additional treatments for PS optoelectronic properties stabilisation

anodic oxidation

Porous silicon biosensor Chalenges (i) integration of biological systems with PS increasing

of the immobilised biomolecule number on the

surface creation of stable covalent bonds

(ii) using micronanoscale materials for amplification of

the detected signal (SERS SEIR SEF)

(iii) using PS as receptor and transducer

(iv) integrating optics with low-power portable devices

PS is RECEPTOR for biological molecules

selective for the analyte (DNA single strand

proteins antibodies enzymes) constitutes the molecular

recognition element

PS is DETECTOR (signal transducer) for biochemical interactions

electrical conductance impedance

(electro)chemical

Optical

1048592 Luminescence

1048592 Phosphorescence

1048592 Internal Reflection Spectroscopy

1048592 Reflectometric Interference Spectroscopy

1048592 Ellipsometry

1048592 Fluorescencemdashintensity lifetime polarization

1048592 Fluorescence resonance energy transfer

1048592 Absorbance

1048592 Raman Scattering SERS

1048592 Surface plasmon resonance

1048592 Interference

1 Fixed position arrays2 Single Particle Encoding

PS host matrixsupport for immobilization

of sensing biomolecule

Techniques for immobilization range from

physical adsorption to the replacement of hydride bonds

with Si alkyls and to antibody bonding at functionalized

PSi surface with subsequent antibody-antigen

interactions

The biological molecules provide the sensor with its selectivity while the transducer determines the extent of the interaction between the biomolecules and the analyte

PS functionalisation

550 600 650 700 750 800 85000

02

04

06

08

10

PL

inte

nsi

ty (

no

rma

lise

d v

alu

es)

Wavelength (nm)

LN3 functLN3

Material in the pores changes the spectral response

PL shift towards smaller energies after functionalisation

SEM image of porous silicon surface before (a) and after (b) BSA deposition

Affinity anchoring This method is commonly used to load proteins -oxidised PS has a negative surface charge - molecules with positive charges will be spontaneously adsorbed on the inner pore walls and surface

Covalent binding To prepare the surface for the capture of BSA proteins the device is first thermally oxidized at 8000C to form a silica-like internal surface The sensor is then treated with 2 amino-propyltrimethoxy-silane to create amino groups on the internal oxide surface for bio-molecule recognition

PCs can be designed to localize the electric field in the low refractive index region (eg air pores) which makes thesensors extremely sensitive to a small refractive index change produced by bio-molecule immobilization on the pore walls

2

2 amino propyltrimethoxy silane

8000C

500 550 600 650 700 750 800 8500

2000

4000

6000

8000

10000

12000

14000

PL

inte

nsi

ty (

au

)

Wavelength (nm)

LN 2

LN 3

LN3 interconnected lines

bull similar recording conditions were (488 nm frequency of excitation and 87

mW nominal power)

bull the intensity of PL emission is three times larger in the case of

semicircular microcavities leading to an important improvement of detection

Si substrate microstructuration and porosification

LN2 pyramidal structures

Silicon substrate was micropatterned prior porosification process as an array of pyramids (right) or as semi-

circular cavities (left)

Si substrate microstructuration and porosification

PL spectra recorded for three array of microcavities 03 06 si 1 microm

1 microm

06 microm

03 microm

The micropatterned substrate is an array of piramidal

cavities which have to act similar to a collimating device

and light emitted from sample traveling in detector

direction to be reflected by the side walls improving the

sensitivity of the biosensor

rarr gold nanoparticles (AuNP) immobilized inside the pores of the porous silicon layer

rarr thiol molecules self assembled on AuNP so that they can recognize a given protein

rarr In the absence of any protein the photoluminescence of the PS is absorbed by the AuNP at a given

wavelength rarr when the protein specifically binds to the AuNP the absorption due to the SPR is modified and

the detected signal is enhanced

Elaboration of PSMetallic plasmonic nanostructures

The combination of the PS with Au-NP

makes possible to design an optical

biosensor where the light source is

the silicon itself (photoluminescence)

and the chemical transducer is the

functionalized AuNP

Functionalised gold nanoparticles act as nano amplifiers

Luminescence of the PS is modified by the SPR of the AuNPs

A specific protein is recognised by the AuNP (specific absorption)

Spherical gold and silver nanoparticles can be used as substrates in SERS-based molecule detection due to their

advantages in -local scattering field enhancing -surface chemical modifications - biocompatibility- well established chemical synthesis process

The intrinsic plasmon resonance of single nanospheres and the plasmon coupling between adjacent

nanospheres are considered as the key and necessary conditions for local field enhancing

Gold PS

X ray diffraction analyses The metallic nanocrystallites orientation on nanostructurated Si

substrates and the influence of additional anealing treatments ndash

The Au (111)nc-Si surface has

a higher density of atoms

comparatively with Au (100)

this favours the attachment

of a higher number of atoms

and bio-molecules on the gold

surface

12 14 16 18 20 22 24 26 28 30 32 34 36102

103

104

Au (311)

Au (220)

Si (400)

Au (222)

Au (200)

Au (111)

I [i

mp

40sec

]

2 [o]

RX3 - as deposited RX1 - T500 RX2 - T900

Au 38 PS

16 18 20 22 24 26 28 30 32 34 36

200

400

600

800

1000

Au (311)

Si (400)

Au (220)

Au (200)

I [im

p4

0sec

]

2 [o]

RX6 - as deposited RX4 - T500 RX5 - T900

Au (111)

Au 60 PS

For AuPS (60) nucleation begin on Au (111) planes more rapid than for AuPS (38)

(i) in the as-deposited mesoporous films the initial nucleation begin on Au (220) planes

(ii) after the thermal annealing at 5000C the crystallisation process on the (111) planes

become dominant and a (111) texture is obtained

(iii) the thermal annealing at 9000C induces an increase of the (111) crystallites indicating a

clear crystallisation on (111) planes

In Au macroporous silicon the Au (111) texture of crystallites became predominant

Au PS

Experimental The AuPSSi

and AgPSSi

nanocomposites layers were

thermal treated at 500 and

9000C in reducing

atmosphere (H2 and N2)

Aumacroporous silicon

Experimental AgNO3 salt and AgNO3 1 ethanolic solution were also deposited on the PSSi substrate

40 50 60 70 80 90 100 110 120

15000

30000

45000

2

Ag(111)

Ag(400)

Si(331)

Co

K

Co KSi(400)Ag(200)

I [

imp

10s

ec]

Ag - RX5 T500 Ag - RX4 As deposited AgPS-Si(400)

40 50 60 70 80 90 100 110 120 1300

10000

20000

30000

40000

50000

60000

2

Si(400)

Ag(400)

Ag(222)

Si(331)

Co

K

Co K

Si(400)

Ag(200)

Ag(111)

I

[imp

10

sec]

Ag - RX4 As deposited AgPS-Si(400) Ag - RX6 T900 AgPS-Si(400) - Fe filter

The Ag as-deposited films obtainrd from diluted solution of AgNO3 are in an initial stage

of crystallisation with a high amorphous content

In the case of AgPS samples the

annealing treatment at T=500oC has no

effect from the point of view of the

microstructure analysis of the Ag films

on PS (crystallization continue on (200)

planes the films have a higher

crystalline content) while annealing at

900o C produces a mixed (111) and

(200) texture with bigger grain sizes

SilverSi nanocomposite layers

Optical microscope image using a UV filter of AgPS

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

OUTLINE

bull Motivationbull Micro- and nanostructured silicon preparationbull PS as sensing element

- Photoluminescence sensors- Microarray substrates for protein detection- DNA detection by Impedance spectroscopy- SERS sensors

bullNanostructured Si as carriers for controlled drug deliverybullConclusions

Motivation

- porous silicon (PS) is a low cost material- PS has controllable pore size- it has high surface area within a small volume (internal surface of 600 m2cm3)- convenient surface chemistry- compatibility with conventional silicon microfabrication technologies - PS is biocompatible [1 2] and bioresorbable (nanometric size Si) with silicic acid releasing without toxic effects for the body [3]

[1] S C Bayliss R Heald D I Fletcher L D Buckberry Adv Mater 11 318 (1999)[2] A Angelescu I Kleps M Miu M Simion T Neghina A Bragaru S Petrescu C Paduraru A Raducanu N Moldovan Rev Adv Mater Sci 5 34-40 (2003)[3] Uracha Rungsardthong Susan I Anderson and Leigh T Canham Chiang Mai J Sci 2005 32(3) 487-494

Micro- and nanostructured silicon preparation

Porous silicon ndash PS - is obtained by electrochemical dissolution of silicon in HF-based

solution

Etching requires holes (electron injection) to break bonds

Resistivity ~ 106 Ωcm similar to intrinsic Si

Schematic view of anodization cell

Si + 4HF + (4-n)h+ rarr SiF4 + 4H+ + ne-

Dissolution chemistries

AMMT etching system for 4rsquorsquo Si wafers with programmable power supply and dedicated software for time-based current profiles

PS morphology ndash SEM characterisation

nano-PS (lt 15 nm)

meso-PS (50- 100 nm)macro-PS (ca 1microm)

Pore size wire size

Electrolyte type HF concentration Doping type and level

Illumination

Experimental ndash PS fabricated by electrochemical etching

p-type silicon (100) with 6 - 10 middotcm resistivity

HF - C2H5OH electrolyte with 15 18 25

concentration

current densities have been 15 mAcm2 and 25 mAcm2

NoConcetration

HF()

Current density

(mAcm2)

Time(min)

Porosity

()

1 25 15 6 58

2 25 20 6 60

3 25 25 6 62

4 18 15 6 65

5 18 20 6 70

6 15 15 6 80

7 15 25 6 88

The orange-red photoluminescence from the porous

silicon is clearly visible when the wafer illuminated by UV

light

550 600 650 700 750 800 850 00

02

04

06

08

10 88 82 70 65 62 60 58

PL

Inte

nsity (n

orm

ate

va

lue

s)

Wavelength (nm)

Photoluminescence spectra of PS Si-p samples with different porosities (58 - 88)

PL

peak

pos

ition

(nm

)

Porosity ()

PL maximum dependence with porosities

The PL emission from PS is observable at

wavelengths ranging from the ultraviolet to

the infrared the normalized spectra recorded

for different experimental samples

demonstrate the dependence on porosity

PL emission from PS

bull the PL peaks for high porosity PS samples are centred around 650-720 nm in visible range due to quantuum confinement and surface states effectsbull a shift of the PL peak position towards high photon energies with the increase of the PS porosity is observed

thermal treatment at different temperatures between

300degC and 750degC

550 600 650 700 750 800 85000

20x103

40x103

60x103

80x103

10x104

12x104

14x104

16x104

18x104

20x104

22x104

24x104

62 PS 62 PS + TT 315oC 62 PS + TT 750oC

Inte

nsita

te P

L (u

a)

Lungime de unda (nm)Wavelength (nm)

PL

inte

nsi

ty (

au

)

550 600 650 700 750 800 8500

10000

20000

30000

40000

50000

676

667

70 PS 70 PS + oxidare anodica

Inte

nsita

te P

L (u

a)

Lungime de unda (nm)

PL

inte

nsi

ty (

au

)

Wavelength (nm)

The PL peaks for high porosity PS samples are centred around 650-720 nm in visible range due to quantuum confinement and surface states effects

bull a shift of the PL peak position towards high photon energies with the increase of the PS porosity is

observed

bull the stabilisation methods lead to a redshift of PL peak when thermal treatments were used and a

blueshift of PL peak when anodic oxidation was used supplementary an improvement of PL intensity was

observed

Additional treatments for PS optoelectronic properties stabilisation

anodic oxidation

Porous silicon biosensor Chalenges (i) integration of biological systems with PS increasing

of the immobilised biomolecule number on the

surface creation of stable covalent bonds

(ii) using micronanoscale materials for amplification of

the detected signal (SERS SEIR SEF)

(iii) using PS as receptor and transducer

(iv) integrating optics with low-power portable devices

PS is RECEPTOR for biological molecules

selective for the analyte (DNA single strand

proteins antibodies enzymes) constitutes the molecular

recognition element

PS is DETECTOR (signal transducer) for biochemical interactions

electrical conductance impedance

(electro)chemical

Optical

1048592 Luminescence

1048592 Phosphorescence

1048592 Internal Reflection Spectroscopy

1048592 Reflectometric Interference Spectroscopy

1048592 Ellipsometry

1048592 Fluorescencemdashintensity lifetime polarization

1048592 Fluorescence resonance energy transfer

1048592 Absorbance

1048592 Raman Scattering SERS

1048592 Surface plasmon resonance

1048592 Interference

1 Fixed position arrays2 Single Particle Encoding

PS host matrixsupport for immobilization

of sensing biomolecule

Techniques for immobilization range from

physical adsorption to the replacement of hydride bonds

with Si alkyls and to antibody bonding at functionalized

PSi surface with subsequent antibody-antigen

interactions

The biological molecules provide the sensor with its selectivity while the transducer determines the extent of the interaction between the biomolecules and the analyte

PS functionalisation

550 600 650 700 750 800 85000

02

04

06

08

10

PL

inte

nsi

ty (

no

rma

lise

d v

alu

es)

Wavelength (nm)

LN3 functLN3

Material in the pores changes the spectral response

PL shift towards smaller energies after functionalisation

SEM image of porous silicon surface before (a) and after (b) BSA deposition

Affinity anchoring This method is commonly used to load proteins -oxidised PS has a negative surface charge - molecules with positive charges will be spontaneously adsorbed on the inner pore walls and surface

Covalent binding To prepare the surface for the capture of BSA proteins the device is first thermally oxidized at 8000C to form a silica-like internal surface The sensor is then treated with 2 amino-propyltrimethoxy-silane to create amino groups on the internal oxide surface for bio-molecule recognition

PCs can be designed to localize the electric field in the low refractive index region (eg air pores) which makes thesensors extremely sensitive to a small refractive index change produced by bio-molecule immobilization on the pore walls

2

2 amino propyltrimethoxy silane

8000C

500 550 600 650 700 750 800 8500

2000

4000

6000

8000

10000

12000

14000

PL

inte

nsi

ty (

au

)

Wavelength (nm)

LN 2

LN 3

LN3 interconnected lines

bull similar recording conditions were (488 nm frequency of excitation and 87

mW nominal power)

bull the intensity of PL emission is three times larger in the case of

semicircular microcavities leading to an important improvement of detection

Si substrate microstructuration and porosification

LN2 pyramidal structures

Silicon substrate was micropatterned prior porosification process as an array of pyramids (right) or as semi-

circular cavities (left)

Si substrate microstructuration and porosification

PL spectra recorded for three array of microcavities 03 06 si 1 microm

1 microm

06 microm

03 microm

The micropatterned substrate is an array of piramidal

cavities which have to act similar to a collimating device

and light emitted from sample traveling in detector

direction to be reflected by the side walls improving the

sensitivity of the biosensor

rarr gold nanoparticles (AuNP) immobilized inside the pores of the porous silicon layer

rarr thiol molecules self assembled on AuNP so that they can recognize a given protein

rarr In the absence of any protein the photoluminescence of the PS is absorbed by the AuNP at a given

wavelength rarr when the protein specifically binds to the AuNP the absorption due to the SPR is modified and

the detected signal is enhanced

Elaboration of PSMetallic plasmonic nanostructures

The combination of the PS with Au-NP

makes possible to design an optical

biosensor where the light source is

the silicon itself (photoluminescence)

and the chemical transducer is the

functionalized AuNP

Functionalised gold nanoparticles act as nano amplifiers

Luminescence of the PS is modified by the SPR of the AuNPs

A specific protein is recognised by the AuNP (specific absorption)

Spherical gold and silver nanoparticles can be used as substrates in SERS-based molecule detection due to their

advantages in -local scattering field enhancing -surface chemical modifications - biocompatibility- well established chemical synthesis process

The intrinsic plasmon resonance of single nanospheres and the plasmon coupling between adjacent

nanospheres are considered as the key and necessary conditions for local field enhancing

Gold PS

X ray diffraction analyses The metallic nanocrystallites orientation on nanostructurated Si

substrates and the influence of additional anealing treatments ndash

The Au (111)nc-Si surface has

a higher density of atoms

comparatively with Au (100)

this favours the attachment

of a higher number of atoms

and bio-molecules on the gold

surface

12 14 16 18 20 22 24 26 28 30 32 34 36102

103

104

Au (311)

Au (220)

Si (400)

Au (222)

Au (200)

Au (111)

I [i

mp

40sec

]

2 [o]

RX3 - as deposited RX1 - T500 RX2 - T900

Au 38 PS

16 18 20 22 24 26 28 30 32 34 36

200

400

600

800

1000

Au (311)

Si (400)

Au (220)

Au (200)

I [im

p4

0sec

]

2 [o]

RX6 - as deposited RX4 - T500 RX5 - T900

Au (111)

Au 60 PS

For AuPS (60) nucleation begin on Au (111) planes more rapid than for AuPS (38)

(i) in the as-deposited mesoporous films the initial nucleation begin on Au (220) planes

(ii) after the thermal annealing at 5000C the crystallisation process on the (111) planes

become dominant and a (111) texture is obtained

(iii) the thermal annealing at 9000C induces an increase of the (111) crystallites indicating a

clear crystallisation on (111) planes

In Au macroporous silicon the Au (111) texture of crystallites became predominant

Au PS

Experimental The AuPSSi

and AgPSSi

nanocomposites layers were

thermal treated at 500 and

9000C in reducing

atmosphere (H2 and N2)

Aumacroporous silicon

Experimental AgNO3 salt and AgNO3 1 ethanolic solution were also deposited on the PSSi substrate

40 50 60 70 80 90 100 110 120

15000

30000

45000

2

Ag(111)

Ag(400)

Si(331)

Co

K

Co KSi(400)Ag(200)

I [

imp

10s

ec]

Ag - RX5 T500 Ag - RX4 As deposited AgPS-Si(400)

40 50 60 70 80 90 100 110 120 1300

10000

20000

30000

40000

50000

60000

2

Si(400)

Ag(400)

Ag(222)

Si(331)

Co

K

Co K

Si(400)

Ag(200)

Ag(111)

I

[imp

10

sec]

Ag - RX4 As deposited AgPS-Si(400) Ag - RX6 T900 AgPS-Si(400) - Fe filter

The Ag as-deposited films obtainrd from diluted solution of AgNO3 are in an initial stage

of crystallisation with a high amorphous content

In the case of AgPS samples the

annealing treatment at T=500oC has no

effect from the point of view of the

microstructure analysis of the Ag films

on PS (crystallization continue on (200)

planes the films have a higher

crystalline content) while annealing at

900o C produces a mixed (111) and

(200) texture with bigger grain sizes

SilverSi nanocomposite layers

Optical microscope image using a UV filter of AgPS

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Motivation

- porous silicon (PS) is a low cost material- PS has controllable pore size- it has high surface area within a small volume (internal surface of 600 m2cm3)- convenient surface chemistry- compatibility with conventional silicon microfabrication technologies - PS is biocompatible [1 2] and bioresorbable (nanometric size Si) with silicic acid releasing without toxic effects for the body [3]

[1] S C Bayliss R Heald D I Fletcher L D Buckberry Adv Mater 11 318 (1999)[2] A Angelescu I Kleps M Miu M Simion T Neghina A Bragaru S Petrescu C Paduraru A Raducanu N Moldovan Rev Adv Mater Sci 5 34-40 (2003)[3] Uracha Rungsardthong Susan I Anderson and Leigh T Canham Chiang Mai J Sci 2005 32(3) 487-494

Micro- and nanostructured silicon preparation

Porous silicon ndash PS - is obtained by electrochemical dissolution of silicon in HF-based

solution

Etching requires holes (electron injection) to break bonds

Resistivity ~ 106 Ωcm similar to intrinsic Si

Schematic view of anodization cell

Si + 4HF + (4-n)h+ rarr SiF4 + 4H+ + ne-

Dissolution chemistries

AMMT etching system for 4rsquorsquo Si wafers with programmable power supply and dedicated software for time-based current profiles

PS morphology ndash SEM characterisation

nano-PS (lt 15 nm)

meso-PS (50- 100 nm)macro-PS (ca 1microm)

Pore size wire size

Electrolyte type HF concentration Doping type and level

Illumination

Experimental ndash PS fabricated by electrochemical etching

p-type silicon (100) with 6 - 10 middotcm resistivity

HF - C2H5OH electrolyte with 15 18 25

concentration

current densities have been 15 mAcm2 and 25 mAcm2

NoConcetration

HF()

Current density

(mAcm2)

Time(min)

Porosity

()

1 25 15 6 58

2 25 20 6 60

3 25 25 6 62

4 18 15 6 65

5 18 20 6 70

6 15 15 6 80

7 15 25 6 88

The orange-red photoluminescence from the porous

silicon is clearly visible when the wafer illuminated by UV

light

550 600 650 700 750 800 850 00

02

04

06

08

10 88 82 70 65 62 60 58

PL

Inte

nsity (n

orm

ate

va

lue

s)

Wavelength (nm)

Photoluminescence spectra of PS Si-p samples with different porosities (58 - 88)

PL

peak

pos

ition

(nm

)

Porosity ()

PL maximum dependence with porosities

The PL emission from PS is observable at

wavelengths ranging from the ultraviolet to

the infrared the normalized spectra recorded

for different experimental samples

demonstrate the dependence on porosity

PL emission from PS

bull the PL peaks for high porosity PS samples are centred around 650-720 nm in visible range due to quantuum confinement and surface states effectsbull a shift of the PL peak position towards high photon energies with the increase of the PS porosity is observed

thermal treatment at different temperatures between

300degC and 750degC

550 600 650 700 750 800 85000

20x103

40x103

60x103

80x103

10x104

12x104

14x104

16x104

18x104

20x104

22x104

24x104

62 PS 62 PS + TT 315oC 62 PS + TT 750oC

Inte

nsita

te P

L (u

a)

Lungime de unda (nm)Wavelength (nm)

PL

inte

nsi

ty (

au

)

550 600 650 700 750 800 8500

10000

20000

30000

40000

50000

676

667

70 PS 70 PS + oxidare anodica

Inte

nsita

te P

L (u

a)

Lungime de unda (nm)

PL

inte

nsi

ty (

au

)

Wavelength (nm)

The PL peaks for high porosity PS samples are centred around 650-720 nm in visible range due to quantuum confinement and surface states effects

bull a shift of the PL peak position towards high photon energies with the increase of the PS porosity is

observed

bull the stabilisation methods lead to a redshift of PL peak when thermal treatments were used and a

blueshift of PL peak when anodic oxidation was used supplementary an improvement of PL intensity was

observed

Additional treatments for PS optoelectronic properties stabilisation

anodic oxidation

Porous silicon biosensor Chalenges (i) integration of biological systems with PS increasing

of the immobilised biomolecule number on the

surface creation of stable covalent bonds

(ii) using micronanoscale materials for amplification of

the detected signal (SERS SEIR SEF)

(iii) using PS as receptor and transducer

(iv) integrating optics with low-power portable devices

PS is RECEPTOR for biological molecules

selective for the analyte (DNA single strand

proteins antibodies enzymes) constitutes the molecular

recognition element

PS is DETECTOR (signal transducer) for biochemical interactions

electrical conductance impedance

(electro)chemical

Optical

1048592 Luminescence

1048592 Phosphorescence

1048592 Internal Reflection Spectroscopy

1048592 Reflectometric Interference Spectroscopy

1048592 Ellipsometry

1048592 Fluorescencemdashintensity lifetime polarization

1048592 Fluorescence resonance energy transfer

1048592 Absorbance

1048592 Raman Scattering SERS

1048592 Surface plasmon resonance

1048592 Interference

1 Fixed position arrays2 Single Particle Encoding

PS host matrixsupport for immobilization

of sensing biomolecule

Techniques for immobilization range from

physical adsorption to the replacement of hydride bonds

with Si alkyls and to antibody bonding at functionalized

PSi surface with subsequent antibody-antigen

interactions

The biological molecules provide the sensor with its selectivity while the transducer determines the extent of the interaction between the biomolecules and the analyte

PS functionalisation

550 600 650 700 750 800 85000

02

04

06

08

10

PL

inte

nsi

ty (

no

rma

lise

d v

alu

es)

Wavelength (nm)

LN3 functLN3

Material in the pores changes the spectral response

PL shift towards smaller energies after functionalisation

SEM image of porous silicon surface before (a) and after (b) BSA deposition

Affinity anchoring This method is commonly used to load proteins -oxidised PS has a negative surface charge - molecules with positive charges will be spontaneously adsorbed on the inner pore walls and surface

Covalent binding To prepare the surface for the capture of BSA proteins the device is first thermally oxidized at 8000C to form a silica-like internal surface The sensor is then treated with 2 amino-propyltrimethoxy-silane to create amino groups on the internal oxide surface for bio-molecule recognition

PCs can be designed to localize the electric field in the low refractive index region (eg air pores) which makes thesensors extremely sensitive to a small refractive index change produced by bio-molecule immobilization on the pore walls

2

2 amino propyltrimethoxy silane

8000C

500 550 600 650 700 750 800 8500

2000

4000

6000

8000

10000

12000

14000

PL

inte

nsi

ty (

au

)

Wavelength (nm)

LN 2

LN 3

LN3 interconnected lines

bull similar recording conditions were (488 nm frequency of excitation and 87

mW nominal power)

bull the intensity of PL emission is three times larger in the case of

semicircular microcavities leading to an important improvement of detection

Si substrate microstructuration and porosification

LN2 pyramidal structures

Silicon substrate was micropatterned prior porosification process as an array of pyramids (right) or as semi-

circular cavities (left)

Si substrate microstructuration and porosification

PL spectra recorded for three array of microcavities 03 06 si 1 microm

1 microm

06 microm

03 microm

The micropatterned substrate is an array of piramidal

cavities which have to act similar to a collimating device

and light emitted from sample traveling in detector

direction to be reflected by the side walls improving the

sensitivity of the biosensor

rarr gold nanoparticles (AuNP) immobilized inside the pores of the porous silicon layer

rarr thiol molecules self assembled on AuNP so that they can recognize a given protein

rarr In the absence of any protein the photoluminescence of the PS is absorbed by the AuNP at a given

wavelength rarr when the protein specifically binds to the AuNP the absorption due to the SPR is modified and

the detected signal is enhanced

Elaboration of PSMetallic plasmonic nanostructures

The combination of the PS with Au-NP

makes possible to design an optical

biosensor where the light source is

the silicon itself (photoluminescence)

and the chemical transducer is the

functionalized AuNP

Functionalised gold nanoparticles act as nano amplifiers

Luminescence of the PS is modified by the SPR of the AuNPs

A specific protein is recognised by the AuNP (specific absorption)

Spherical gold and silver nanoparticles can be used as substrates in SERS-based molecule detection due to their

advantages in -local scattering field enhancing -surface chemical modifications - biocompatibility- well established chemical synthesis process

The intrinsic plasmon resonance of single nanospheres and the plasmon coupling between adjacent

nanospheres are considered as the key and necessary conditions for local field enhancing

Gold PS

X ray diffraction analyses The metallic nanocrystallites orientation on nanostructurated Si

substrates and the influence of additional anealing treatments ndash

The Au (111)nc-Si surface has

a higher density of atoms

comparatively with Au (100)

this favours the attachment

of a higher number of atoms

and bio-molecules on the gold

surface

12 14 16 18 20 22 24 26 28 30 32 34 36102

103

104

Au (311)

Au (220)

Si (400)

Au (222)

Au (200)

Au (111)

I [i

mp

40sec

]

2 [o]

RX3 - as deposited RX1 - T500 RX2 - T900

Au 38 PS

16 18 20 22 24 26 28 30 32 34 36

200

400

600

800

1000

Au (311)

Si (400)

Au (220)

Au (200)

I [im

p4

0sec

]

2 [o]

RX6 - as deposited RX4 - T500 RX5 - T900

Au (111)

Au 60 PS

For AuPS (60) nucleation begin on Au (111) planes more rapid than for AuPS (38)

(i) in the as-deposited mesoporous films the initial nucleation begin on Au (220) planes

(ii) after the thermal annealing at 5000C the crystallisation process on the (111) planes

become dominant and a (111) texture is obtained

(iii) the thermal annealing at 9000C induces an increase of the (111) crystallites indicating a

clear crystallisation on (111) planes

In Au macroporous silicon the Au (111) texture of crystallites became predominant

Au PS

Experimental The AuPSSi

and AgPSSi

nanocomposites layers were

thermal treated at 500 and

9000C in reducing

atmosphere (H2 and N2)

Aumacroporous silicon

Experimental AgNO3 salt and AgNO3 1 ethanolic solution were also deposited on the PSSi substrate

40 50 60 70 80 90 100 110 120

15000

30000

45000

2

Ag(111)

Ag(400)

Si(331)

Co

K

Co KSi(400)Ag(200)

I [

imp

10s

ec]

Ag - RX5 T500 Ag - RX4 As deposited AgPS-Si(400)

40 50 60 70 80 90 100 110 120 1300

10000

20000

30000

40000

50000

60000

2

Si(400)

Ag(400)

Ag(222)

Si(331)

Co

K

Co K

Si(400)

Ag(200)

Ag(111)

I

[imp

10

sec]

Ag - RX4 As deposited AgPS-Si(400) Ag - RX6 T900 AgPS-Si(400) - Fe filter

The Ag as-deposited films obtainrd from diluted solution of AgNO3 are in an initial stage

of crystallisation with a high amorphous content

In the case of AgPS samples the

annealing treatment at T=500oC has no

effect from the point of view of the

microstructure analysis of the Ag films

on PS (crystallization continue on (200)

planes the films have a higher

crystalline content) while annealing at

900o C produces a mixed (111) and

(200) texture with bigger grain sizes

SilverSi nanocomposite layers

Optical microscope image using a UV filter of AgPS

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Micro- and nanostructured silicon preparation

Porous silicon ndash PS - is obtained by electrochemical dissolution of silicon in HF-based

solution

Etching requires holes (electron injection) to break bonds

Resistivity ~ 106 Ωcm similar to intrinsic Si

Schematic view of anodization cell

Si + 4HF + (4-n)h+ rarr SiF4 + 4H+ + ne-

Dissolution chemistries

AMMT etching system for 4rsquorsquo Si wafers with programmable power supply and dedicated software for time-based current profiles

PS morphology ndash SEM characterisation

nano-PS (lt 15 nm)

meso-PS (50- 100 nm)macro-PS (ca 1microm)

Pore size wire size

Electrolyte type HF concentration Doping type and level

Illumination

Experimental ndash PS fabricated by electrochemical etching

p-type silicon (100) with 6 - 10 middotcm resistivity

HF - C2H5OH electrolyte with 15 18 25

concentration

current densities have been 15 mAcm2 and 25 mAcm2

NoConcetration

HF()

Current density

(mAcm2)

Time(min)

Porosity

()

1 25 15 6 58

2 25 20 6 60

3 25 25 6 62

4 18 15 6 65

5 18 20 6 70

6 15 15 6 80

7 15 25 6 88

The orange-red photoluminescence from the porous

silicon is clearly visible when the wafer illuminated by UV

light

550 600 650 700 750 800 850 00

02

04

06

08

10 88 82 70 65 62 60 58

PL

Inte

nsity (n

orm

ate

va

lue

s)

Wavelength (nm)

Photoluminescence spectra of PS Si-p samples with different porosities (58 - 88)

PL

peak

pos

ition

(nm

)

Porosity ()

PL maximum dependence with porosities

The PL emission from PS is observable at

wavelengths ranging from the ultraviolet to

the infrared the normalized spectra recorded

for different experimental samples

demonstrate the dependence on porosity

PL emission from PS

bull the PL peaks for high porosity PS samples are centred around 650-720 nm in visible range due to quantuum confinement and surface states effectsbull a shift of the PL peak position towards high photon energies with the increase of the PS porosity is observed

thermal treatment at different temperatures between

300degC and 750degC

550 600 650 700 750 800 85000

20x103

40x103

60x103

80x103

10x104

12x104

14x104

16x104

18x104

20x104

22x104

24x104

62 PS 62 PS + TT 315oC 62 PS + TT 750oC

Inte

nsita

te P

L (u

a)

Lungime de unda (nm)Wavelength (nm)

PL

inte

nsi

ty (

au

)

550 600 650 700 750 800 8500

10000

20000

30000

40000

50000

676

667

70 PS 70 PS + oxidare anodica

Inte

nsita

te P

L (u

a)

Lungime de unda (nm)

PL

inte

nsi

ty (

au

)

Wavelength (nm)

The PL peaks for high porosity PS samples are centred around 650-720 nm in visible range due to quantuum confinement and surface states effects

bull a shift of the PL peak position towards high photon energies with the increase of the PS porosity is

observed

bull the stabilisation methods lead to a redshift of PL peak when thermal treatments were used and a

blueshift of PL peak when anodic oxidation was used supplementary an improvement of PL intensity was

observed

Additional treatments for PS optoelectronic properties stabilisation

anodic oxidation

Porous silicon biosensor Chalenges (i) integration of biological systems with PS increasing

of the immobilised biomolecule number on the

surface creation of stable covalent bonds

(ii) using micronanoscale materials for amplification of

the detected signal (SERS SEIR SEF)

(iii) using PS as receptor and transducer

(iv) integrating optics with low-power portable devices

PS is RECEPTOR for biological molecules

selective for the analyte (DNA single strand

proteins antibodies enzymes) constitutes the molecular

recognition element

PS is DETECTOR (signal transducer) for biochemical interactions

electrical conductance impedance

(electro)chemical

Optical

1048592 Luminescence

1048592 Phosphorescence

1048592 Internal Reflection Spectroscopy

1048592 Reflectometric Interference Spectroscopy

1048592 Ellipsometry

1048592 Fluorescencemdashintensity lifetime polarization

1048592 Fluorescence resonance energy transfer

1048592 Absorbance

1048592 Raman Scattering SERS

1048592 Surface plasmon resonance

1048592 Interference

1 Fixed position arrays2 Single Particle Encoding

PS host matrixsupport for immobilization

of sensing biomolecule

Techniques for immobilization range from

physical adsorption to the replacement of hydride bonds

with Si alkyls and to antibody bonding at functionalized

PSi surface with subsequent antibody-antigen

interactions

The biological molecules provide the sensor with its selectivity while the transducer determines the extent of the interaction between the biomolecules and the analyte

PS functionalisation

550 600 650 700 750 800 85000

02

04

06

08

10

PL

inte

nsi

ty (

no

rma

lise

d v

alu

es)

Wavelength (nm)

LN3 functLN3

Material in the pores changes the spectral response

PL shift towards smaller energies after functionalisation

SEM image of porous silicon surface before (a) and after (b) BSA deposition

Affinity anchoring This method is commonly used to load proteins -oxidised PS has a negative surface charge - molecules with positive charges will be spontaneously adsorbed on the inner pore walls and surface

Covalent binding To prepare the surface for the capture of BSA proteins the device is first thermally oxidized at 8000C to form a silica-like internal surface The sensor is then treated with 2 amino-propyltrimethoxy-silane to create amino groups on the internal oxide surface for bio-molecule recognition

PCs can be designed to localize the electric field in the low refractive index region (eg air pores) which makes thesensors extremely sensitive to a small refractive index change produced by bio-molecule immobilization on the pore walls

2

2 amino propyltrimethoxy silane

8000C

500 550 600 650 700 750 800 8500

2000

4000

6000

8000

10000

12000

14000

PL

inte

nsi

ty (

au

)

Wavelength (nm)

LN 2

LN 3

LN3 interconnected lines

bull similar recording conditions were (488 nm frequency of excitation and 87

mW nominal power)

bull the intensity of PL emission is three times larger in the case of

semicircular microcavities leading to an important improvement of detection

Si substrate microstructuration and porosification

LN2 pyramidal structures

Silicon substrate was micropatterned prior porosification process as an array of pyramids (right) or as semi-

circular cavities (left)

Si substrate microstructuration and porosification

PL spectra recorded for three array of microcavities 03 06 si 1 microm

1 microm

06 microm

03 microm

The micropatterned substrate is an array of piramidal

cavities which have to act similar to a collimating device

and light emitted from sample traveling in detector

direction to be reflected by the side walls improving the

sensitivity of the biosensor

rarr gold nanoparticles (AuNP) immobilized inside the pores of the porous silicon layer

rarr thiol molecules self assembled on AuNP so that they can recognize a given protein

rarr In the absence of any protein the photoluminescence of the PS is absorbed by the AuNP at a given

wavelength rarr when the protein specifically binds to the AuNP the absorption due to the SPR is modified and

the detected signal is enhanced

Elaboration of PSMetallic plasmonic nanostructures

The combination of the PS with Au-NP

makes possible to design an optical

biosensor where the light source is

the silicon itself (photoluminescence)

and the chemical transducer is the

functionalized AuNP

Functionalised gold nanoparticles act as nano amplifiers

Luminescence of the PS is modified by the SPR of the AuNPs

A specific protein is recognised by the AuNP (specific absorption)

Spherical gold and silver nanoparticles can be used as substrates in SERS-based molecule detection due to their

advantages in -local scattering field enhancing -surface chemical modifications - biocompatibility- well established chemical synthesis process

The intrinsic plasmon resonance of single nanospheres and the plasmon coupling between adjacent

nanospheres are considered as the key and necessary conditions for local field enhancing

Gold PS

X ray diffraction analyses The metallic nanocrystallites orientation on nanostructurated Si

substrates and the influence of additional anealing treatments ndash

The Au (111)nc-Si surface has

a higher density of atoms

comparatively with Au (100)

this favours the attachment

of a higher number of atoms

and bio-molecules on the gold

surface

12 14 16 18 20 22 24 26 28 30 32 34 36102

103

104

Au (311)

Au (220)

Si (400)

Au (222)

Au (200)

Au (111)

I [i

mp

40sec

]

2 [o]

RX3 - as deposited RX1 - T500 RX2 - T900

Au 38 PS

16 18 20 22 24 26 28 30 32 34 36

200

400

600

800

1000

Au (311)

Si (400)

Au (220)

Au (200)

I [im

p4

0sec

]

2 [o]

RX6 - as deposited RX4 - T500 RX5 - T900

Au (111)

Au 60 PS

For AuPS (60) nucleation begin on Au (111) planes more rapid than for AuPS (38)

(i) in the as-deposited mesoporous films the initial nucleation begin on Au (220) planes

(ii) after the thermal annealing at 5000C the crystallisation process on the (111) planes

become dominant and a (111) texture is obtained

(iii) the thermal annealing at 9000C induces an increase of the (111) crystallites indicating a

clear crystallisation on (111) planes

In Au macroporous silicon the Au (111) texture of crystallites became predominant

Au PS

Experimental The AuPSSi

and AgPSSi

nanocomposites layers were

thermal treated at 500 and

9000C in reducing

atmosphere (H2 and N2)

Aumacroporous silicon

Experimental AgNO3 salt and AgNO3 1 ethanolic solution were also deposited on the PSSi substrate

40 50 60 70 80 90 100 110 120

15000

30000

45000

2

Ag(111)

Ag(400)

Si(331)

Co

K

Co KSi(400)Ag(200)

I [

imp

10s

ec]

Ag - RX5 T500 Ag - RX4 As deposited AgPS-Si(400)

40 50 60 70 80 90 100 110 120 1300

10000

20000

30000

40000

50000

60000

2

Si(400)

Ag(400)

Ag(222)

Si(331)

Co

K

Co K

Si(400)

Ag(200)

Ag(111)

I

[imp

10

sec]

Ag - RX4 As deposited AgPS-Si(400) Ag - RX6 T900 AgPS-Si(400) - Fe filter

The Ag as-deposited films obtainrd from diluted solution of AgNO3 are in an initial stage

of crystallisation with a high amorphous content

In the case of AgPS samples the

annealing treatment at T=500oC has no

effect from the point of view of the

microstructure analysis of the Ag films

on PS (crystallization continue on (200)

planes the films have a higher

crystalline content) while annealing at

900o C produces a mixed (111) and

(200) texture with bigger grain sizes

SilverSi nanocomposite layers

Optical microscope image using a UV filter of AgPS

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

PS morphology ndash SEM characterisation

nano-PS (lt 15 nm)

meso-PS (50- 100 nm)macro-PS (ca 1microm)

Pore size wire size

Electrolyte type HF concentration Doping type and level

Illumination

Experimental ndash PS fabricated by electrochemical etching

p-type silicon (100) with 6 - 10 middotcm resistivity

HF - C2H5OH electrolyte with 15 18 25

concentration

current densities have been 15 mAcm2 and 25 mAcm2

NoConcetration

HF()

Current density

(mAcm2)

Time(min)

Porosity

()

1 25 15 6 58

2 25 20 6 60

3 25 25 6 62

4 18 15 6 65

5 18 20 6 70

6 15 15 6 80

7 15 25 6 88

The orange-red photoluminescence from the porous

silicon is clearly visible when the wafer illuminated by UV

light

550 600 650 700 750 800 850 00

02

04

06

08

10 88 82 70 65 62 60 58

PL

Inte

nsity (n

orm

ate

va

lue

s)

Wavelength (nm)

Photoluminescence spectra of PS Si-p samples with different porosities (58 - 88)

PL

peak

pos

ition

(nm

)

Porosity ()

PL maximum dependence with porosities

The PL emission from PS is observable at

wavelengths ranging from the ultraviolet to

the infrared the normalized spectra recorded

for different experimental samples

demonstrate the dependence on porosity

PL emission from PS

bull the PL peaks for high porosity PS samples are centred around 650-720 nm in visible range due to quantuum confinement and surface states effectsbull a shift of the PL peak position towards high photon energies with the increase of the PS porosity is observed

thermal treatment at different temperatures between

300degC and 750degC

550 600 650 700 750 800 85000

20x103

40x103

60x103

80x103

10x104

12x104

14x104

16x104

18x104

20x104

22x104

24x104

62 PS 62 PS + TT 315oC 62 PS + TT 750oC

Inte

nsita

te P

L (u

a)

Lungime de unda (nm)Wavelength (nm)

PL

inte

nsi

ty (

au

)

550 600 650 700 750 800 8500

10000

20000

30000

40000

50000

676

667

70 PS 70 PS + oxidare anodica

Inte

nsita

te P

L (u

a)

Lungime de unda (nm)

PL

inte

nsi

ty (

au

)

Wavelength (nm)

The PL peaks for high porosity PS samples are centred around 650-720 nm in visible range due to quantuum confinement and surface states effects

bull a shift of the PL peak position towards high photon energies with the increase of the PS porosity is

observed

bull the stabilisation methods lead to a redshift of PL peak when thermal treatments were used and a

blueshift of PL peak when anodic oxidation was used supplementary an improvement of PL intensity was

observed

Additional treatments for PS optoelectronic properties stabilisation

anodic oxidation

Porous silicon biosensor Chalenges (i) integration of biological systems with PS increasing

of the immobilised biomolecule number on the

surface creation of stable covalent bonds

(ii) using micronanoscale materials for amplification of

the detected signal (SERS SEIR SEF)

(iii) using PS as receptor and transducer

(iv) integrating optics with low-power portable devices

PS is RECEPTOR for biological molecules

selective for the analyte (DNA single strand

proteins antibodies enzymes) constitutes the molecular

recognition element

PS is DETECTOR (signal transducer) for biochemical interactions

electrical conductance impedance

(electro)chemical

Optical

1048592 Luminescence

1048592 Phosphorescence

1048592 Internal Reflection Spectroscopy

1048592 Reflectometric Interference Spectroscopy

1048592 Ellipsometry

1048592 Fluorescencemdashintensity lifetime polarization

1048592 Fluorescence resonance energy transfer

1048592 Absorbance

1048592 Raman Scattering SERS

1048592 Surface plasmon resonance

1048592 Interference

1 Fixed position arrays2 Single Particle Encoding

PS host matrixsupport for immobilization

of sensing biomolecule

Techniques for immobilization range from

physical adsorption to the replacement of hydride bonds

with Si alkyls and to antibody bonding at functionalized

PSi surface with subsequent antibody-antigen

interactions

The biological molecules provide the sensor with its selectivity while the transducer determines the extent of the interaction between the biomolecules and the analyte

PS functionalisation

550 600 650 700 750 800 85000

02

04

06

08

10

PL

inte

nsi

ty (

no

rma

lise

d v

alu

es)

Wavelength (nm)

LN3 functLN3

Material in the pores changes the spectral response

PL shift towards smaller energies after functionalisation

SEM image of porous silicon surface before (a) and after (b) BSA deposition

Affinity anchoring This method is commonly used to load proteins -oxidised PS has a negative surface charge - molecules with positive charges will be spontaneously adsorbed on the inner pore walls and surface

Covalent binding To prepare the surface for the capture of BSA proteins the device is first thermally oxidized at 8000C to form a silica-like internal surface The sensor is then treated with 2 amino-propyltrimethoxy-silane to create amino groups on the internal oxide surface for bio-molecule recognition

PCs can be designed to localize the electric field in the low refractive index region (eg air pores) which makes thesensors extremely sensitive to a small refractive index change produced by bio-molecule immobilization on the pore walls

2

2 amino propyltrimethoxy silane

8000C

500 550 600 650 700 750 800 8500

2000

4000

6000

8000

10000

12000

14000

PL

inte

nsi

ty (

au

)

Wavelength (nm)

LN 2

LN 3

LN3 interconnected lines

bull similar recording conditions were (488 nm frequency of excitation and 87

mW nominal power)

bull the intensity of PL emission is three times larger in the case of

semicircular microcavities leading to an important improvement of detection

Si substrate microstructuration and porosification

LN2 pyramidal structures

Silicon substrate was micropatterned prior porosification process as an array of pyramids (right) or as semi-

circular cavities (left)

Si substrate microstructuration and porosification

PL spectra recorded for three array of microcavities 03 06 si 1 microm

1 microm

06 microm

03 microm

The micropatterned substrate is an array of piramidal

cavities which have to act similar to a collimating device

and light emitted from sample traveling in detector

direction to be reflected by the side walls improving the

sensitivity of the biosensor

rarr gold nanoparticles (AuNP) immobilized inside the pores of the porous silicon layer

rarr thiol molecules self assembled on AuNP so that they can recognize a given protein

rarr In the absence of any protein the photoluminescence of the PS is absorbed by the AuNP at a given

wavelength rarr when the protein specifically binds to the AuNP the absorption due to the SPR is modified and

the detected signal is enhanced

Elaboration of PSMetallic plasmonic nanostructures

The combination of the PS with Au-NP

makes possible to design an optical

biosensor where the light source is

the silicon itself (photoluminescence)

and the chemical transducer is the

functionalized AuNP

Functionalised gold nanoparticles act as nano amplifiers

Luminescence of the PS is modified by the SPR of the AuNPs

A specific protein is recognised by the AuNP (specific absorption)

Spherical gold and silver nanoparticles can be used as substrates in SERS-based molecule detection due to their

advantages in -local scattering field enhancing -surface chemical modifications - biocompatibility- well established chemical synthesis process

The intrinsic plasmon resonance of single nanospheres and the plasmon coupling between adjacent

nanospheres are considered as the key and necessary conditions for local field enhancing

Gold PS

X ray diffraction analyses The metallic nanocrystallites orientation on nanostructurated Si

substrates and the influence of additional anealing treatments ndash

The Au (111)nc-Si surface has

a higher density of atoms

comparatively with Au (100)

this favours the attachment

of a higher number of atoms

and bio-molecules on the gold

surface

12 14 16 18 20 22 24 26 28 30 32 34 36102

103

104

Au (311)

Au (220)

Si (400)

Au (222)

Au (200)

Au (111)

I [i

mp

40sec

]

2 [o]

RX3 - as deposited RX1 - T500 RX2 - T900

Au 38 PS

16 18 20 22 24 26 28 30 32 34 36

200

400

600

800

1000

Au (311)

Si (400)

Au (220)

Au (200)

I [im

p4

0sec

]

2 [o]

RX6 - as deposited RX4 - T500 RX5 - T900

Au (111)

Au 60 PS

For AuPS (60) nucleation begin on Au (111) planes more rapid than for AuPS (38)

(i) in the as-deposited mesoporous films the initial nucleation begin on Au (220) planes

(ii) after the thermal annealing at 5000C the crystallisation process on the (111) planes

become dominant and a (111) texture is obtained

(iii) the thermal annealing at 9000C induces an increase of the (111) crystallites indicating a

clear crystallisation on (111) planes

In Au macroporous silicon the Au (111) texture of crystallites became predominant

Au PS

Experimental The AuPSSi

and AgPSSi

nanocomposites layers were

thermal treated at 500 and

9000C in reducing

atmosphere (H2 and N2)

Aumacroporous silicon

Experimental AgNO3 salt and AgNO3 1 ethanolic solution were also deposited on the PSSi substrate

40 50 60 70 80 90 100 110 120

15000

30000

45000

2

Ag(111)

Ag(400)

Si(331)

Co

K

Co KSi(400)Ag(200)

I [

imp

10s

ec]

Ag - RX5 T500 Ag - RX4 As deposited AgPS-Si(400)

40 50 60 70 80 90 100 110 120 1300

10000

20000

30000

40000

50000

60000

2

Si(400)

Ag(400)

Ag(222)

Si(331)

Co

K

Co K

Si(400)

Ag(200)

Ag(111)

I

[imp

10

sec]

Ag - RX4 As deposited AgPS-Si(400) Ag - RX6 T900 AgPS-Si(400) - Fe filter

The Ag as-deposited films obtainrd from diluted solution of AgNO3 are in an initial stage

of crystallisation with a high amorphous content

In the case of AgPS samples the

annealing treatment at T=500oC has no

effect from the point of view of the

microstructure analysis of the Ag films

on PS (crystallization continue on (200)

planes the films have a higher

crystalline content) while annealing at

900o C produces a mixed (111) and

(200) texture with bigger grain sizes

SilverSi nanocomposite layers

Optical microscope image using a UV filter of AgPS

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Experimental ndash PS fabricated by electrochemical etching

p-type silicon (100) with 6 - 10 middotcm resistivity

HF - C2H5OH electrolyte with 15 18 25

concentration

current densities have been 15 mAcm2 and 25 mAcm2

NoConcetration

HF()

Current density

(mAcm2)

Time(min)

Porosity

()

1 25 15 6 58

2 25 20 6 60

3 25 25 6 62

4 18 15 6 65

5 18 20 6 70

6 15 15 6 80

7 15 25 6 88

The orange-red photoluminescence from the porous

silicon is clearly visible when the wafer illuminated by UV

light

550 600 650 700 750 800 850 00

02

04

06

08

10 88 82 70 65 62 60 58

PL

Inte

nsity (n

orm

ate

va

lue

s)

Wavelength (nm)

Photoluminescence spectra of PS Si-p samples with different porosities (58 - 88)

PL

peak

pos

ition

(nm

)

Porosity ()

PL maximum dependence with porosities

The PL emission from PS is observable at

wavelengths ranging from the ultraviolet to

the infrared the normalized spectra recorded

for different experimental samples

demonstrate the dependence on porosity

PL emission from PS

bull the PL peaks for high porosity PS samples are centred around 650-720 nm in visible range due to quantuum confinement and surface states effectsbull a shift of the PL peak position towards high photon energies with the increase of the PS porosity is observed

thermal treatment at different temperatures between

300degC and 750degC

550 600 650 700 750 800 85000

20x103

40x103

60x103

80x103

10x104

12x104

14x104

16x104

18x104

20x104

22x104

24x104

62 PS 62 PS + TT 315oC 62 PS + TT 750oC

Inte

nsita

te P

L (u

a)

Lungime de unda (nm)Wavelength (nm)

PL

inte

nsi

ty (

au

)

550 600 650 700 750 800 8500

10000

20000

30000

40000

50000

676

667

70 PS 70 PS + oxidare anodica

Inte

nsita

te P

L (u

a)

Lungime de unda (nm)

PL

inte

nsi

ty (

au

)

Wavelength (nm)

The PL peaks for high porosity PS samples are centred around 650-720 nm in visible range due to quantuum confinement and surface states effects

bull a shift of the PL peak position towards high photon energies with the increase of the PS porosity is

observed

bull the stabilisation methods lead to a redshift of PL peak when thermal treatments were used and a

blueshift of PL peak when anodic oxidation was used supplementary an improvement of PL intensity was

observed

Additional treatments for PS optoelectronic properties stabilisation

anodic oxidation

Porous silicon biosensor Chalenges (i) integration of biological systems with PS increasing

of the immobilised biomolecule number on the

surface creation of stable covalent bonds

(ii) using micronanoscale materials for amplification of

the detected signal (SERS SEIR SEF)

(iii) using PS as receptor and transducer

(iv) integrating optics with low-power portable devices

PS is RECEPTOR for biological molecules

selective for the analyte (DNA single strand

proteins antibodies enzymes) constitutes the molecular

recognition element

PS is DETECTOR (signal transducer) for biochemical interactions

electrical conductance impedance

(electro)chemical

Optical

1048592 Luminescence

1048592 Phosphorescence

1048592 Internal Reflection Spectroscopy

1048592 Reflectometric Interference Spectroscopy

1048592 Ellipsometry

1048592 Fluorescencemdashintensity lifetime polarization

1048592 Fluorescence resonance energy transfer

1048592 Absorbance

1048592 Raman Scattering SERS

1048592 Surface plasmon resonance

1048592 Interference

1 Fixed position arrays2 Single Particle Encoding

PS host matrixsupport for immobilization

of sensing biomolecule

Techniques for immobilization range from

physical adsorption to the replacement of hydride bonds

with Si alkyls and to antibody bonding at functionalized

PSi surface with subsequent antibody-antigen

interactions

The biological molecules provide the sensor with its selectivity while the transducer determines the extent of the interaction between the biomolecules and the analyte

PS functionalisation

550 600 650 700 750 800 85000

02

04

06

08

10

PL

inte

nsi

ty (

no

rma

lise

d v

alu

es)

Wavelength (nm)

LN3 functLN3

Material in the pores changes the spectral response

PL shift towards smaller energies after functionalisation

SEM image of porous silicon surface before (a) and after (b) BSA deposition

Affinity anchoring This method is commonly used to load proteins -oxidised PS has a negative surface charge - molecules with positive charges will be spontaneously adsorbed on the inner pore walls and surface

Covalent binding To prepare the surface for the capture of BSA proteins the device is first thermally oxidized at 8000C to form a silica-like internal surface The sensor is then treated with 2 amino-propyltrimethoxy-silane to create amino groups on the internal oxide surface for bio-molecule recognition

PCs can be designed to localize the electric field in the low refractive index region (eg air pores) which makes thesensors extremely sensitive to a small refractive index change produced by bio-molecule immobilization on the pore walls

2

2 amino propyltrimethoxy silane

8000C

500 550 600 650 700 750 800 8500

2000

4000

6000

8000

10000

12000

14000

PL

inte

nsi

ty (

au

)

Wavelength (nm)

LN 2

LN 3

LN3 interconnected lines

bull similar recording conditions were (488 nm frequency of excitation and 87

mW nominal power)

bull the intensity of PL emission is three times larger in the case of

semicircular microcavities leading to an important improvement of detection

Si substrate microstructuration and porosification

LN2 pyramidal structures

Silicon substrate was micropatterned prior porosification process as an array of pyramids (right) or as semi-

circular cavities (left)

Si substrate microstructuration and porosification

PL spectra recorded for three array of microcavities 03 06 si 1 microm

1 microm

06 microm

03 microm

The micropatterned substrate is an array of piramidal

cavities which have to act similar to a collimating device

and light emitted from sample traveling in detector

direction to be reflected by the side walls improving the

sensitivity of the biosensor

rarr gold nanoparticles (AuNP) immobilized inside the pores of the porous silicon layer

rarr thiol molecules self assembled on AuNP so that they can recognize a given protein

rarr In the absence of any protein the photoluminescence of the PS is absorbed by the AuNP at a given

wavelength rarr when the protein specifically binds to the AuNP the absorption due to the SPR is modified and

the detected signal is enhanced

Elaboration of PSMetallic plasmonic nanostructures

The combination of the PS with Au-NP

makes possible to design an optical

biosensor where the light source is

the silicon itself (photoluminescence)

and the chemical transducer is the

functionalized AuNP

Functionalised gold nanoparticles act as nano amplifiers

Luminescence of the PS is modified by the SPR of the AuNPs

A specific protein is recognised by the AuNP (specific absorption)

Spherical gold and silver nanoparticles can be used as substrates in SERS-based molecule detection due to their

advantages in -local scattering field enhancing -surface chemical modifications - biocompatibility- well established chemical synthesis process

The intrinsic plasmon resonance of single nanospheres and the plasmon coupling between adjacent

nanospheres are considered as the key and necessary conditions for local field enhancing

Gold PS

X ray diffraction analyses The metallic nanocrystallites orientation on nanostructurated Si

substrates and the influence of additional anealing treatments ndash

The Au (111)nc-Si surface has

a higher density of atoms

comparatively with Au (100)

this favours the attachment

of a higher number of atoms

and bio-molecules on the gold

surface

12 14 16 18 20 22 24 26 28 30 32 34 36102

103

104

Au (311)

Au (220)

Si (400)

Au (222)

Au (200)

Au (111)

I [i

mp

40sec

]

2 [o]

RX3 - as deposited RX1 - T500 RX2 - T900

Au 38 PS

16 18 20 22 24 26 28 30 32 34 36

200

400

600

800

1000

Au (311)

Si (400)

Au (220)

Au (200)

I [im

p4

0sec

]

2 [o]

RX6 - as deposited RX4 - T500 RX5 - T900

Au (111)

Au 60 PS

For AuPS (60) nucleation begin on Au (111) planes more rapid than for AuPS (38)

(i) in the as-deposited mesoporous films the initial nucleation begin on Au (220) planes

(ii) after the thermal annealing at 5000C the crystallisation process on the (111) planes

become dominant and a (111) texture is obtained

(iii) the thermal annealing at 9000C induces an increase of the (111) crystallites indicating a

clear crystallisation on (111) planes

In Au macroporous silicon the Au (111) texture of crystallites became predominant

Au PS

Experimental The AuPSSi

and AgPSSi

nanocomposites layers were

thermal treated at 500 and

9000C in reducing

atmosphere (H2 and N2)

Aumacroporous silicon

Experimental AgNO3 salt and AgNO3 1 ethanolic solution were also deposited on the PSSi substrate

40 50 60 70 80 90 100 110 120

15000

30000

45000

2

Ag(111)

Ag(400)

Si(331)

Co

K

Co KSi(400)Ag(200)

I [

imp

10s

ec]

Ag - RX5 T500 Ag - RX4 As deposited AgPS-Si(400)

40 50 60 70 80 90 100 110 120 1300

10000

20000

30000

40000

50000

60000

2

Si(400)

Ag(400)

Ag(222)

Si(331)

Co

K

Co K

Si(400)

Ag(200)

Ag(111)

I

[imp

10

sec]

Ag - RX4 As deposited AgPS-Si(400) Ag - RX6 T900 AgPS-Si(400) - Fe filter

The Ag as-deposited films obtainrd from diluted solution of AgNO3 are in an initial stage

of crystallisation with a high amorphous content

In the case of AgPS samples the

annealing treatment at T=500oC has no

effect from the point of view of the

microstructure analysis of the Ag films

on PS (crystallization continue on (200)

planes the films have a higher

crystalline content) while annealing at

900o C produces a mixed (111) and

(200) texture with bigger grain sizes

SilverSi nanocomposite layers

Optical microscope image using a UV filter of AgPS

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

thermal treatment at different temperatures between

300degC and 750degC

550 600 650 700 750 800 85000

20x103

40x103

60x103

80x103

10x104

12x104

14x104

16x104

18x104

20x104

22x104

24x104

62 PS 62 PS + TT 315oC 62 PS + TT 750oC

Inte

nsita

te P

L (u

a)

Lungime de unda (nm)Wavelength (nm)

PL

inte

nsi

ty (

au

)

550 600 650 700 750 800 8500

10000

20000

30000

40000

50000

676

667

70 PS 70 PS + oxidare anodica

Inte

nsita

te P

L (u

a)

Lungime de unda (nm)

PL

inte

nsi

ty (

au

)

Wavelength (nm)

The PL peaks for high porosity PS samples are centred around 650-720 nm in visible range due to quantuum confinement and surface states effects

bull a shift of the PL peak position towards high photon energies with the increase of the PS porosity is

observed

bull the stabilisation methods lead to a redshift of PL peak when thermal treatments were used and a

blueshift of PL peak when anodic oxidation was used supplementary an improvement of PL intensity was

observed

Additional treatments for PS optoelectronic properties stabilisation

anodic oxidation

Porous silicon biosensor Chalenges (i) integration of biological systems with PS increasing

of the immobilised biomolecule number on the

surface creation of stable covalent bonds

(ii) using micronanoscale materials for amplification of

the detected signal (SERS SEIR SEF)

(iii) using PS as receptor and transducer

(iv) integrating optics with low-power portable devices

PS is RECEPTOR for biological molecules

selective for the analyte (DNA single strand

proteins antibodies enzymes) constitutes the molecular

recognition element

PS is DETECTOR (signal transducer) for biochemical interactions

electrical conductance impedance

(electro)chemical

Optical

1048592 Luminescence

1048592 Phosphorescence

1048592 Internal Reflection Spectroscopy

1048592 Reflectometric Interference Spectroscopy

1048592 Ellipsometry

1048592 Fluorescencemdashintensity lifetime polarization

1048592 Fluorescence resonance energy transfer

1048592 Absorbance

1048592 Raman Scattering SERS

1048592 Surface plasmon resonance

1048592 Interference

1 Fixed position arrays2 Single Particle Encoding

PS host matrixsupport for immobilization

of sensing biomolecule

Techniques for immobilization range from

physical adsorption to the replacement of hydride bonds

with Si alkyls and to antibody bonding at functionalized

PSi surface with subsequent antibody-antigen

interactions

The biological molecules provide the sensor with its selectivity while the transducer determines the extent of the interaction between the biomolecules and the analyte

PS functionalisation

550 600 650 700 750 800 85000

02

04

06

08

10

PL

inte

nsi

ty (

no

rma

lise

d v

alu

es)

Wavelength (nm)

LN3 functLN3

Material in the pores changes the spectral response

PL shift towards smaller energies after functionalisation

SEM image of porous silicon surface before (a) and after (b) BSA deposition

Affinity anchoring This method is commonly used to load proteins -oxidised PS has a negative surface charge - molecules with positive charges will be spontaneously adsorbed on the inner pore walls and surface

Covalent binding To prepare the surface for the capture of BSA proteins the device is first thermally oxidized at 8000C to form a silica-like internal surface The sensor is then treated with 2 amino-propyltrimethoxy-silane to create amino groups on the internal oxide surface for bio-molecule recognition

PCs can be designed to localize the electric field in the low refractive index region (eg air pores) which makes thesensors extremely sensitive to a small refractive index change produced by bio-molecule immobilization on the pore walls

2

2 amino propyltrimethoxy silane

8000C

500 550 600 650 700 750 800 8500

2000

4000

6000

8000

10000

12000

14000

PL

inte

nsi

ty (

au

)

Wavelength (nm)

LN 2

LN 3

LN3 interconnected lines

bull similar recording conditions were (488 nm frequency of excitation and 87

mW nominal power)

bull the intensity of PL emission is three times larger in the case of

semicircular microcavities leading to an important improvement of detection

Si substrate microstructuration and porosification

LN2 pyramidal structures

Silicon substrate was micropatterned prior porosification process as an array of pyramids (right) or as semi-

circular cavities (left)

Si substrate microstructuration and porosification

PL spectra recorded for three array of microcavities 03 06 si 1 microm

1 microm

06 microm

03 microm

The micropatterned substrate is an array of piramidal

cavities which have to act similar to a collimating device

and light emitted from sample traveling in detector

direction to be reflected by the side walls improving the

sensitivity of the biosensor

rarr gold nanoparticles (AuNP) immobilized inside the pores of the porous silicon layer

rarr thiol molecules self assembled on AuNP so that they can recognize a given protein

rarr In the absence of any protein the photoluminescence of the PS is absorbed by the AuNP at a given

wavelength rarr when the protein specifically binds to the AuNP the absorption due to the SPR is modified and

the detected signal is enhanced

Elaboration of PSMetallic plasmonic nanostructures

The combination of the PS with Au-NP

makes possible to design an optical

biosensor where the light source is

the silicon itself (photoluminescence)

and the chemical transducer is the

functionalized AuNP

Functionalised gold nanoparticles act as nano amplifiers

Luminescence of the PS is modified by the SPR of the AuNPs

A specific protein is recognised by the AuNP (specific absorption)

Spherical gold and silver nanoparticles can be used as substrates in SERS-based molecule detection due to their

advantages in -local scattering field enhancing -surface chemical modifications - biocompatibility- well established chemical synthesis process

The intrinsic plasmon resonance of single nanospheres and the plasmon coupling between adjacent

nanospheres are considered as the key and necessary conditions for local field enhancing

Gold PS

X ray diffraction analyses The metallic nanocrystallites orientation on nanostructurated Si

substrates and the influence of additional anealing treatments ndash

The Au (111)nc-Si surface has

a higher density of atoms

comparatively with Au (100)

this favours the attachment

of a higher number of atoms

and bio-molecules on the gold

surface

12 14 16 18 20 22 24 26 28 30 32 34 36102

103

104

Au (311)

Au (220)

Si (400)

Au (222)

Au (200)

Au (111)

I [i

mp

40sec

]

2 [o]

RX3 - as deposited RX1 - T500 RX2 - T900

Au 38 PS

16 18 20 22 24 26 28 30 32 34 36

200

400

600

800

1000

Au (311)

Si (400)

Au (220)

Au (200)

I [im

p4

0sec

]

2 [o]

RX6 - as deposited RX4 - T500 RX5 - T900

Au (111)

Au 60 PS

For AuPS (60) nucleation begin on Au (111) planes more rapid than for AuPS (38)

(i) in the as-deposited mesoporous films the initial nucleation begin on Au (220) planes

(ii) after the thermal annealing at 5000C the crystallisation process on the (111) planes

become dominant and a (111) texture is obtained

(iii) the thermal annealing at 9000C induces an increase of the (111) crystallites indicating a

clear crystallisation on (111) planes

In Au macroporous silicon the Au (111) texture of crystallites became predominant

Au PS

Experimental The AuPSSi

and AgPSSi

nanocomposites layers were

thermal treated at 500 and

9000C in reducing

atmosphere (H2 and N2)

Aumacroporous silicon

Experimental AgNO3 salt and AgNO3 1 ethanolic solution were also deposited on the PSSi substrate

40 50 60 70 80 90 100 110 120

15000

30000

45000

2

Ag(111)

Ag(400)

Si(331)

Co

K

Co KSi(400)Ag(200)

I [

imp

10s

ec]

Ag - RX5 T500 Ag - RX4 As deposited AgPS-Si(400)

40 50 60 70 80 90 100 110 120 1300

10000

20000

30000

40000

50000

60000

2

Si(400)

Ag(400)

Ag(222)

Si(331)

Co

K

Co K

Si(400)

Ag(200)

Ag(111)

I

[imp

10

sec]

Ag - RX4 As deposited AgPS-Si(400) Ag - RX6 T900 AgPS-Si(400) - Fe filter

The Ag as-deposited films obtainrd from diluted solution of AgNO3 are in an initial stage

of crystallisation with a high amorphous content

In the case of AgPS samples the

annealing treatment at T=500oC has no

effect from the point of view of the

microstructure analysis of the Ag films

on PS (crystallization continue on (200)

planes the films have a higher

crystalline content) while annealing at

900o C produces a mixed (111) and

(200) texture with bigger grain sizes

SilverSi nanocomposite layers

Optical microscope image using a UV filter of AgPS

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Porous silicon biosensor Chalenges (i) integration of biological systems with PS increasing

of the immobilised biomolecule number on the

surface creation of stable covalent bonds

(ii) using micronanoscale materials for amplification of

the detected signal (SERS SEIR SEF)

(iii) using PS as receptor and transducer

(iv) integrating optics with low-power portable devices

PS is RECEPTOR for biological molecules

selective for the analyte (DNA single strand

proteins antibodies enzymes) constitutes the molecular

recognition element

PS is DETECTOR (signal transducer) for biochemical interactions

electrical conductance impedance

(electro)chemical

Optical

1048592 Luminescence

1048592 Phosphorescence

1048592 Internal Reflection Spectroscopy

1048592 Reflectometric Interference Spectroscopy

1048592 Ellipsometry

1048592 Fluorescencemdashintensity lifetime polarization

1048592 Fluorescence resonance energy transfer

1048592 Absorbance

1048592 Raman Scattering SERS

1048592 Surface plasmon resonance

1048592 Interference

1 Fixed position arrays2 Single Particle Encoding

PS host matrixsupport for immobilization

of sensing biomolecule

Techniques for immobilization range from

physical adsorption to the replacement of hydride bonds

with Si alkyls and to antibody bonding at functionalized

PSi surface with subsequent antibody-antigen

interactions

The biological molecules provide the sensor with its selectivity while the transducer determines the extent of the interaction between the biomolecules and the analyte

PS functionalisation

550 600 650 700 750 800 85000

02

04

06

08

10

PL

inte

nsi

ty (

no

rma

lise

d v

alu

es)

Wavelength (nm)

LN3 functLN3

Material in the pores changes the spectral response

PL shift towards smaller energies after functionalisation

SEM image of porous silicon surface before (a) and after (b) BSA deposition

Affinity anchoring This method is commonly used to load proteins -oxidised PS has a negative surface charge - molecules with positive charges will be spontaneously adsorbed on the inner pore walls and surface

Covalent binding To prepare the surface for the capture of BSA proteins the device is first thermally oxidized at 8000C to form a silica-like internal surface The sensor is then treated with 2 amino-propyltrimethoxy-silane to create amino groups on the internal oxide surface for bio-molecule recognition

PCs can be designed to localize the electric field in the low refractive index region (eg air pores) which makes thesensors extremely sensitive to a small refractive index change produced by bio-molecule immobilization on the pore walls

2

2 amino propyltrimethoxy silane

8000C

500 550 600 650 700 750 800 8500

2000

4000

6000

8000

10000

12000

14000

PL

inte

nsi

ty (

au

)

Wavelength (nm)

LN 2

LN 3

LN3 interconnected lines

bull similar recording conditions were (488 nm frequency of excitation and 87

mW nominal power)

bull the intensity of PL emission is three times larger in the case of

semicircular microcavities leading to an important improvement of detection

Si substrate microstructuration and porosification

LN2 pyramidal structures

Silicon substrate was micropatterned prior porosification process as an array of pyramids (right) or as semi-

circular cavities (left)

Si substrate microstructuration and porosification

PL spectra recorded for three array of microcavities 03 06 si 1 microm

1 microm

06 microm

03 microm

The micropatterned substrate is an array of piramidal

cavities which have to act similar to a collimating device

and light emitted from sample traveling in detector

direction to be reflected by the side walls improving the

sensitivity of the biosensor

rarr gold nanoparticles (AuNP) immobilized inside the pores of the porous silicon layer

rarr thiol molecules self assembled on AuNP so that they can recognize a given protein

rarr In the absence of any protein the photoluminescence of the PS is absorbed by the AuNP at a given

wavelength rarr when the protein specifically binds to the AuNP the absorption due to the SPR is modified and

the detected signal is enhanced

Elaboration of PSMetallic plasmonic nanostructures

The combination of the PS with Au-NP

makes possible to design an optical

biosensor where the light source is

the silicon itself (photoluminescence)

and the chemical transducer is the

functionalized AuNP

Functionalised gold nanoparticles act as nano amplifiers

Luminescence of the PS is modified by the SPR of the AuNPs

A specific protein is recognised by the AuNP (specific absorption)

Spherical gold and silver nanoparticles can be used as substrates in SERS-based molecule detection due to their

advantages in -local scattering field enhancing -surface chemical modifications - biocompatibility- well established chemical synthesis process

The intrinsic plasmon resonance of single nanospheres and the plasmon coupling between adjacent

nanospheres are considered as the key and necessary conditions for local field enhancing

Gold PS

X ray diffraction analyses The metallic nanocrystallites orientation on nanostructurated Si

substrates and the influence of additional anealing treatments ndash

The Au (111)nc-Si surface has

a higher density of atoms

comparatively with Au (100)

this favours the attachment

of a higher number of atoms

and bio-molecules on the gold

surface

12 14 16 18 20 22 24 26 28 30 32 34 36102

103

104

Au (311)

Au (220)

Si (400)

Au (222)

Au (200)

Au (111)

I [i

mp

40sec

]

2 [o]

RX3 - as deposited RX1 - T500 RX2 - T900

Au 38 PS

16 18 20 22 24 26 28 30 32 34 36

200

400

600

800

1000

Au (311)

Si (400)

Au (220)

Au (200)

I [im

p4

0sec

]

2 [o]

RX6 - as deposited RX4 - T500 RX5 - T900

Au (111)

Au 60 PS

For AuPS (60) nucleation begin on Au (111) planes more rapid than for AuPS (38)

(i) in the as-deposited mesoporous films the initial nucleation begin on Au (220) planes

(ii) after the thermal annealing at 5000C the crystallisation process on the (111) planes

become dominant and a (111) texture is obtained

(iii) the thermal annealing at 9000C induces an increase of the (111) crystallites indicating a

clear crystallisation on (111) planes

In Au macroporous silicon the Au (111) texture of crystallites became predominant

Au PS

Experimental The AuPSSi

and AgPSSi

nanocomposites layers were

thermal treated at 500 and

9000C in reducing

atmosphere (H2 and N2)

Aumacroporous silicon

Experimental AgNO3 salt and AgNO3 1 ethanolic solution were also deposited on the PSSi substrate

40 50 60 70 80 90 100 110 120

15000

30000

45000

2

Ag(111)

Ag(400)

Si(331)

Co

K

Co KSi(400)Ag(200)

I [

imp

10s

ec]

Ag - RX5 T500 Ag - RX4 As deposited AgPS-Si(400)

40 50 60 70 80 90 100 110 120 1300

10000

20000

30000

40000

50000

60000

2

Si(400)

Ag(400)

Ag(222)

Si(331)

Co

K

Co K

Si(400)

Ag(200)

Ag(111)

I

[imp

10

sec]

Ag - RX4 As deposited AgPS-Si(400) Ag - RX6 T900 AgPS-Si(400) - Fe filter

The Ag as-deposited films obtainrd from diluted solution of AgNO3 are in an initial stage

of crystallisation with a high amorphous content

In the case of AgPS samples the

annealing treatment at T=500oC has no

effect from the point of view of the

microstructure analysis of the Ag films

on PS (crystallization continue on (200)

planes the films have a higher

crystalline content) while annealing at

900o C produces a mixed (111) and

(200) texture with bigger grain sizes

SilverSi nanocomposite layers

Optical microscope image using a UV filter of AgPS

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

PS functionalisation

550 600 650 700 750 800 85000

02

04

06

08

10

PL

inte

nsi

ty (

no

rma

lise

d v

alu

es)

Wavelength (nm)

LN3 functLN3

Material in the pores changes the spectral response

PL shift towards smaller energies after functionalisation

SEM image of porous silicon surface before (a) and after (b) BSA deposition

Affinity anchoring This method is commonly used to load proteins -oxidised PS has a negative surface charge - molecules with positive charges will be spontaneously adsorbed on the inner pore walls and surface

Covalent binding To prepare the surface for the capture of BSA proteins the device is first thermally oxidized at 8000C to form a silica-like internal surface The sensor is then treated with 2 amino-propyltrimethoxy-silane to create amino groups on the internal oxide surface for bio-molecule recognition

PCs can be designed to localize the electric field in the low refractive index region (eg air pores) which makes thesensors extremely sensitive to a small refractive index change produced by bio-molecule immobilization on the pore walls

2

2 amino propyltrimethoxy silane

8000C

500 550 600 650 700 750 800 8500

2000

4000

6000

8000

10000

12000

14000

PL

inte

nsi

ty (

au

)

Wavelength (nm)

LN 2

LN 3

LN3 interconnected lines

bull similar recording conditions were (488 nm frequency of excitation and 87

mW nominal power)

bull the intensity of PL emission is three times larger in the case of

semicircular microcavities leading to an important improvement of detection

Si substrate microstructuration and porosification

LN2 pyramidal structures

Silicon substrate was micropatterned prior porosification process as an array of pyramids (right) or as semi-

circular cavities (left)

Si substrate microstructuration and porosification

PL spectra recorded for three array of microcavities 03 06 si 1 microm

1 microm

06 microm

03 microm

The micropatterned substrate is an array of piramidal

cavities which have to act similar to a collimating device

and light emitted from sample traveling in detector

direction to be reflected by the side walls improving the

sensitivity of the biosensor

rarr gold nanoparticles (AuNP) immobilized inside the pores of the porous silicon layer

rarr thiol molecules self assembled on AuNP so that they can recognize a given protein

rarr In the absence of any protein the photoluminescence of the PS is absorbed by the AuNP at a given

wavelength rarr when the protein specifically binds to the AuNP the absorption due to the SPR is modified and

the detected signal is enhanced

Elaboration of PSMetallic plasmonic nanostructures

The combination of the PS with Au-NP

makes possible to design an optical

biosensor where the light source is

the silicon itself (photoluminescence)

and the chemical transducer is the

functionalized AuNP

Functionalised gold nanoparticles act as nano amplifiers

Luminescence of the PS is modified by the SPR of the AuNPs

A specific protein is recognised by the AuNP (specific absorption)

Spherical gold and silver nanoparticles can be used as substrates in SERS-based molecule detection due to their

advantages in -local scattering field enhancing -surface chemical modifications - biocompatibility- well established chemical synthesis process

The intrinsic plasmon resonance of single nanospheres and the plasmon coupling between adjacent

nanospheres are considered as the key and necessary conditions for local field enhancing

Gold PS

X ray diffraction analyses The metallic nanocrystallites orientation on nanostructurated Si

substrates and the influence of additional anealing treatments ndash

The Au (111)nc-Si surface has

a higher density of atoms

comparatively with Au (100)

this favours the attachment

of a higher number of atoms

and bio-molecules on the gold

surface

12 14 16 18 20 22 24 26 28 30 32 34 36102

103

104

Au (311)

Au (220)

Si (400)

Au (222)

Au (200)

Au (111)

I [i

mp

40sec

]

2 [o]

RX3 - as deposited RX1 - T500 RX2 - T900

Au 38 PS

16 18 20 22 24 26 28 30 32 34 36

200

400

600

800

1000

Au (311)

Si (400)

Au (220)

Au (200)

I [im

p4

0sec

]

2 [o]

RX6 - as deposited RX4 - T500 RX5 - T900

Au (111)

Au 60 PS

For AuPS (60) nucleation begin on Au (111) planes more rapid than for AuPS (38)

(i) in the as-deposited mesoporous films the initial nucleation begin on Au (220) planes

(ii) after the thermal annealing at 5000C the crystallisation process on the (111) planes

become dominant and a (111) texture is obtained

(iii) the thermal annealing at 9000C induces an increase of the (111) crystallites indicating a

clear crystallisation on (111) planes

In Au macroporous silicon the Au (111) texture of crystallites became predominant

Au PS

Experimental The AuPSSi

and AgPSSi

nanocomposites layers were

thermal treated at 500 and

9000C in reducing

atmosphere (H2 and N2)

Aumacroporous silicon

Experimental AgNO3 salt and AgNO3 1 ethanolic solution were also deposited on the PSSi substrate

40 50 60 70 80 90 100 110 120

15000

30000

45000

2

Ag(111)

Ag(400)

Si(331)

Co

K

Co KSi(400)Ag(200)

I [

imp

10s

ec]

Ag - RX5 T500 Ag - RX4 As deposited AgPS-Si(400)

40 50 60 70 80 90 100 110 120 1300

10000

20000

30000

40000

50000

60000

2

Si(400)

Ag(400)

Ag(222)

Si(331)

Co

K

Co K

Si(400)

Ag(200)

Ag(111)

I

[imp

10

sec]

Ag - RX4 As deposited AgPS-Si(400) Ag - RX6 T900 AgPS-Si(400) - Fe filter

The Ag as-deposited films obtainrd from diluted solution of AgNO3 are in an initial stage

of crystallisation with a high amorphous content

In the case of AgPS samples the

annealing treatment at T=500oC has no

effect from the point of view of the

microstructure analysis of the Ag films

on PS (crystallization continue on (200)

planes the films have a higher

crystalline content) while annealing at

900o C produces a mixed (111) and

(200) texture with bigger grain sizes

SilverSi nanocomposite layers

Optical microscope image using a UV filter of AgPS

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

500 550 600 650 700 750 800 8500

2000

4000

6000

8000

10000

12000

14000

PL

inte

nsi

ty (

au

)

Wavelength (nm)

LN 2

LN 3

LN3 interconnected lines

bull similar recording conditions were (488 nm frequency of excitation and 87

mW nominal power)

bull the intensity of PL emission is three times larger in the case of

semicircular microcavities leading to an important improvement of detection

Si substrate microstructuration and porosification

LN2 pyramidal structures

Silicon substrate was micropatterned prior porosification process as an array of pyramids (right) or as semi-

circular cavities (left)

Si substrate microstructuration and porosification

PL spectra recorded for three array of microcavities 03 06 si 1 microm

1 microm

06 microm

03 microm

The micropatterned substrate is an array of piramidal

cavities which have to act similar to a collimating device

and light emitted from sample traveling in detector

direction to be reflected by the side walls improving the

sensitivity of the biosensor

rarr gold nanoparticles (AuNP) immobilized inside the pores of the porous silicon layer

rarr thiol molecules self assembled on AuNP so that they can recognize a given protein

rarr In the absence of any protein the photoluminescence of the PS is absorbed by the AuNP at a given

wavelength rarr when the protein specifically binds to the AuNP the absorption due to the SPR is modified and

the detected signal is enhanced

Elaboration of PSMetallic plasmonic nanostructures

The combination of the PS with Au-NP

makes possible to design an optical

biosensor where the light source is

the silicon itself (photoluminescence)

and the chemical transducer is the

functionalized AuNP

Functionalised gold nanoparticles act as nano amplifiers

Luminescence of the PS is modified by the SPR of the AuNPs

A specific protein is recognised by the AuNP (specific absorption)

Spherical gold and silver nanoparticles can be used as substrates in SERS-based molecule detection due to their

advantages in -local scattering field enhancing -surface chemical modifications - biocompatibility- well established chemical synthesis process

The intrinsic plasmon resonance of single nanospheres and the plasmon coupling between adjacent

nanospheres are considered as the key and necessary conditions for local field enhancing

Gold PS

X ray diffraction analyses The metallic nanocrystallites orientation on nanostructurated Si

substrates and the influence of additional anealing treatments ndash

The Au (111)nc-Si surface has

a higher density of atoms

comparatively with Au (100)

this favours the attachment

of a higher number of atoms

and bio-molecules on the gold

surface

12 14 16 18 20 22 24 26 28 30 32 34 36102

103

104

Au (311)

Au (220)

Si (400)

Au (222)

Au (200)

Au (111)

I [i

mp

40sec

]

2 [o]

RX3 - as deposited RX1 - T500 RX2 - T900

Au 38 PS

16 18 20 22 24 26 28 30 32 34 36

200

400

600

800

1000

Au (311)

Si (400)

Au (220)

Au (200)

I [im

p4

0sec

]

2 [o]

RX6 - as deposited RX4 - T500 RX5 - T900

Au (111)

Au 60 PS

For AuPS (60) nucleation begin on Au (111) planes more rapid than for AuPS (38)

(i) in the as-deposited mesoporous films the initial nucleation begin on Au (220) planes

(ii) after the thermal annealing at 5000C the crystallisation process on the (111) planes

become dominant and a (111) texture is obtained

(iii) the thermal annealing at 9000C induces an increase of the (111) crystallites indicating a

clear crystallisation on (111) planes

In Au macroporous silicon the Au (111) texture of crystallites became predominant

Au PS

Experimental The AuPSSi

and AgPSSi

nanocomposites layers were

thermal treated at 500 and

9000C in reducing

atmosphere (H2 and N2)

Aumacroporous silicon

Experimental AgNO3 salt and AgNO3 1 ethanolic solution were also deposited on the PSSi substrate

40 50 60 70 80 90 100 110 120

15000

30000

45000

2

Ag(111)

Ag(400)

Si(331)

Co

K

Co KSi(400)Ag(200)

I [

imp

10s

ec]

Ag - RX5 T500 Ag - RX4 As deposited AgPS-Si(400)

40 50 60 70 80 90 100 110 120 1300

10000

20000

30000

40000

50000

60000

2

Si(400)

Ag(400)

Ag(222)

Si(331)

Co

K

Co K

Si(400)

Ag(200)

Ag(111)

I

[imp

10

sec]

Ag - RX4 As deposited AgPS-Si(400) Ag - RX6 T900 AgPS-Si(400) - Fe filter

The Ag as-deposited films obtainrd from diluted solution of AgNO3 are in an initial stage

of crystallisation with a high amorphous content

In the case of AgPS samples the

annealing treatment at T=500oC has no

effect from the point of view of the

microstructure analysis of the Ag films

on PS (crystallization continue on (200)

planes the films have a higher

crystalline content) while annealing at

900o C produces a mixed (111) and

(200) texture with bigger grain sizes

SilverSi nanocomposite layers

Optical microscope image using a UV filter of AgPS

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Si substrate microstructuration and porosification

PL spectra recorded for three array of microcavities 03 06 si 1 microm

1 microm

06 microm

03 microm

The micropatterned substrate is an array of piramidal

cavities which have to act similar to a collimating device

and light emitted from sample traveling in detector

direction to be reflected by the side walls improving the

sensitivity of the biosensor

rarr gold nanoparticles (AuNP) immobilized inside the pores of the porous silicon layer

rarr thiol molecules self assembled on AuNP so that they can recognize a given protein

rarr In the absence of any protein the photoluminescence of the PS is absorbed by the AuNP at a given

wavelength rarr when the protein specifically binds to the AuNP the absorption due to the SPR is modified and

the detected signal is enhanced

Elaboration of PSMetallic plasmonic nanostructures

The combination of the PS with Au-NP

makes possible to design an optical

biosensor where the light source is

the silicon itself (photoluminescence)

and the chemical transducer is the

functionalized AuNP

Functionalised gold nanoparticles act as nano amplifiers

Luminescence of the PS is modified by the SPR of the AuNPs

A specific protein is recognised by the AuNP (specific absorption)

Spherical gold and silver nanoparticles can be used as substrates in SERS-based molecule detection due to their

advantages in -local scattering field enhancing -surface chemical modifications - biocompatibility- well established chemical synthesis process

The intrinsic plasmon resonance of single nanospheres and the plasmon coupling between adjacent

nanospheres are considered as the key and necessary conditions for local field enhancing

Gold PS

X ray diffraction analyses The metallic nanocrystallites orientation on nanostructurated Si

substrates and the influence of additional anealing treatments ndash

The Au (111)nc-Si surface has

a higher density of atoms

comparatively with Au (100)

this favours the attachment

of a higher number of atoms

and bio-molecules on the gold

surface

12 14 16 18 20 22 24 26 28 30 32 34 36102

103

104

Au (311)

Au (220)

Si (400)

Au (222)

Au (200)

Au (111)

I [i

mp

40sec

]

2 [o]

RX3 - as deposited RX1 - T500 RX2 - T900

Au 38 PS

16 18 20 22 24 26 28 30 32 34 36

200

400

600

800

1000

Au (311)

Si (400)

Au (220)

Au (200)

I [im

p4

0sec

]

2 [o]

RX6 - as deposited RX4 - T500 RX5 - T900

Au (111)

Au 60 PS

For AuPS (60) nucleation begin on Au (111) planes more rapid than for AuPS (38)

(i) in the as-deposited mesoporous films the initial nucleation begin on Au (220) planes

(ii) after the thermal annealing at 5000C the crystallisation process on the (111) planes

become dominant and a (111) texture is obtained

(iii) the thermal annealing at 9000C induces an increase of the (111) crystallites indicating a

clear crystallisation on (111) planes

In Au macroporous silicon the Au (111) texture of crystallites became predominant

Au PS

Experimental The AuPSSi

and AgPSSi

nanocomposites layers were

thermal treated at 500 and

9000C in reducing

atmosphere (H2 and N2)

Aumacroporous silicon

Experimental AgNO3 salt and AgNO3 1 ethanolic solution were also deposited on the PSSi substrate

40 50 60 70 80 90 100 110 120

15000

30000

45000

2

Ag(111)

Ag(400)

Si(331)

Co

K

Co KSi(400)Ag(200)

I [

imp

10s

ec]

Ag - RX5 T500 Ag - RX4 As deposited AgPS-Si(400)

40 50 60 70 80 90 100 110 120 1300

10000

20000

30000

40000

50000

60000

2

Si(400)

Ag(400)

Ag(222)

Si(331)

Co

K

Co K

Si(400)

Ag(200)

Ag(111)

I

[imp

10

sec]

Ag - RX4 As deposited AgPS-Si(400) Ag - RX6 T900 AgPS-Si(400) - Fe filter

The Ag as-deposited films obtainrd from diluted solution of AgNO3 are in an initial stage

of crystallisation with a high amorphous content

In the case of AgPS samples the

annealing treatment at T=500oC has no

effect from the point of view of the

microstructure analysis of the Ag films

on PS (crystallization continue on (200)

planes the films have a higher

crystalline content) while annealing at

900o C produces a mixed (111) and

(200) texture with bigger grain sizes

SilverSi nanocomposite layers

Optical microscope image using a UV filter of AgPS

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

rarr gold nanoparticles (AuNP) immobilized inside the pores of the porous silicon layer

rarr thiol molecules self assembled on AuNP so that they can recognize a given protein

rarr In the absence of any protein the photoluminescence of the PS is absorbed by the AuNP at a given

wavelength rarr when the protein specifically binds to the AuNP the absorption due to the SPR is modified and

the detected signal is enhanced

Elaboration of PSMetallic plasmonic nanostructures

The combination of the PS with Au-NP

makes possible to design an optical

biosensor where the light source is

the silicon itself (photoluminescence)

and the chemical transducer is the

functionalized AuNP

Functionalised gold nanoparticles act as nano amplifiers

Luminescence of the PS is modified by the SPR of the AuNPs

A specific protein is recognised by the AuNP (specific absorption)

Spherical gold and silver nanoparticles can be used as substrates in SERS-based molecule detection due to their

advantages in -local scattering field enhancing -surface chemical modifications - biocompatibility- well established chemical synthesis process

The intrinsic plasmon resonance of single nanospheres and the plasmon coupling between adjacent

nanospheres are considered as the key and necessary conditions for local field enhancing

Gold PS

X ray diffraction analyses The metallic nanocrystallites orientation on nanostructurated Si

substrates and the influence of additional anealing treatments ndash

The Au (111)nc-Si surface has

a higher density of atoms

comparatively with Au (100)

this favours the attachment

of a higher number of atoms

and bio-molecules on the gold

surface

12 14 16 18 20 22 24 26 28 30 32 34 36102

103

104

Au (311)

Au (220)

Si (400)

Au (222)

Au (200)

Au (111)

I [i

mp

40sec

]

2 [o]

RX3 - as deposited RX1 - T500 RX2 - T900

Au 38 PS

16 18 20 22 24 26 28 30 32 34 36

200

400

600

800

1000

Au (311)

Si (400)

Au (220)

Au (200)

I [im

p4

0sec

]

2 [o]

RX6 - as deposited RX4 - T500 RX5 - T900

Au (111)

Au 60 PS

For AuPS (60) nucleation begin on Au (111) planes more rapid than for AuPS (38)

(i) in the as-deposited mesoporous films the initial nucleation begin on Au (220) planes

(ii) after the thermal annealing at 5000C the crystallisation process on the (111) planes

become dominant and a (111) texture is obtained

(iii) the thermal annealing at 9000C induces an increase of the (111) crystallites indicating a

clear crystallisation on (111) planes

In Au macroporous silicon the Au (111) texture of crystallites became predominant

Au PS

Experimental The AuPSSi

and AgPSSi

nanocomposites layers were

thermal treated at 500 and

9000C in reducing

atmosphere (H2 and N2)

Aumacroporous silicon

Experimental AgNO3 salt and AgNO3 1 ethanolic solution were also deposited on the PSSi substrate

40 50 60 70 80 90 100 110 120

15000

30000

45000

2

Ag(111)

Ag(400)

Si(331)

Co

K

Co KSi(400)Ag(200)

I [

imp

10s

ec]

Ag - RX5 T500 Ag - RX4 As deposited AgPS-Si(400)

40 50 60 70 80 90 100 110 120 1300

10000

20000

30000

40000

50000

60000

2

Si(400)

Ag(400)

Ag(222)

Si(331)

Co

K

Co K

Si(400)

Ag(200)

Ag(111)

I

[imp

10

sec]

Ag - RX4 As deposited AgPS-Si(400) Ag - RX6 T900 AgPS-Si(400) - Fe filter

The Ag as-deposited films obtainrd from diluted solution of AgNO3 are in an initial stage

of crystallisation with a high amorphous content

In the case of AgPS samples the

annealing treatment at T=500oC has no

effect from the point of view of the

microstructure analysis of the Ag films

on PS (crystallization continue on (200)

planes the films have a higher

crystalline content) while annealing at

900o C produces a mixed (111) and

(200) texture with bigger grain sizes

SilverSi nanocomposite layers

Optical microscope image using a UV filter of AgPS

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Gold PS

X ray diffraction analyses The metallic nanocrystallites orientation on nanostructurated Si

substrates and the influence of additional anealing treatments ndash

The Au (111)nc-Si surface has

a higher density of atoms

comparatively with Au (100)

this favours the attachment

of a higher number of atoms

and bio-molecules on the gold

surface

12 14 16 18 20 22 24 26 28 30 32 34 36102

103

104

Au (311)

Au (220)

Si (400)

Au (222)

Au (200)

Au (111)

I [i

mp

40sec

]

2 [o]

RX3 - as deposited RX1 - T500 RX2 - T900

Au 38 PS

16 18 20 22 24 26 28 30 32 34 36

200

400

600

800

1000

Au (311)

Si (400)

Au (220)

Au (200)

I [im

p4

0sec

]

2 [o]

RX6 - as deposited RX4 - T500 RX5 - T900

Au (111)

Au 60 PS

For AuPS (60) nucleation begin on Au (111) planes more rapid than for AuPS (38)

(i) in the as-deposited mesoporous films the initial nucleation begin on Au (220) planes

(ii) after the thermal annealing at 5000C the crystallisation process on the (111) planes

become dominant and a (111) texture is obtained

(iii) the thermal annealing at 9000C induces an increase of the (111) crystallites indicating a

clear crystallisation on (111) planes

In Au macroporous silicon the Au (111) texture of crystallites became predominant

Au PS

Experimental The AuPSSi

and AgPSSi

nanocomposites layers were

thermal treated at 500 and

9000C in reducing

atmosphere (H2 and N2)

Aumacroporous silicon

Experimental AgNO3 salt and AgNO3 1 ethanolic solution were also deposited on the PSSi substrate

40 50 60 70 80 90 100 110 120

15000

30000

45000

2

Ag(111)

Ag(400)

Si(331)

Co

K

Co KSi(400)Ag(200)

I [

imp

10s

ec]

Ag - RX5 T500 Ag - RX4 As deposited AgPS-Si(400)

40 50 60 70 80 90 100 110 120 1300

10000

20000

30000

40000

50000

60000

2

Si(400)

Ag(400)

Ag(222)

Si(331)

Co

K

Co K

Si(400)

Ag(200)

Ag(111)

I

[imp

10

sec]

Ag - RX4 As deposited AgPS-Si(400) Ag - RX6 T900 AgPS-Si(400) - Fe filter

The Ag as-deposited films obtainrd from diluted solution of AgNO3 are in an initial stage

of crystallisation with a high amorphous content

In the case of AgPS samples the

annealing treatment at T=500oC has no

effect from the point of view of the

microstructure analysis of the Ag films

on PS (crystallization continue on (200)

planes the films have a higher

crystalline content) while annealing at

900o C produces a mixed (111) and

(200) texture with bigger grain sizes

SilverSi nanocomposite layers

Optical microscope image using a UV filter of AgPS

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Experimental AgNO3 salt and AgNO3 1 ethanolic solution were also deposited on the PSSi substrate

40 50 60 70 80 90 100 110 120

15000

30000

45000

2

Ag(111)

Ag(400)

Si(331)

Co

K

Co KSi(400)Ag(200)

I [

imp

10s

ec]

Ag - RX5 T500 Ag - RX4 As deposited AgPS-Si(400)

40 50 60 70 80 90 100 110 120 1300

10000

20000

30000

40000

50000

60000

2

Si(400)

Ag(400)

Ag(222)

Si(331)

Co

K

Co K

Si(400)

Ag(200)

Ag(111)

I

[imp

10

sec]

Ag - RX4 As deposited AgPS-Si(400) Ag - RX6 T900 AgPS-Si(400) - Fe filter

The Ag as-deposited films obtainrd from diluted solution of AgNO3 are in an initial stage

of crystallisation with a high amorphous content

In the case of AgPS samples the

annealing treatment at T=500oC has no

effect from the point of view of the

microstructure analysis of the Ag films

on PS (crystallization continue on (200)

planes the films have a higher

crystalline content) while annealing at

900o C produces a mixed (111) and

(200) texture with bigger grain sizes

SilverSi nanocomposite layers

Optical microscope image using a UV filter of AgPS

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Experimental

bull macroporous silicon p-type (100) Si (5ndash10 Ω cm) 4 HF

in DMF 77 mAcm2 current density

bull 100 nm gold layer was deposited by cathodic sputtering

bull 11-mercaptoundecanoic acid (11-MUA) was auto-

assembled on all investigated substrates by their immersion

in 2mM MUA in ethanol solution

SEM image of PVD-evaporated gold on macroPS substrateSEM image of macroPS layer

Raman measurements on the AumacroPS emphasised higher sensitivity of the organic molecule representative picks than the commercial substrate Moreover the Aumacroporous Si is a suitable substrate for (bio)sensors organic molecule conformation on the solid surface being achieved

SERS spectra of 11- MUA adsorbed on different investigated substrates

600 800 1000 1200 1400 1600 1800-2000

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Co

unts

Raman shiftcm-1

11-MUA on commercial-Klarite substrate 11-MUA on gold-PVD on porous silicon 11-MUA on flat PVD-gold substrate 11-MUA on thermal treated PVD-goldporous silicon

substrate substrate

Nanostructured AuSi substrate for organic molecule SERS detection

In the normal resonance Raman the molecule

interacts directly with the electromagnetic field

associated with the traveling wave In SERS this

field is already modulated by the electron cloud

oscillation in the metal and the molecule experience

an enhanced field

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

rarr Nanocrystalline Au(111)PS substrates have important applications in

biochemistry especially in self-assembly of the thiol-end DNA molecule (SAM) such as HS-(CH2)6-5prime-GGC-CAT-CGT-TGA-AGA-TGC-CTC-TGC-C-3prime

Immobilisation of a fluorescent oligonucleotide on AuPs (A ndash 20times B ndash 40times Nikon fluorescence

microscope)

The difference between the fluorescence (FL) emission of PS and fluorophors (Trp and NADH) from NutMix culture medium allowed us to investigate the modifications induced on the PS spectra by NutMix neurons interaction with chip FL spectra reveal a red shifts of the optical signal recorded on the PS - NutMix neurons sample

NADH is a Co-Enzyme naturally present in ALL living cells and is NECESSARY for Cellular development and Energy production TRYPSIN is a serine protease from the pancreas of vertebrates Cleaves peptide bonds involving the amino groups of lysine or arginine

AuPS enhance fluorescence

PS as sensitive element for neurons in NutMix culture

Main applications

bull immunologic biosensors

bull protein and DNA microarray technology

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Investigation of the CHO cells immobilised in PS microreactor

PS as sensitive element for CHO cells investigation

Emission spectra recorded at 280 nm wavelength excitation

PS has an intrinsic fluorescence spectrum and the emission is

modified ndash suffer a peak shift ndash after the PS treatment for cell

growth and their process of fixing

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Microarray technology and immunologic sensors

PS substrates advantages

bull low surface wetting (providing mild immobilisation

conditions ndash a hydrophilic surface at the molecular level)

bull small spots (increased immobilisation density

over a spot improved reaction kinetics high density

arraying)

bull homogeneous probe molecule coverage (uniform

fluorescence intensity over a spot improved data

quality)

bull low fluorescence background (auto-fluorescence)

bull biocompatibility (immobilisation andor adsorption of

biospecific binders with maintained affinity and

selectivity allowing protein digestion to be performed

directly on the surface allowing complex sample analysis

such as blood and tissue lysates)

The need to measure multiple parameters was

solved by bundling several sensors together in

order to multiplex them

Today arrays with tens of thousands and even

hundreds of thousands of features are realised in the

DNAprotein microarray technology

The microarrays use fluorescence signals as the

transduction mechanism

Main requirements for microarray substrates

bull inert and resistent to non-specific adsorption surface

bull the surface should contain functional groups for the facile

immobilization of protein molecules of interest

bull bonding between a protein molecule and a solid surface

should to be strong enough to retain the molecule on the

surface but also sufficiently non intrusive to have minimal

effect on the 3D structure

bull the linking chemistry should control of protein orientation

bull the local chemical environment favors the immobilized

protein molecules to retain their native conformation

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technologyExperimental microarray technique for printing and characterization

-PS and AuPS substrates were investigated for BSA immobilization- each sample was spotted with bovine serum albumin (BSA) - the spots contain Cy3 fluorophores ranging from 2-9 to 1 fluorsmicrom2 - after 24 hours of incubation at 40C they were laser scanned - in order to check if protein is completely bonded on the PS substrate the samples were washed in PBS several times - after that the samples were scanned again in the same conditions - the fluorescence intensity remains almost constant after each washing run

Conclusion Small Cy3 fluorophore concentration of the spoted protein are well defined on the PS array

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Electrical Impedance detection

Binding of DNA inside the PSi matrix induces a change in capacitance and conductanceMott-Schottky plots for different concentration of DNA in electrolyte solution

The capacitance of the ElectrolytePSSi

has been measured as function of DC

potential and the data recorded for

different concentration of DNA in

electrolyte solution are presented The

figure illustrate that the test structure

capacitance plots suffer modifications

by adding different concentrations of

DNA in electrolyte solution the flat

band potential is shifted to lower

values as DNA immobilization starts

from 031V to 024V and 021V

respectively

Experimental dataPhosphate buffered saline- PBS) is a buffer solution commonly used in biological research It is a salty solution containing sodium chloride sodium phosphate and (in some formulations) potassium chloride and potassium phosphate The buffer helps to maintain a constant pH DNA 1 1 microM 50 ml PBS solutionDNA 2 2 microM50ml PBS solution

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Bode (a b) and Nyquist (c) plots for different concentration of DNA in electrolyte solution

The Bode phase angle plots (a) reveal that phase angle attained a maximum at the value of -30deg at

higher frequency region which tends to -20deg as the concentration of DNA is increased opposing in

lower frequencies domain the maximum is initially around -70deg and its value increases to -80deg going

concomitantly with a gradual shift to lower frequencies proving that the interface phenomena

became dominant The corresponding impedance module plots fig (b) show a similar behavior with

a more evident decrease of values in low frequencies domain The Nyquist graphs presented in fig (c)

indicate that the data are not well separated above 200 Hz and clear distinct below with a dominant

interface capacitance

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Nanostructured Si as carriers for controlled drug delivery

AIM different methods for fabrication of PS based

nanostructured microparticles as well as their loading

with organic molecule or anorganic nanoparticles with

therapeutic effect were experimented

Schematic representation of the pn Si structure fabrication in view of porosification

Optical image of a Si microparticle

obtained by porosification using Si3N4

layer as mask followed by an ultrasonation process

SEM images of nanostructured Si microparticles

obtained by selective porosification (a) n-epip+

Si process and (b) n-diffusion in p+Si process

Si pn junctions selective porosification

Si porosification using Si3N4 mask

Si porous multilayers

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Nanostructured Si multilayers as carriers

PS multilayers obtained on p+ Si

Nanoporous Si obtained on p+ Si (50 nm pore

size)

Microparticles with nanoporous structure lower than 8 microm

Current-potential-time diagram for 30 cicles(0570 A 100 sec and 3000 A 100 sec)

Ball mill (1h)

- The alternance of ultrathin layers with different morphologies and corresponding pore diameters ranging from few nanometers to tens of nanometers determine a cleavage phenomenon when a simple ultrasonation treatment is applied - The dimensions of Si microparticles are given by the time of each step and we have used a 10 sec time interval for each porosification step

Current-potential-time diagram for 150 cicles(0554 A 10 sec 2825 A 4 sec)

PS multilayers obtained on p+ Si

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Loading of molecule of therapeutic interest

Chondroitin sulfate (CS) (sulfated glycosaminoglycan -

GAG)

Lactoferrin (immunomodulatory compound-

globular protein with antimicrobial activity)

N-butyl-deoxynojirimycin (NB-DNJ)(an imino sugar that inhibits the

growth of the CT-2A brain tumour)

Ellipsometric parameters Delta and Psi for PSSi APTSPSSi and

NB-DNJAPTSPSSi

Mouse melanoma B16 F10 cells proliferation on different APTSPS devices A- control cells B-

APTSPS-CS C-APTSPS-Lf D- APTSPS-NB-DNJ

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Magnetite nanoparticles in PS carriers

Gold silver and iron oxides were chemically or

deposited by evaporation on porous silicon in

order to assure biocompatibility targeting

antimicrobial and terapeutic properties

SEM images of iron oxide nanoparticles on PS and EDAX analysis of the ironiron oxide on the PS

substrate

P S + F e 2 + F e 3

+ + N H 4 O H F e 3 O 4 P S

FePS for drug delivery

- Two steps (deposition ndash diffusion) process of iron deposited from corresponding salts was realized - Nitrate Fe(NO3)39H2O - and sulfonate - Fe(SO4)7H2O ndash were deposited on PS surface from saturated solutions- Annealing temperatures 1 h or in oven at 650C or 900C - Iron drive-into the PS skeleton was performed at high temperature 6000C in inert (Ar) or reducing (H2N2) atmosphere

Atomic absorption spectroscopy (AAS) measurements for investigation of Fe release in SBF solution

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

Gold nanoparticles on porous silicon carriers

5 mercaptopropyl trimetoxysilane

MPTS

SEM image of gold nanoparticles (7 nm) self-assembled on PS

Optical microscope image using a UV filter of AgPS

Silanization protocol for thiol groups

attachement (Anal Chem 2001 73 2476-

2483)

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

CONCLUSION

1 Medical diagnosis ndash Bionanodetection

Biosensors

Microarray technology

2 Medical therapy Carriers for controlled drug delivery

bull The present study demonstrates that porous silicon can combine the properties of a chemically stable high surface area

host material with the function of an optical transducer which makes it an ideal material for biosensing applications

bull We have improved the PL sensitivity of the biosensor by Si substrate micropatterning before the porosification process

obtaining an effect like in a collimating device where light emitted from sample travelling in detector direction is reflected

by the side walls

bull PS substrate has been used for optimisation microarray imaging technique parameters

bull We have shown how electrochemical impedance spectroscopy (EIS) next to surface plasmon resonance (SPR) could

become a reliable technique for analyzing the changes in interfacial properties of modified active surfaces induced by the

binding of charged molecules

bull Other challenging field of research presented herewith was related to nanostructured silicon particles which can

combine optical with drug delivery properties

Micronanoporous silicon technology is very promising to develop novel devices and

systems that have a biomedical impact

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction

IMT-Nanotechnology Laboratory TeamIrina Kleps Project coordinatorMihaela Miu Impedance SpectroscopyMonica Simion Microarray TechnologyTeodora Ignat SERS substratesFlorea Craciunoiu Microparticle preparationAdina Bragaru Porous silicon fabricationMihai Danila X-ray diffraction

• OUTLINE
• Motivation
• Micro- and nanostructured silicon preparation
• PS morphology ndash SEM characterisation
• Folie 6
• Folie 7
• Porous silicon biosensor
• PS functionalisation
• Folie 10
• Folie 11
• Folie 12
• Folie 13
• Folie 14
• Folie 15
• Folie 16
• Investigation of the CHO cells immobilised in PS microreactor
• Microarray technology and immunologic sensors
• PS and AuPS as substrates for protein immobilisation and fluorescent detection in microarray technology
• Electrical Impedance detection
• Folie 21
• Nanostructured Si as carriers for controlled drug delivery
• Nanostructured Si multilayers as carriers
• Loading of molecule of therapeutic interest
• Magnetite nanoparticles in PS carriers
• Gold nanoparticles on porous silicon carriers
• Folie 27
• IMT-Nanotechnology Laboratory Team Irina Kleps Project coordinator Mihaela Miu Impedance Spectroscopy Monica Simion Microarray Technology Teodora Ignat SERS substrates Florea Craciunoiu Microparticle preparation Adina Bragaru Porous silicon fabrication Mihai Danila X-ray diffraction