Tehnologii laser pulsate pentru transferulpentru transferul,

nanostructurarea si modificarea controlata a

materialelormaterialelorIon N Mihailescu

National Institute for Lasers, Plasma and Radiation Physics, Lasers Department, 409 Atomistilor, PO Box MG-

Ion N. Mihailescu

Physics, Lasers Department, 409 Atomistilor, PO Box MG54, RO-077125, Bucharest-Magurele, Romania

ion.mihailescu@inflpr.ro , lspi.inflpr.ro

Specialization:p- main field: Laser interactions; lasers and plasma physics; nanostructured thin films technology, nano-powders generation and characterization, surface physics and engineering, laser spectroscopy.

other fields: Biophysics and biomedicine; nano bio technologies gas and bio- other fields: Biophysics and biomedicine; nano- bio- technologies, gas- and bio-sensors, plasma and laser theory. - current research interests: Pulsed laser deposition (PLD), modification and characterization of nanostructured thin coatings; matrix assisted pulsed laser evaporation (MAPLE); laser surface studies and processing; biomaterials thin layers; tissue engineering; biomimetic metallic implants; optoelectronics and sensors.

Publications and patents:Publications and patents:- Books: 8 (2 in Romania, 6 abroad)- Papers in refereed journals: 320 in international regular journals; 224 in proceedings of international meetings- Communications to scientific meetings (international conferences, symposiums, workshops, schools): invited lectures: 73; oral contributions: 80; posters: 131; invited seminars: 30 in Romania, 117 abroad - Citations in international journals and books: 1728, h index 21 (according to ISI WebCitations in international journals and books: 1728, h index 21 (according to ISI Web of Knowledge, Web of Science )-Patents: 10 (8 in Romania, 2 abroad)

Pulsed Laser Deposition (PLD)Pulsed Laser Deposition (PLD)What is PLD?

• Laser evaporation• Laser assisted deposition & annealing• Laser flash evaporation• Laser MBE• Hydrodynamic sputtering• Laser ablation

L bl ti d iti• Laser ablation deposition• Photonic sputtering

Characteristics:- Short laser pulse strongly absorbed in solid and / or liquid- Material ejected and collected on nearby substrate- Energetic plasma conductive to good film growth

Experimental PLD/RPLD setupLaser parameters:Excimerλ = 248 nm (308 nm 193 nm)nm, 193 nm)τFWHM ~ 25 nsrep. rate 2 - 50 HzF= (0,2 – 10 ) J/cm2

PLD/RPLD set-up in LSPI lab

Laser – Target Interactiong

Charged ParticlesCharged Particles(e-, Ions, Plasma)

ThermalWave

Laser Pulse

Photons

Vapor

Shock

Vapor

CraterShockWave

First attemptFirst attempt

•Ruby laser, 1 ms/pulse, 3 J/pulse•PbCl2, MoO2, CdTe, PbTe, ZnTe•“Laser beam produced only rough and optically unsatisfactory films”

Explanation of the failureExplanation of the failure• Too long laser pulses (ms in free-running regime)g p ( g g )• Improper laser wavelength (VIS, IR)• 1964 – 1984: technological accumulations

Ad t f hi h i t it li bl l i UV (λ <• Advent of high intensity, reliable laser sources in UV (λ <330 nm)

• Generation of very short laser pulses (ns, ps, fs, as) atreasonable frequency repetition rates (~ 1 kHz)

• The most used laser sources now are:– KrF* (248 nm) XeCl* (308 nm) ArF* (193 nm)KrF (248 nm), XeCl (308 nm), ArF (193 nm)– 4th harmonic of Nd:YAG (266 nm)

Periodic table of the elementsPeriodic table of the elements1H

2He

3Li

4Be

5B

6C

7N

8O

9F

10Ne

11Na

12Mg

13Al

14Si

15P

16S

17Cl

18Ar

19K

20Ca

21Sc

22Ti

23V

24Cr

25Mn

26Fe

27Co

28Ni

29Cu

30Zn

31Ga

32Ge

33As

34Se

35Br

36Kr

37Rb

38Sr

39Y

40Zr

41Nb

42Mo

43Tc

44Ru

45Rh

46Pd

47Ag

48Cd

49In

50Sn

51Sb

52Te

53I

54Xe

55Cs

56Ba *

71Lu

72Hf

73Ta

74W

75Re

76Os

77Ir

78Pt

79Au

80Hg

81Tl

82Pb

83Bi

84Po

85At

86Rn

87Fr

88Ra

**

103Lr

104Rf

105Db

106Sg

107Bh

108Hs

109Mt

110Ds

111Rg

112Uub

113Uut

114Uuq

115Uup

116Uuh

117Uus

118Uuo

*Lanthanoids *57La

58Ce

59Pr

60Nd

61Pm

62Sm

63Eu

64Gd

65Tb

66Dy

67Ho

68Er

69Tm

70Yb

89 90 91 92 93 94 95 96 97 98 99 100 101 102**Actinoids **

89Ac

90Th

91Pa

92U

93Np

94Pu

95Am

96Cm

97Bk

98Cf

99Es

100Fm

101Md

102No

Reactive PLD (RPLD)Reactive PLD (RPLD)

Wh PLD i d t d i th bi• When PLD is conducted in the ambienceof a chemically active gas, a differentcompound as compared to the target iscompound as compared to the target isdeposited. This process is usually calledReactive PLDReactive PLD.

• RPLD is important whenever thesynthesized compound has better qualitiessynthesized compound has better qualitiesand/or is more expensive than thebase/raw material.

PLD: advantagesPLD: advantages• accurate control of the stoichiometry of the deposited material y p

(congruent ablation);

• reduced film contamination due to the use of laser light;

• promotion of the growth of crystalline structures for desired applications;

• energy source independent of the deposition environment;

• relative simplicity of the growth facility offering great experimental ( )versatility (multilayers, doping);

• control of the film thickness (with a precision of > 10-2 Å/pulse).

Working procedureWorking procedureTARGETS AND SUBSTRATE PREPARATION

1) Targets can be manufactured by pressing and sintering from any powder material1) Targets can be manufactured by pressing and sintering from any powder material2) Substrates are cleaned in alcohol or other very pure reagents using an ultrasonic bath and dried with N2;3) Substrate is directly attached to heater;

OPTICS AND CHAMBER PREPARATION1) Target and substrate are placed inside the chamber;2) Laser spot is focused on the target and spot area is measured;3) The substrate is placed parallel normal or oblique to the target;3) The substrate is placed parallel, normal or oblique to the target;4) The thermocouple is fixed to the heater;5) The chamber is evacuated preliminary vacuum (< 10-1 Pa)

(rotary pump)high vacuum (<10-4 Pa)high vacuum ( 10 Pa)

(turbomolecular pump);

6) The substrate is heated under control up to the working temperature (up to1000°C).

Main parameters determining the h t i ti f bt i d t tcharacteristics of obtained structures

• Incident laser fluence, F = E0/Ss

A bi t t d• Ambient gas nature and pressure

• Deposition geometryDeposition geometry

• Cleaning and heating of the collectorg g

• Possible application of the external electric and magnetic fieldsmagnetic fields

Crater formation: Ti targetCrater formation: Ti target

P 1 3 P NP0 = 1.3 Pa N2

-Pronounced micro-relief with asymmetric waves, parallel to borders

-Overall depth of 10 μm, after 104 subsequent laser pulses (f = 3 Hz), quasi-regular periodicity ith l th f 30with a wavelength of 30 μm

-Regular steps are observed

These features are essential for supporting plasma expansion normal to target surface.

Role of the gas pressure: synthesis f TiN b RPLD f Ti i Nof TiN by RPLD of Ti in N2

Pressure (Pa) Description of the deposited filmsPressure (Pa) Description of the deposited films0.06 Traces only of amorphous TiN embedded in amorphous

silicon

0.2 Mixture of amorphous TiN and amorphous silicon

0.7 Polycrystalline f.c.c. TiN with crystallites of 10-15 nm1.33.37.013 0 Oxidized polycrystalline TiN most probably oxy nitride of the13.0 Oxidized polycrystalline TiN, most probably oxy-nitride of the

type TiNxO1-x with crystallites of 5–10 nm

33.0 Mixture of amorphized TiN and amorphized TiO

1000 Poorly crystallized TiO2 with crystallites of a few nm

Existence of an optimum pressure range!

Plasma rolePlasma rolePlasma was considered for long time only a supplementary loss channel.

Absence of plasma

Well developed plasmaplasma plasma

Very low deposition rates

Larger deposition rates

Ablated material ReducedAblated material spread over the entire

surface

Reduced contamination of the

reaction chamber

Non-stoichiometric Usually stoichiometric layers films

Peel rather easily Adherent layers

Practical criterion: good quality adherent thin films are obtained whenever theplasma column length is equal to the target – collector separation distance

Plasma lengthPlasma length

( )[ ] ( ) γγγγγ 3131311 −−= PVEAL *( )[ ] ( ) γγγγ 01−= PVEAL p

- Lp is plasma length;Lp is plasma length;- A is a geometrical factor related with the shape of the laser spot onto targetsurface,- γ ratio of specific heats,

E l- E0 laser energy,- P gas pressure, and- V initial volume of the plasma (V = v0τlasSc, v0 initial speed of the species, τlaslaser pulse duration, and Sc laser spot area).p , c p )

For a given geometry, plasma length is governed by E0 and P. Whenever P is increasing, Lp decreases, while any amplification of E0 induces an increase of Lp.

* This relation applies if we consider an adiabatically expansion.

Plasma expansionPlasma expansion

Plasma expansionPlasma expansionEssential characteristics: initialKnudsen flow of the vapor / plasmacloudThis renders possible the furthercompact e pansion of plasmacompact expansion of plasmaconfined around the normal to thetarget surface.

Z0 – initial length of the plasma

R0= X0/2 – initial width of the plasma which practically coincides with the laser spot dimension

θ - radial angle

h(z θ) – profile of the depositedh(zs, θ) – profile of the deposited film

Experimental evidence and l l icalculations

• h(zs) ~ zs-2

• Deposition rotated with 90°• h(θ) ~ (1+k2tg2θ)-3/2, for θ around the normal h(θ) ~ cosn θ

k0 0.001 0.003 0.01 0.03 0.1 0.3

k(∞) 35.8 20.6 11.3 6.5 3.5 1.9

• K0 = Z0/X0

• k(∞) = Z(∞)/X(∞), i.e. at the moment of the impact on the collector• An extended initial plasma ends in spherical expansion, while a short initial

plasma evolves to a highly forwarded expansion• It is generally erroneous to compare results obtained with different focal

spots (X )spots (X0)

Energy balanceEnergy balance• Es = E0e-α;s 0• AEs = Ea + Et;• Er = (1-A)E0e-2α;

E E E E E• E0 = Er + Epl + Ea + Et

– E0 – laser pulse energyE i id t th t t f– Es – energy incident on the target surface

– Epl – energy absorbed in the plasma– Ea – energy effectively used for ablation– Et - energy dissipated in the target (heat)t gy p g ( )– Er – energy which leaves the interaction zone– A – effective absorptivity on the target– α - optical thickness of the plasma

Δm ablation rate– Δm – ablation rate

Plasma radiation loss and plasma heat transfer to the target are neglected.

Typical exampleTypical example• Al, 308 nm, 100 mJ/pulse, 25 ns, Δm = 0.8 μm/pulse

Et/E0 Er/E0 Ea/E0 Epl/E0 A α

• E + E l ~ 50% of E0

0.2 0.3 0.3 0.2 0.6 0.2

Ea + Epl 50% of E0• Epl ~ 20% of E0• α = 0.2, plasma is fairly transparent to the laser radiation. We note that in

visible and IR typically α>1 (opaque plasma). This makes indispensable thef UV di ti f PLD RPLD d l i th f il f fi t PLDuse of UV radiation for PLD, RPLD and explain the failure of first PLD

attempt by Smith and Turner.

⇒ UV generated plasma is described as “cold” plasma!⇒ UV generated plasma is described as cold plasma!

Illustrative results: PLD of SnO2 in O2Illustrative results: PLD of SnO2 in O2

Particulate free film, with stoichiometric composition

400

(400

)

S O O ount

s)300

SnO

2 (2

11)

Si

SnO2 target, 10 Pa O2, RT, 6 cm, 10 J/cm2, 308 nm Li

n (C

o

100

200

SnO

2 (1

10)

SnO

2 (1

01)

SnO

2 (2

00)

2 (2

20)

(002

)

O2

(221

)

SnO

2 (3

32)

(321

)

0

2-Theta - Scale10 20 30 40 50 60 70 80 90 10

SnO

2

SnO

2

SnO

SnO

2 (

Illustrative results: multilayersIllustrative results: multilayers

AmorphousHA layer

0.812 nm(100)

HA

TiN

XTEM micro-graphXTEM micro graph

HRTEM micrograph showing the interface film HA-TiN buffer layer

TEM studiesMaterials transferred by PLD must resemble the biological ones incomposition, structure, morphology, and functionality.

Characteristic TEM images of (A) HA films deposited on Al2O3 substrates sintered at 1500 ºC and (B) bone nanocrystals

A B

A CB

TEM histograms of HA nanocrystals deposited on Al2O3 substrates sintered at (A) 1400 ºC, (B) 1500 ºC and (C) 1600 ºC

XRD spectra for Mn-CHA films deposited and treated at350°C (# 1), 400°C (# 2), 450°C (# 3)

El t i t di (SAED d HRTEM) l fi d th f ti f HAElectron microscopy studies (SAED and HRTEM) also confirmed the formation of HA. (good accordance with C. F. Koch et al., Mat. Sc. Eng. C, 2006 : XRD showed that crystallization in HA-PLD thin films deposited at RT occurs after annealing at ~ 340 °C)

OCP and Mn-CHA structures exhibit different morphologiesmorphologies

b- OCP: porous, tree-like morphology

dropletsb

- Mn-CHA:

c d

Mn CHA: granular, more compact morphology

Sr-HA structures exhibit rather porous morphologies

c d

SEM micrographs of thin films deposited from (a) Sr0; (b) Sr10 samples.Scale bars = 1 μm

Larger Sr doping induces an increase of porosityLarger Sr doping induces an increase of porosity

Sr distribution in HA coatingsSr distribution in HA coatings

EDS maps recorded from the coatings: (a) TiSr1; (b) TiSr5; and (c) TiSr10

Increase of Sr doping confirmed by EDS

Sr-red, HA blue.

Biocompatibility Tests - Cell Cultures

Human primary osteoblasts (hOB) were cultured on OCP-coated Ti, Mn-CHA –

(Proliferation and Viability)

Human primary osteoblasts (hOB) were cultured on OCP coated Ti, Mn CHA coated Ti, bare Ti

hOB response: SEM micrographs- on bare Ti: (a) after 7 days, (b) after 21 days

Elongated, rod-like morphology

hOB response: SEM micrographs

- OCP coatings: (c) after 7 days, (d) after 21 days

Over time, the cells spread and expand with flattened, polyhedral-morphology.morphology.

Numerous cytoplasmatic extensions → firm attachment

hOB response: SEM micrographs

- Mn-CHA coatings: (e) after 7 days, (f) after 21 days

The cells spread and expand overtime, showing a flattened, polyhedral-morphology;

Fewer cytoplasmatic extensions

hOB response: SEM micrographs

- Sr-HA coatings: SEM micrographs of osteoblasts after 21 days of culture on: (a) TiHA; (b) TiSr5; and (c) TiSr10. Scale bars = 50 μm.

hOB response: SEM micrographs

( ) ; ( ) ; ( ) μ

→ Ti/HA: hOB were flattened, with polygonal configuration and dorsal ruffles; wellattached to the substrate by cellular extensions.→ Ti/Sr doped HA: hOB appear much more flattened and better spread across thep pp psurface.

Florescence microscopy images of hOB S HA tihOB on Sr-HA coatings

P t f t bl t dh i 1 h ft di ( ) TiHAPercentage of osteoblast adhesion 1 hour after seeding on (a) TiHA,(42±4%); (b) TiSr1, (48±8%); (c) TiSr5, (58±5%); and (d) TiSr10, (71±13%*).

Bar: 50 μm.

Proliferation of osteoclast (hOC) culture on Sr HA coatings: 21 dayson Sr-HA coatings: 21 days

(a) TiHA (3.285±0.021); (b) TiSr1 (3.252±0.047); (c) TiSr5(3.211±0.008*); and (d) TiSr10 (3.193±0.019*). Bar: 50 μm.

hOC percentage decreases while cells peel!

DEGRADATION TESTS

SBF composition Na+ K+ Mg2+ Ca2+ Cl- HPO42- SO4

2- HCO3-

concentration mM 142 5 1.5 2.5 103 1 0.5 27

OCP ti di l d di l t t t ll ft 7 d f i i i SBF

Mn-CHA coatings remain almost intact after 7days of SBF immersion.

OCP coatings dissolve and disappear almost totally after 7 days of immersion in SBF.

XPS spectrum of OCP before (OCP3) and after (OCP2) degradation tests

XPS spectrum of Mn-CHA before (HA) and after (HA1) degradation tests

ALKALINE PHOSPHATASE ACTIVITY (ALP)ALP level is an early index of cell activation and differentiation. yThe mineralization stage correlates with a reduced ALP activity.

Alkaline Phosphatase/cell number control Ti Ha MnHa OCP

10,0

12,0

14,0

16,0

ml)

2 0

4,0

6,0

8,0

ALP

(IU

/m

0,0

2,0

3 7 14 21 days

increase, days 3 to 14 ⇒ a shift to a more differentiated state;

slight decrease, days 14 to 21 ⇒ the mineralization matrix is formed;

higher values for CaP coatings ⇒ coatings are capable of improving tissue integrationg g g p p g g

CaP coatings activate osteoblast metabolism and differentiation, as shown by the increased values of ALP!

Osteoblast proliferation and activity after 7, 14, and 21 days of culture on Sr-HA, ALP

test

7 days: n.s.; 14 days: **TiSr5 versus TiHA; **TiSr10 versus TiSr1, ***TiSr10 versus TiHA; 21 days: *TiSr5 versus TiHA, **TiSr10 versus TiHA, TiSr1.-Similar time evolutions - mineralization stage correlates with a reduced ALP activity;Higher values after doping with Sr – further improvement of tissue integration!

Collagen type I (CICP):Collagen type I is synthesized by osteoblasts as the major organicCollagen type I is synthesized by osteoblasts as the major organic

macromolecule in the extracellular bone matrix.

Procollagen type I/cell number control Ti Ha MnHa OCP

200

250)

50

100

150

CIC

P (n

g/m

l)

03 7 14 21 days

the values for polystyrene (control) and Ti were highest on day 3; they gradually decreased during days 7 to 21;

on OCP and Mn-CHA coatings, an increase from days 3 to 7 was followed by a decrease after day 14

TRANSFORMING GROWTH FACTOR BETA 1 (TGF β1):( β )

TGF-β1 protein, synthesized by osteoblasts, modulates cell proliferation and differentiation and enhances the deposition of extracellular matrix

Transforming growth factor/cell number

550

600

650

control Ti Ha MnHa OCP

350

400

450

500

550

TGFb

(pg/

ml)

150

200

250

300

3 7 14 21 days

Values for Control (polystyrene) and Ti peaked after 7 days and then constantlydecreased;

3 7 14 21 days

decreased;

TGF-β1 of coated materials increased from day 7 to day 21, indicating bonegrowth 3 weeks after implantation

IN VIVO – PULL OUT TESTS

• Pull out test discriminates between different implant attachmentmechanisms The model involves the use of a flat coin shaped implantmechanisms. The model involves the use of a flat coin shaped implantplaced on the cortical bone of rabbit tibia.

• New Zealand White adult female rabbits 8 months 3000-3500 gNew Zealand White adult female rabbits, 8 months, 3000 3500 gweight

• Moderate Ti substrate roughness was chosen:Moderate Ti substrate roughness was chosen:

- High enough to stimulate bone repair and growth ; but

- low enough to allow separation of biological effects;

- threated on reverse side

Surgical procedure (a - g)PULL OUT PROCEDURE

a b c d

e f g

T il t t d (h k)

PULL OUT PROCEDURETensile test procedure (h - k)

• Pullout test conducted after 8-week healing time;

h

• Cross head speed was set to 1,0 mm/min

h

j

i

k

Average pull out force for CaPs vs. control

11.2412

9.11 8.458

10

Control

5.324

6Newton

ControlGroup Ia, OCPGroup Ib, HA:MnGroup Ic, HA

2

4

0

All CaP PLD-coated Ti implants reveal enhanced bone healing/repairing ( pull out force), about twoAll CaP PLD coated Ti implants reveal enhanced bone healing/repairing ( pull out force), about two times better than in the case of control machined-Ti implants.

New CaPs (OCP and Mn-CHA) lead to significant increases in osteointegration efficiency, higher pullout forces (up to ¼ of maximum value).

Drawbacks of PLDDrawbacks of PLD

• existence of particulates of variousdimensions and compositiondimensions and composition

• limited application range to compoundshi h ll iti twhich usually are very sensitive to

temperature decomposition and damaged UV lunder UV laser exposure

ParticulatesParticulatesSEM images of the deposited SiNxA SEM images of the deposited SiNx

film with embedded droplets. A –image in plan view at normalorientation and B – a similar image

A

taken in 45° oblique view.

The large droplets aresplashed over the film surface.In some cases, atB In some cases, atimpact, second generations ofsmall droplets are formed anddispersed on the film

B

dispersed on the film.

XTEM image of a large droplet b i d i h filburied in the film

All the investigated dropletg pcross-sections show adouble morphologyresulting from theresulting from thecrystallization process:the first layer, about 100

thi k i t t ithnm thick, in contact witheither the collector or theearlier depositedfilm, consists ofnanometric-sizedcrystallites This proves incrystallites. This proves inour opinion that thecrystallization processt k l idl

SEM images

(a) Al target

KrF* excimer laser source

(a)

(λ=248 nm, τFWHM=450 fs)

(a) 5 Pa N2( ) 2

(b) 50 Pa N2

(b)(b)

Formation mechanismsFormation mechanismsThe particulates present on the deposited filmp p p

surface can be formed by:1. explosive dislocation of the substance caused by the

subsurface overheating of the targetsubsurface overheating of the target2. gas phase condensation of the evaporated material

(clustering)3. liquid phase expulsion under the action of the recoil

pressure of the ablated substance4 blast-wave explosion at the liquid (melt) – solid4. blast wave explosion at the liquid (melt) solid

interface5. hydrodinamic instabilities at the target surface

Possible solutionsPossible solutions

• To avoid the presence of the liquid inside theTo avoid the presence of the liquid inside the crater

• Proper choice of laser wavelengthp g• To set the incident laser fluence at a level high

enough to vaporize all the melted substanceg p• Application of electric and/or magnetic fields

normally to expansion• Intersection and elimination of particulates by a

second laser beam

Experimental set-upExperimental set up

Experimental details• Laser used for the ablation of Ta targets:

- KrF* (λ=248 nm, τFWHM∼12 ns) R i i 10 H- Repetition rate: 10 Hz.

- The laser beam incidence angle onto the target: ~ 45°- Laser fluence at the target position: 3.3 J/cm2

• IR beam parallel to the target surface: Nd:YAG laser (λ= 1 064• IR beam parallel to the target surface: Nd:YAG laser (λ= 1.064 μm, τFWHM∼10 ns)

• Distance above the focus of the first ablating laser beam: 2 mm • Nd:YAG laser fluence: 3.8–4.6 J/cm2

• Time delay between the UV and IR laser pulses: in the range 0 to 1500 μs - EG&G Mo. DDG 9650 high precision digital delay generator - fast photodiode (rise time < 1 ns) - 350 MHz Tektronix oscilloscope

• Vacuum chamber residual pressure: 7 x 10-4 PaDi t b t th t t d l lid ll t 35• Distance between the target and glass slides collector: 35 mm

• Scanning electron microscopy (SEM) investigations : Cambridge S120 and JEOL TEM Scan 200 CX instruments.

Results

(a) SEM image of a reference thin film deposited by KrF* laser irradiation

with a fluence of 3.3 J/cm2

(b) SEM image at higher magnification of the same thin film

SEM image of thin films deposited by KrF* laser irradiation with a fluence of 3 3 J/cm2laser irradiation with a fluence of 3.3 J/cm2

The time delay of thesecond IR Nd:YAGlaser pulses from theUV KrF* laser pulses:(a) 1000 μs, (b) 230μs, (c) 100 μs, (d) 10μs

Completely particulates-free thin films were deposited with time delays of 100 μs, or 10 μs.

MAPLEMAPLEMain differences PLD vs MAPLE: target preparation and interaction mechanismsmechanisms

th it t t i bt i d b i i l t ith i t i l• the composite target is obtained by mixing a solvent with an organic material• the mixture is frozen at liquid nitrogen temperature (77K)• during the deposition the target is kept at low temperature using a cooler

MAPLE

Simplified schematic of the MAPLE pdesorption process

1.The active substance (solute) is dispersed in a large excess of matrix material which will strongly absorb the incident laser light when in frozen state2. Short pulses of UV laser light focused onto the target cause volatilization of the solute and matrix;solute and matrix;3. The formed active ions are transferred to a collector.

MAPLE

Photographs of MAPLE targets, (left) before the laser strikes the target, and (right) after deposition, showing the eroded area on the target corresponding to the region where the laser hit.

*protein in deionized water, 248 nm

General observations:

• laser fluence must have proper values, lower than in PLD• incident laser energy must be majoritary absorbed by solvent molecules and not by organic molecules of the base material (0.5 - 10) %• frozen solvent must be characterized by a high absorption at working laser wavelength• solvent has to be selected so that organic material presents a good solubility• solvent has to present a high freezing point • solvent must not produce chemical reaction under laser radiation exposure

Drug delivery systems: Triacetate pullulan polysaccharideTriacetate-pullulan polysaccharide

Major differences between starting material and the PLD (248 nm, 390 mJ/cm2 ) film which demonstrate the degradation of the structure duringdegradation of the structure during PLD.

a. Pullulan dropcast b. 2% pullulan in distilled water c 2% pullulan in 3-butanolc. 2% pullulan in 3-butanol d. filtered 2% pullulan in 3-butanole. filtered 2% pullulan in DMSOLaser fluence 240 mJ/cm2

No decomposition by MAPLE of 2% solutions in distilled water and 3-butanol!

Biosensors: Creatinine (248 nm 2 or 5 wt% in deionized water)

creatinine base material

thin film obtained from the 2 wt.%concentration target

Creatinine (248 nm, 2 or 5 wt% in deionized water)

Creatinine level isi di i f kidindicative for kidneyfunction

Flaser2 wt. %5 wt. %

-bands intensity increases with creatinine concentration and incident laser fluence-challenge: get the best compromise between film biological quality and smoothness

Papain : 10% in distilled water; 248 nm- treats ulcers, reduces fever after surgery, is active against gram- positive and gram-negative bacteria, and has beneficial effects in systemic enzyme therapy in oncology

FTIR spectra of the (a) glass substrate and (b) base papain powder used for

the targets’ preparation.

FTIR spectra of thin films obtained from 10 wt % concentration frozen composite targets and

(a) 0.2, (b) 0.4 as well as (c) 0.6 J /cm2

incident laser fluenceincident laser fluence

FTIR spectra of the films reproduce all the characteristic bands of the p ppapain powder and no shifts in the bands were observed. The thin films preserved the primary structure of the base material used for the targets

preparation.

Urease: 1 or 10% in deionized water; 248 nmUrease: 1 or 10% in deionized water; 248 nm

FTIR t f b t i l d f th tiFTIR spectra of urease base material used for the preparation of the composite targets (Reference) and urease thin film

deposited at 0.4 J/cm2 laser fluence from the (a) 1 and (b) 10 wt.% urease concentration

Chemical composition and structure are preserved after the thin films growth process (in particular for 10% urease concentration)!

Urease thin film morphology

Tilted (a), detailed view (b) AFM micrographs, and corresponding surface profiles (c,d) of urease thi fil d it d t 400 J/ 2 l flthin film deposited at 400 mJ/cm2 laser fluence.

Interconnected island-like morphology, with an average height of ~ 240 nm could benoticed.Minimum root-mean square surface roughness was ~100 nm.Morphology can be considered a real advantage, because it provides a considerablybroader active area as recognition element in biosensor devices.

Urease enzymatic activity evaluationThe hydrolysis of urea was measured by coupling ammonia to NADH

(nicotinamide adenine dinucleotide) oxidation reaction

Urease enzymatic activity and kinetics => determined by Worthington assay method

ureaseurea [H2N-CO-NH2] + H2O 2NH4

+ + CO2 (1)

2NH4+ + 2α-Ketoglutarate + 2NADH 2Glutamate (C5H9NO4) + 2NAD+ +

2H O (2)

glutamate dehydrogenase

2H2O (2)

Reaction (1) is catalyzed by urease and reaction (2) is catalyzed by glutamate dehydrogenase

The NADH molecule (reduced NAD) is oxidized to NAD+. NADH absorbs UV light at 340 nm but NAD+ does not.

If the number of NADH molecules drops, the absorbance at 340 nm decreases – application to urea monitoring

Urease enzymatic activity evaluation1. Thin films were immersed in the solutions and mixed with 1.8 M urea in 0.1

M potassium phosphate buffer.

3. We compared the kinetic assay slope of the catalized reaction with the blank solution slope.

2. Kinetic analyses were performed by measuring the absorbance of the obtained solution at 340 nm over one hour.

Kinetic assay of urease thin film after incubation with 1.8M urea for (a) 5, (b) 15, and (c) 30 minutes y ( ) , ( ) , ( )

Increase of slope variation of absorbance with incubation time: laser pimmobilised enzyme active in urea breaking down / diagnostic of urea!

HA –AL: XRD investigations

Powder X-ray diffraction patterns of the thin films deposited from: (a) HA, (b) HA-AL7(4%), (c) HA-AL28(7%)

Alendronate

The slight increase of the broadening of the diffraction peaks whenincreasing alendronate concentration is indicative for a modest decreaseof the length of the crystalline domains as the alendronate content in theof the length of the crystalline domains as the alendronate content in theapatite nanocrystals increases up to 7.1%.

* 3.9 or 7% AL with HA; 0.25 g of HA-AL powder suspended in 5 ml deionized water, 248 nm, 750 mJ/cm2

SEM and AFM studies

SEM micrographs of thin films deposited from: (a) HA, (b) HA-AL7, (c)SEM micrographs of thin films deposited from: (a) HA, (b) HA AL7, (c) HA-AL28. Bars ¼ 2 mm. (d) AFM image of the surface of a thin film

deposited from HA.

The films exhibit a porous-like structure (similar to human bone) with poresThe films exhibit a porous like structure (similar to human bone), with poresdimension of 2–4 μm, while only few grains are visible.AFM analyses are quite similar for the different coatings.

Florescence microscopy images of hOB on alendronate HA coatingsalendronate-HA coatings

cba

Phallodin staining of culture after 24 hour from seeding: (a) Ti, (b) HA, (c) HA-AL7. Bars = 20 µm.

Presence of alendronate in HA thin films enhances osteointegration and b ti !bone regeneration!

Proliferation of osteoclast (hOC) culture on alendronate-HA coatings: 21 daysalendronate-HA coatings: 21 days

Phallodin staining of culture after 21 days from seeding: (a) Ti, (b) HA, (c) HA-AL7. Bars = 20 µm.

Presence of alendronate prevents the undesirable bone resorption!

In vitro studies

- ALP activity showed no differences among groups at 7 days, while at 14 days theTi t d i ifi tl l l th th thTi group presented significantly lower values than the others;-The production of CICP was significantly higher for both HA-AL7 and HA-AL28groups as compared to HA and Ti (after 7 days);-The level of OC after 14 days was significantly higher for both HA-AL7 and HA-y g y gAL28 than for HA and Ti groups

O t bl t h hi h t f lif ti d li diff ti ti iOsteoblasts show a higher rate of proliferation and earlier differentiation in the presence of alendronate!

BG-PMMAMetals and alloys used in implants are susceptible to corrosion in body fluids release of ions that accumulate in vital organs

Most common corrosion types of metallic medical devices:- pitting - galvanicg- crevice

Proposed solution: apply protective BG-PMMA coatingPMMA insulator and barrier against ions

(C5O2H8)nPMMA insulator and barrier against ions

Newly formed bioapatite

Metallic substrate Metallic substrate Metallic substrate

Newly formed bioapatite

BG+PMMA compositeImmersion in body fluids Continuous , compact layer of PMMA

Experimental details

• Target preparation:0 6 PMMA 0 08 BG i 19 3 l hl f0.6g PMMA +0.08g BG in 19.3 ml chloroform

• Two types of BG :6P61: SiO 61 1% Na O 10 3% K O 2 8% CaO 12 6% MgO 7 2% P O 6%6P61: SiO2 61,1%, Na2O 10.3%, K2O 2.8%, CaO 12.6%, MgO, 7.2%, P2O5 6%

6P57: SiO2 57%, Na2O 11%, K2O 3%, CaO 15%, MgO 8%, P2O5 6%

Deposition substrates: Ti gr.4 disks

L t 2Laser parameters: λ=248 nm, τ= 25 ns, F=0.55J/cm2

Immersion in SBF: 28 days (for 6P57) or 42 days (for 6P61)

Results

40

50

PMMA powder 6P57-PMMA thin film

banc

e [a

.u.]

a 2s

C 1

s

O 1

s

O K

LLNa

1s

C K

LL

6P57_PMMA sample

u.)

0

10

20

30

Abso

rb

Ti 3

pN

a 2s

Si 2

pS

i 2s

P 2s

Ca

N 1

s Ca

2pTi 2

p

C

Inte

nsity

(a

. u

SEM micrograph showing a typical surface morphology of a 6P57-PMMA coating

1000 1500 2000 2500

Wawenumber (cm-1)

FT-IR spectra of PMMA powders and of a 6P57-PMMA coating obtained by MAPLE

1200 1000 800 600 400 200 0

Binding energy (eV)

XPS survey spectrum of the 6P57-PMMA coating

The film contains PMMA and bioglass.Biological properties similar to simple 6P57Biological properties similar to simple 6P57 and 6P61 bioactive glass coatings

Fluorescence microscopy of cells cultured on MAPLE deposited structures 6P57 – PMMA

Corrosion measurements

Material Ecorr icorr Ebd Epas

Corrosion parameters for the bioglass-polymer samples determined from the polarization curves

Corrosion measurements

corr

(mV)

corr

(µA/cm2)

bd

(mV)

pas

(mV)

Ti -357 1.320 141 541

BG57+PMMA/Ti -251 0.053 1274 1192

BG61+PMMA/Ti -224 0.022 1454 1512

Ecorr – corrosion potential; icorr – corrosion current density ~ corrosion rate;E breakdown potential;

The corrosion rate (given by i ) dropped 25 times for BG57 + PMMA and 60 times

Ebd - breakdown potential;Epas – passivation potential ~ transpassive region dimension

The corrosion rate (given by icorr) dropped 25 times for BG57 + PMMA and 60 timesin case of BG61+PMMA => the bioglass-polymer nanocomposite coatings protectvery well the titanium implant against corrosion.Ebd increases about 10 times after coating => significant increase of shielding effect

HEK293 on Ti/HA–MP coatings

composite target HA-MP: 0.2% or 1% solution of 80% HA + 20% maleic anhydride copolymer (MP) in isopropanol

H b i kid (HEK293) ll

- cell morphology: polyhedralgood spreading establishing cell cell

Human embryonic kidney (HEK293) cells

- good spreading, establishing cell-cell contacts, tendency to occupy the entire surface

A B A B

The actin filament pattern ofcytoskeleton of cells on HA-MP →indicative of biocompatibilityp y

A - Hek293 cells grown on HA - maleic anhidride copolymer; B - Hek293 cells grown on standard

glass material

HEK293 on Ti/HA–MP coatings by cytoskeleton labelling

Fluorescence microscopy images

by cytoskeleton labelling

a b

a - Hek293 cells grown on HA - maleic anhidride copolymer; b - Hek293 cells grown on HA

Polymer enhances adhesion/proliferation qualities of the biomaterial coating surface

HEK cells preferred HA–MP coatings to HA as demonstrated by cell morphology and actin pattern!

HA-ECM proteins

ECM proteins: Fibronectin, vitronectin

Fibronectin structureFibronectin structure

Protein investigations: FT-IR studies

0,4 saline buffer

Fibronectin (1631 cm-1)

MAPLE FN dropcast FN Si

Fibronectin

0,3

Fibronectin

Fibronectin3186 cm-1(NH stretch)C=O, CN stretch, NH bending

0,2saline buffer

Fibronectin2990 cm-1

Fibronectin (1631 cm-1)

bsor

banc

e

ν(CH3), ν(CH2)

0,1

Ab

saline buffer

1500 2000 2500 3000 3500 40000,0

Wavenumber (cm-1)

IR absorption bands of FN structures obtained on silicon substrates

* 1.8 mg/ml in deionized water based saline buffer, 700 mJ/cm2

Protein investigations: FT-IR studiesVN MAPLE

0,5

0,6

saline bufferVitronectin2990 cm-1

Vitronectin3186 cm-1

VN MAPLE VN dropcast Si

(NH stretch)

0 3

0,4 Vitronectin (1631 cm-1)

2990 cm

ance

C O CN h NH b diν(CH3), ν(CH2)

0,2

0,3saline buffer

saline buffer

Abs

orba C=O, CN stretch, NH bending

0,0

0,1

saline buffer

IR absorption bands of VN structures obtained on silicon substrates

1500 2000 2500 3000 3500 4000

Wavenumber (cm-1)

The peaks of proteins deposited by MAPLE are matching very well the ones from dropcast!

Antibody stainingExperiments with anti-human FN and anti-human VN rabbit polyclonal serum. The secondary antibody for FN and VN was an FITC-conjugated anti-rabbit IgG.

Immunofluorescence detection of fibronectin structures

Fibrilar structure of FN and necklace-like confined structure in case of

Immunofluorescence detection of vitronectin structures

Fibrilar structure of FN and necklace-like confined structure in case of VN were evidenced – FN and VN in MAPLE films are FUNCTIONAL!

HOB precursor cells: Actin and nuclei staining 7 daysTi/HA Ti/HA/BSA Ti/HA/FN Ti/HA/VN

10 X 10 X 10 X 10 XHigher potential for adhesion and spreading in the case of Ti/HA/FN and

Ti/HA/VN structures as compared to Ti/HA and Ti/HA/BSA

Ti/HA Ti/HA/BSAScanning electron microscopy studies 7 days

Ti/HA/VN Ti/HA/FN

Flat cell morphology, intimate contacts and long filopodia were visualized for FN and VN covered structures as compared to Ti/HA and Ti/HA/BSA controls.

Cell culture – human osteoprogenitor cells: MTS assayMTS assay

0,120

0,100Day 1

0,060

0,080Day 3

0 020

0,040Day 7

Day 14

0,000

0,020 Day 14

HA BSA FN VNFN VN

In-vitro tests demonstrate a much larger bioactivity of Ti/HA/FN and Ti/HA/VN as compared toTi/HA or Ti/HA/BSA.

Laser direct write - LDWLaser direct write LDW

N.T. Kattamis et al., Appl. Phys Lett 91, 171120 (2007)

FN transfer by LDWFN transfer by LDW

λ=248 nmλ 248 nmτ=25 nsS=32 mm²E=40-100 mJ

40 mJ 50 mJ 60 mJ 70 mJ 80 mJ 90 mJ 100 mJ40 mJ 50 mJ 60 mJ 70 mJ 80 mJ 90 mJ 100 mJ

FN transfer by LDWFN transfer by LDW

40 mJ 70 mJ 80 mJ 90 mJ 100 mJ40 mJ 70 mJ 80 mJ 90 mJ 100 mJ

Experimental conditionsNd:YAP laser, 5 ns @ 1078 nm;

(240 – 900) mJ/cm2

for (50 – 1500) nm Ti interlayer, 5 multilayers of TPP-multilayers of TPP-SA (Mesotetraphenylporphyrin (TPP) and stearic acid (SA) dissolved in chloroform (both 10-

3 M); the ratio of

Fluence decreases

M); the ratio of TPP:SA = 1:1);

receiving substrate silica glass) at 10Fluence decreases silica glass) at 10 µm

Microstructures of mesotetraphenylporphyrin (TPP)mesotetraphenylporphyrin (TPP)

A series of successfully transferred material on the red fluorescent channelcorresponds to the second and third columns The highest fluence generatescorresponds to the second and third columns. The highest fluence generatessplashing of protein which seems to be aggregated because of the increased amountof material. Lower fluences are optimal for obtaining discrete geometrical structureswith a remarkable precision.

Illustrative resultsIllustrative results

Fractal antenna pattern deposited onto the abdomen of a honey bee

A rectangular area of hair was first removed by laser micromachining,and then laser forward transfer was used to deposit the silver pattern.The resonant frequency is estimated to be 54 GHz.

Laser Transfer of Live E. Coli Bacteria

800 microns

Spot size < 10μm

Bacteria CompositeViewed With Visible Light

Bacteria CompositeViewed With UV Light

150 μm 150 μm

Green fluorescence demonstrates the successfultransfer!

Combinatorial Pulsed Laser Deposition (C-PLD) Setup

Carousel system in C-PLD setupy p

XRR investigationsXRR investigations

XRR spectra acquired from pure and mixed films:XRR spectra acquired from pure and mixed films:

the critical angle region and the whole spectra

X-ray Diffraction Investigations

XRD spectra acquired from a mixed ITO-ZnO film, on various position along the p q , p gtransversal axis: control of the lattice parameter between that of ITO and ZnO,

depending on the position (i.e. composition) of the sample

Conclusions• PLD/RPLD are universal techniques: they can be applied to

any compound existing in nature or predicted by theoreticalmodelsmodels

• PLD/RPLD are rather simple, economic, low-thermal budgetand ambient preserving techniquesThe plasma plays an essential role in PLD/RPLD; it acts as• The plasma plays an essential role in PLD/RPLD; it acts asa “piston” pushing the ablated substance from target tocollector, intervenes in the chemical reaction and provideshot particles which result in the formation of very adherenthot particles which result in the formation of very adherentdeposited layer

• PLD/RPLD are rather efficient processes: more than 50% ofthe incident laser energy is spent in ablation and plasmathe incident laser energy is spent in ablation and plasmaheating. The efficiency can be improved by using a targetwith poor thermal conductivity and/or external mirrors

• Particulates of various shapes and dimensions representp pthe major drawback in PLD/RPLD; however there are nowwell established methods to decrease their density down tocomplete elimination

Conclusions• Nevertheless, we can deposit either a thin film or independentnanoparticles by the appropriate choice of the ablationparameters (laser wavelength laser fluence ambient gasparameters (laser wavelength, laser fluence, ambient gasnature and pressure, deposition geometry, substrate heating)•MAPLE has been successfully used for many “delicate”

i & bi t i lorganics & biomaterials• LDW has been successfully applied for active proteins andcells• C-PLD can transfer data bases of compounds with finecontrolled change of composition• It was demonstrated that the deposited thin films and• It was demonstrated that the deposited thin films andstructures are identical to the starting material, preserving theirchemical structure and very likely their functionality and biologic

ti itactivity

List of relevant publications1. Composite biocompatible hydroxyapatite–silk fibroin coatings for medical implants obtained by Matrix Assisted Pulsed Laser

Evaporation, F.M. Miroiu , G. Socol, A. Visan, N. Stefan, D. Craciun, V. Craciun, G. Dorcioman, I.N. Mihailescu, L.E. Sima, S.M.Petrescu A Andronie I Stamatin S Moga and C Ducu Materials Science and Engineering B 169 151–158 2010Petrescu, A. Andronie, I. Stamatin, S. Moga and C. Ducu, Materials Science and Engineering B 169, 151–158, 2010

2. Biomolecular urease thin films grown by laser techniques for blood diagnostic applications, E. Gyorgy, F. Sima, I. N. Mihailescu, T.Smauz, B. Hopp, D. Predoi, S. Ciuca, L. E. Sima, S. M. Petrescu, Materials Science and Engineering C, 30 (2010) 537–541

3. Functional porphyrin thin films deposited by matrix assisted pulsed laser evaporation, R. Cristescu, C. Popescu, A.C. Popescu, I.N.Mihailescu, A.A. Ciucu, A. Andronie, S. Iordache, I. Stamatin, E. Fagadar-Cosma, D.B. Chrisey, Mat. Sci. Eng. B 169, 106–110 (2010)

4. Application of clean laser transfer for porphyrin micropatterning, T.V. Kononenko, I.A. Nagovitsyn, G.K. Chudinova, I.N. Mihailescu, AppliedSurface Science 256 (2010) 2803 2808Surface Science 256 (2010) 2803–2808

5. Comparative study on Pulsed Laser Deposition and Matrix Assisted Pulsed Laser Evaporation of urease thin films, T. Smausz, G.Megyeri, R. Kékesi, C. Vass, E. György, F. Sima, I. N. Mihailescu, B. Hopp, Thin Solid Films, 517 (15), 4299-4302 , 2009

6. Specific biofunctional performances of the hydroxyapatite-sodium maleate copolymer hybrid coating nanostructures evaluated by in vitrostudies, L. E. Sima, A. Filimon, R.M. Piticescu, G.C. Chitanu, D.M. Suflet, M. Miroiu, G. Socol, I. N. Mihailescu, J. Neamtu, G.Negroiu, Journal of Materials Science: Materials in Medicine, 20, 2305-2316, 2009

7 Bi l l thi ti bt i d b MAPLE f ti f i l t F Si C Ri t A P I N7. Bioglass –polymer thin coatings obtained by MAPLE for a new generation of implants, F. Sima, C. Ristoscu, A. Popescu, I. N.Mihailescu, T. Kononenko, S. Simon, T. Radu, O. Ponta, R. Mustata, L. E. Sima, S. M. Petrescu, Journal of Optoelectronics and AdvancedMaterials, 11(9), 1170 – 1174 (2009)

8. Immobilization of urease by laser techniques: synthesis and application to urea biosensors, György E, Sima F, Mihailescu IN, SmauszT, Megyeri G, Kékesi R, Hopp B, Zdrentu L, Petrescu SM., Journal of Biomedical Materials Research: 89A: 186–191, 2009

9. Biofunctional alendronate–Hydroxyapatite thin films deposited by Matrix Assisted Pulsed Laser Evaporation, A. Bigi, E. Boanini, C.Capuccini, M. Fini, I. N. Mihailescu, C. Ristoscu, F. Sima, P. Torricelli, Biomaterials, Volume 30, Issue 31, October 2009, Pages 6168-6177

10. Thin Films of Polymer Mimics of Cross-Linking Mussel Adhesive Proteins Deposited by Matrix Assisted Pulsed Laser Evaporation, RCristescu, I.N. Mihailescu, I. Stamatin, A. Doraiswamy, R.J. Narayan, G. Westwood, J.J. Wilker, S. Stafslien, B. Chisholm, D.B.Chrisey, Applied Surface Science, 255 (2009) 5496–5498

11. Functional polyethylene glycol derivatives nanostructured thin films synthesized by matrix-assisted pulsed laser evaporation, R.Cristescu, C. Popescu, A. Popescu, S. Grigorescu, I.N. Mihailescu, D. Mihaiescu, S.D. Gittard, R.J. Narayan, T. Buruiana, I. Stamatin, D.B.p p g yChrisey, Applied Surface Science 255 (2009) 9873–9876

12. Functionalized Polyvinyl Alcohol Derivatives Thin Films for Controlled Drug Release and Targeting Systems: MAPLE Deposition andMorphological, Chemical and In Vitro Characterization, R. Cristescu, C. Cojanu, A. Popescu, S. Grigorescu, L. Duta, G. Caraene, A.Ionescu, D. Mihaiescu, R. Albulescu, T. Buruiana, A. Andronie, I. Stamatin, I. N. Mihailescu, D. B. Chrisey, Appl. Surf. Sci. 255 (2009)5600–5604

13. Laser Processing of Polyethylene Glycol Derivative and Block Copolymer Thin Films, R. Cristescu, C. Cojanu, A. Popescu, S.13. Laser Processing of Polyethylene Glycol Derivative and Block Copolymer Thin Films, R. Cristescu, C. Cojanu, A. Popescu, S.Grigorescu, L. Duta, O. Ionescu, D. Mihaiescu, T. Buruiana, A. Andronie, I. Stamatin, I, N. Mihailescu, D. B. Chrisey, Applied SurfaceScience, 255 (2009) 5605–5610

14. Creatinine biomaterial thin films grown by laser techniques, E.Gyorgy, E. Axente, I. N. Mihailescu, D. Predoi, S. Ciuca, J. Neamtu, Journalof Material Science: Materials in Medicine,19(3), 1335-1339, 2008

15. Biocompatibility evaluation of a novel hydroxyapatite-polymer coating for medical implants (in vitro tests), G.Negroiu, R.M. Piticescu, G.C.Chitanu, I.N. Mihailescu, L. Zdrentu, M.Miroiu, Journal of Materials Science: Materials in Medicine19 (4) 2008 1537-1544

16. Laser Processing of Natural Mussel Adhesive Protein Thin Films, A. Doraiswamy, R.J. Narayan, R. Cristescu, I.N. Mihailescu, D.B.Chrisey, Materials Science and Engineering: C 27(3), (2007) 409-413

17. MAPLE Applications in Studying Organic Thin Films, M. Jelinek, T. Kocourek, J. Remsa, R. Cristescu, I.N. Mihailescu, D.B. Chrisey, LaserPhysics 17(2) (2007) 66-70(5)Physics 17(2), (2007) 66-70(5)

18. Thin Films Growth Parameters in MAPLE; Application to Fibrinogen, M. Jelinek, R. Cristescu, T. Kocourek, V. Vorliček, J Remsa, L.Stamatin, D. Mihaiescu, I. Stamatin, I.N. Mihailescu, D.B. Chrisey, Journal of Physics: Conference Series 59, (2007) 22-27

19. Matrix Assisted Pulsed Laser Evaporation of Pullulan Tailor-Made Biomaterials Thin Films for Controlled Drug Delivery Systems, R.Cristescu, M. Jelinek, T. Kocourek, E. Axente, S. Grigorescu, A. Moldovan, D.E. Mihaiescu, M. Albulescu, T. Buruiana, J. Dybal, I.Stamatin, I.N. Mihailescu, D.B. Chrisey, Journal of Physics: Conference Series 59, (2007) 144-149

20 Biomolecular papain thin films growth by laser techniques E Gyorgy J Santiso A Figueras G Socol I N Mihailescu Journal of Materials20. Biomolecular papain thin films growth by laser techniques, E. Gyorgy, J. Santiso, A. Figueras, G. Socol, I. N. Mihailescu, Journal of MaterialsScience: Materials in Medicine 18, 8, 1471 - 1663, 2007

21. Laser Processing of DOPA modified PEG Mussel Adhesive Protein Analog Thin Films, A. Doraiswamy, R.J. Narayan, C. Dinu, R.Cristescu, P.B. Messersmith, S. Stafslien, D.B. Chrisey, Journal of Adhesion Science & Technology, 21(3-4), (2007) 287-299(13)

22. Processing of poly(1,3-bis-(p-carboxyphenoxy propane)-co-(sebacic anhydride)) 20:80 (P(CPP:SA)20:80) by matrix-assisted pulsed laserevaporation for drug delivery systems, R. Cristescu, C. Cojanu, A. Popescu, S. Grigorescu, C. Nastase, F. Nastase, A. Doraiswamy, R.J.N I St ti I N Mih il d D B Ch i A li d S f S i 254(4) 1169 1173 (2007)Narayan, I. Stamatin, I.N. Mihailescu and D.B. Chrisey Applied Surface Science 254(4) 1169 - 1173 (2007)

23. Matrix Assisted Pulsed Laser Evaporation of Cinnamate- and Tosylate-Pullulan Polysaccharide Derivative Thin Films for PharmaceuticalApplications M Jelinek, R Cristescu, E. Axente, T Kocourek, J Dybal, J Remsa, J Plestil, D. Mihaiescu, M. Albulescu, T. Buruiana, I.Stamatin, I N Mihailescu, D B Chrisey, Applied Surface Science, 253(19), (2007) 7755-7760

24. Matrix Assisted Pulsed Laser Evaporation of Poly(D,L-Lactide) Thin Films for Controlled-Release Drug Systems, R. Cristescu, A.Doraiswamy, T. Patz, G. Socol, S. Grigorescu, E. Axente, F. Sima, R.J. Narayan, D. Mihaiescu, A. Moldovan, I. Stamatin, I.N. Mihailescu, B.J. Chislom, D.B. Chrisey, Applied Surface Science, 253(19), (2007) 7702–7706

25. Polycaprolactone Biopolymer Thin Films Obtained by Matrix Assisted Pulsed Laser Evaporation, R. Cristescu, A. Doraiswamy, G. Socol, S.Grigorescu, E. Axente, F. Sima, R. J. Narayan, D. Mihaiescu, A. Moldovan, I. Stamatin, I. N. Mihailescu, B. J. Chisholm, D. B.Chrisey, Applied Surface Science, 253 (2007), 6476–6479

26. MAPLE Applications in Studying Organic Thin Films M. Jelinek, T. Kocourek, J. Remsa, R. Cristescu, I.N. Mihailescu, D.B. Chrisey, LaserPhysics 17 (1), (2007) 1-5.y ( ) ( )

27. Matrix Assisted Pulsed Laser Evaporation Processing of Triacetate-Pullulan Polysaccharides Thin Films for Drug Delivery Systems, R.Cristescu, G. Dorcioman, C. Ristoscu, E. Axente, S. Grigorescu, A. Moldovan, I.N. Mihailescu, T. Kocourek, M. Jelinek, M. Albulescu, T.Buruiana, D. Mihaiescu, I. Stamatin, D.B. Chrisey, Applied Surface Science 252(13), (2006) 4647-4651

28. Matrix Assisted Pulsed Laser Evaporation of Azo-Polyurethane Thin Films R. Cristescu, E. Axente, G. Socol, A. Moldovan, D. Mihaiescu, I.Stamatin, T. Buruiana, M. Jelinek, I. N. Mihailescu, D.B. Chrisey, Laser Physics 15 12, (2005)

29. Laser deposition of fibrinogen blood proteins thin films by matrix assisted pulsed laser evaporation, L. Stamatin, R. Cristescu, G. Socol, A.29. Laser deposition of fibrinogen blood proteins thin films by matrix assisted pulsed laser evaporation, L. Stamatin, R. Cristescu, G. Socol, A.Moldovan, D. Mihaiescu, I. Stamatin, I. N. Mihailescu, D.B. Chrisey, Applied Surface Science 248 422-427 (2005)

30. Processing of mussel-adhesive protein analog copolymer thin films by matrix-assisted pulsed laser evaporation, T. Patz, R. Cristescu, R.Narayan, N. Menegazzo, B. Mizaikoff, P.B. Messersmith, I. Stamatin, I. N. Mihailescu, D.B. Chrisey Appl. Surf. Sci. 248, 416-421 (2005)

31. Processing of mussel adhesive protein analog thin films by matrix assisted pulsed laser evaporation, R. Cristescu ,T. Patz, R.J. Narayan, N.Menegazzo, B. Mizaikoff , D.E. Mihaiescu , P.B. Messersmith, I. Stamatin, I. N. Mihailescu, D.B. Chrisey, Appl. Surf. Sci. 247 217-224 (2005)

“Nanostructured Thin Optical Sensors For Detection Of Gas Traces”, C. Ristoscu, I. N. Mihailescu, D. Caiteanu, C. N. Mihailescu, Th.Mazingue, L. Escoubas, A. Perrone, H. Du, “Functionalized Nanoscale Materials, Devices, & Systems”, Proceedings ofNATO AdvancedStudy Institute “Functionalized nanoscale materials, devices, and systems for chem.-bio sensors, photonics, and energy generation andstorage”, June 4-15, 2007, Sinaia, Romania, Edited by A. Vaseashta, and I. N. Mihailescu, SPRINGER SCIENCE + BUSINESS MEDIAB.V., (2008), p. 27 - 50

"Enhanced gas sensing of Au nanocluster-doped or -coated zinc oxide thin films“, G. Socol, E. Axente, C. Ristoscu, F. Sima, A. Popescu, N.Stefan I N Mihailescu L Escoubas J Ferreira A Szekeres S Bakalova J Appl Phys 102 083103 (2007)Stefan, I. N. Mihailescu, L. Escoubas, J. Ferreira, A. Szekeres, S. Bakalova, J. Appl. Phys., 102, 083103 (2007),

“Structural and optical characterization of undoped, doped, and clustered ZnO thin films obtained by PLD for gas sensing applications”, C.Ristoscu, D. Caiteanu, G. Prodan, G. Socol, S. Grigorescu, E. Axente, N. Stefan, V. Ciupina, G. Aldica, I.N. Mihailescu, Applied SurfaceScience 253 (2007) 6499–6503

“Optical characterizations of ZnO, SnO2, and TiO2 thin films for butane detection”, T. Mazingue, L. Escoubas, F. Flory, P. Jacquouton, A.Perrone, E. Kaminska, A. Piotrowska, I. Mihailescu, P. Atanasov, Applied Optics, 45(7), 1425 (2006)

“Nanostructured ZnO coatings grown by pulsed laser deposition for optical gas sensing of butane”, T. Mazingue, L. Escoubas, L. Spalluto, F.Flory, G. Socol, C. Ristoscu, E. Axente, S. Grigorescu, I.N. Mihailescu, N. A. Vainos, J.Appl. Phys. 98, 074312 (2005)

“Morphology evolution and local electric properties of Au nanoparticles on ZnO thin films”, E. György, J. Santiso, A. Figueras, A.Giannoudakos, M. Kompitsas, I. N. Mihailescu, Journal of Applied Physics 98, 084302 (2005)

“Anatase phase TiO2 thin films obtained by pulsed laser deposition for gas sensing applications”, E. Gyorgy, G. Socol, E. Axente, I. N.Mih il C D S Ci A li d S f S i 247 429 433 (2005)Mihailescu, C. Ducu, S. Ciuca, Applied Surface Science, 247, 429-433, (2005)

“Structural and optical characterization of WO3 thin films for gas sensor applications”, E. György, G. Socol, I. N. Mihailescu, C. Ducu, S.Ciuca, Journal of Applied Physics 97, 093527-1_4 (2005)

“Doped thin metal oxide films for catalytic gas sensors,” E. Gyorgy, E. Axente, I. N. Mihailescu, C. Ducu and H. Du, Applied SurfaceScience, 252(13), 30 April 2006, Pages 4578-4581

“SnO nanostructured films obtained by pulsed laser ablation deposition” C Ristoscu L Cultrera A Dima A Perrone R Cutting H L Du ASnO2 nanostructured films obtained by pulsed laser ablation deposition , C. Ristoscu, L. Cultrera, A. Dima, A. Perrone, R. Cutting, H. L. Du, A.Busiakiewicz, Z. Klusek, S. Datta, S. Rose, Applied Surface Science, 247(1-4), 95-100 (2005)

“Functional nanostructured metal oxide thin films for applications in optical gas detection”, G. Socol, I. N. Mihailescu, E. Axente, C. Ristoscu, E.Gyorgy, D. Stanoi, S. Grigorescu, L. Escoubas, T. Mazingue, NATO Science Series by Springer Science and Business Media, Eds. CyrilPopov and Wilhelm Kulisch (NATO-ASI “Functional properties of nanostructured materials”, 3-14 June 2005, Sozopol, Bulgaria), 363-366

“Diffractive optical elements for photonic gas sensors”, N. Madamopoulos, G. Siganakis, A. Tsigara, L. Athanasekos, S. Pispas, N. Vainos, E.Kaminska, A. Piotrowska, A. Perrone, C. Ristoscu, K. Kibasi, Nanosensing: Materials and Devices II, M. Saif Islam, Achyut K.Dutta, Editors, Proceedings of SPIE - 6008, 60081C (2005)

•“Advanced biomimetic implants based on nanostructured coatings synthesized by pulsed laser technologies” Ion N. Mihailescu, CarmenRistoscu, Adriana Bigi, Isaac Mayer, Chapter 10 in “Laser-Surface Interactions for New Materials Production Tailoring Structure andProperties”, Series: Springer Series in Materials Science, Vol. 130, Miotello, Antonio; Ossi, Paolo M. (Eds.), 2010, pp. 235 – 260•Biocompatibility and Bioactivity Enhancement of Ce Stabilized ZrO2 Doped HA Coatings by Controlled Porosity Change of Al2O3Substrates, F. Sima, C. Ristoscu, D. Caiteanu, N. Stefan, C. N. Mihailescu, I. N. Mihailescu, G. Prodan, V. Ciupina, E. Palcevskis, J.Krastins I Zalite L E Sima S M Petrescu Journal of Biomedical Materials Research: Part B - Applied Biomaterials DOI:Krastins, I. Zalite, L. E. Sima, S. M. Petrescu, Journal of Biomedical Materials Research: Part B Applied Biomaterials, DOI:10.1002/jbm.b.31755•Shallow hydroxyapatite coatings pulsed laser deposited onto Al2O3 substrates with controlled porosity: correlation of morphologicalcharacteristics with in vitro testing results, F. Sima, C. Ristoscu, N. Stefan, G. Dorcioman, I.N. Mihailescu, L.E. Sima, S.M. Petrescu, E.Palcevskis, J. Krastins, I. Zalite, Applied Surface Science 255 (2009) 5312–5317;•Processing of dense nanostructured HAP ceramics by sintering and hot pressing, Dj. Veljovic, B. Jokic, R. Petrovic, E. Palcevskis, A.Dindune,I.N. Mihailescu, Dj. Janackovic, Ceramics International 35 (2009) 1407–1413;Bi tibl d bi ti t t d l ti th i d b l d l d iti I it bi l i l t t A C•Biocompatible and bioactive nanostructured glass coatings synthesized by pulsed laser deposition: In vitro biological tests, A.C.

Popescu, F. Sima, L. Duta, C. Popescu, I.N. Mihailescu, D. Capitanu, R. Mustata, L.E. Sima, S.M. Petrescu and D. Janackovic, AppliedSurface Science , Applied Surface 255 (2009) 5486–5490;•Nanostructured bioglass thin films synthesized by pulsed laser deposition: CSLM, FTIR investigations and in vitro biotests, L. Floroian, B.Savu, G. Stanciu, A. C. Popescu, F. Sima, I.N. Mihailescu, R. Mustata, L.E.Sima, S.M. Petrescu, D. Tanaskovic, Dj. Janackovic, AppliedSurface Science, 255 (2008), 5, 3056-3062;•Double-layer Bioactive Glass Coatings Obtained by Pulsed Laser Deposition, D. Tanaskovic, Dj. Veljković, R. Petrović, Dj. Janaćković,y g y p j j jM.Mitrić, C. Cojanu, C. Ristoscu, I.N. Mihailescu, Key Engin. Mater., 361-363 (2008) 277-280;•Bioactive glass and hydroxyapatite thin films obtained by pulsed laser deposition, E. Gyorgy, S. Grigorescu, G. Socol, I. N. Mihailescu, A.Figueras, D. Janackovic, E. Palcevskis, L. E. Zdrentu, S. Petrescu, Applied Surface Science (19), 253 (2007) 7981 – 7986;•Synthesis of functionally graded bioactive glass - apatite multistructures on Ti substrates by pulsed laser deposition, D. Tanaskovic, B.Jokic, G. Socol, A. Popescu, I. Mihailescu, R. Petrovic, Dj. Janackovic, Applied Surface Science, 254 (2007), 4, 1279-1282;•Synthesis and characterization of bioglass thin films, L. Floroian, B. Savu , F. Sima, I. N. Mihailescu, D. Tanaskovic, D. Janackovic,Digest Journal of Nanomaterials and Biostructures vol 2 no 3 2007 p 285-291Digest Journal of Nanomaterials and Biostructures, vol.2, no.3, 2007, p.285-291•Tailoring immobilization of immunoglobulin by excimer laser for biosensor applications, Felix Sima, Emanuel Axente, Carmen Ristoscu,Ion N. Mihailescu, Taras V. Kononenko, Ilya A. Nagovitsin, Galina Chudinova, Vitaly I. Konov, Marcela Socol, Ionut Enculescu, Livia E.Sima, Stefana M. Petrescu, Accepted for publication to Journal of Biomedical Materials Research: Part A, October 2010•"Pulsed Laser Deposition: an Overview" I. N. Mihailescu, Eniko Gyorgy 4-th International Commission for Optics (ICO) Book"International Trends in Optics and Photonics", Ed. T. Asakura (ICO President), pp. 201-214 (1999)• Pulsed laser deposition of biomedical materials" V. Nelea, M. Jelinek, I. N. Mihailescu, Chapter 9 of Pulsed laser deposition of

l i fil " 265 311 l 2 S i O l i M i l d D i 2005optoelectronic films" pag. 265-311, vol. 2, Series: Optoelectronic Materials and Devices, 2005• Biomaterials : new issues and breakthroughs for biomedical applications" V. Nelea, M. Jelinek, I. N. Mihailescu, Chapter 18 in PulsedLaser Deposition of thin films: applications-lead growth of functional materials", Wiley & Sons, 2007

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