Chemical sensors (Chemosensors) “A chemosensor is molecule … lezione... · Chemical sensors...

Chemical sensors (Chemosensors)“A chemosensor is molecule of abiotic origin that signals the presence of matter or energy”

(A. W. Czarnick)

Working mode

UNITA’ DI SEGNALAZIONE

SITO DIRICONOSCIMENTO

ANALITASEGNALE

UNITA’ DI TRASDUZIONE

• A receptor capable to selectively bind the analyte• A site with some tunable moelcular property• A transduction mechanism that converts the recongintion into a

modification of the tunable property signal

In principle, any measurable molecular property can be used

Chemical sensors (Chemosensors)

Fluorescent chemosensorIt is a chemosensor that generate a fluorescence signal

Why fluorescence?

Sensitivity (even single molecule detection is possible) High spatial and temporal resolution Low cost and easily performed instrumentations

Most used:• Redox potential• Absorbance (color)• Luminescence (fluorescence)• NMR relaxation times (recent)

Sensor: device that interacts reversibly with an analyte with measurable signal generationA chemosensor is not a sensor, strictly speaking, as it is not a device, but it can be the active part of the device.

Which signal do we measure with fluorescent chemosensors? Fluorescence quenching (ON-OFF) Fluorescence increase (OFF-ON) Emission spectrum shape modification (ratiometric) Life-time Emission anisotropy

Photoluminescence

Emission of photons by molecules as a consequence of electronic transitions

INTRINSECO

On Off

STRATEGIE PER LA PROGETTAZIONE DI UN SENSORE SUPRAMOLECOLARE

CONIUGATO

AUTOASSEMBLATO +

Esempi di sensori intrinseci

O

N

O

OO

N N

COO-

COO--OOCCOO-

COO-

N

N

H3CO

O N

H3C

COO-

COO-

COO-

COO-

FURA-2(Tsien 1980)

Quin-2(Tsien 1985)

Ca2+

COO-

NH

NO COO-

COO-COO-

Mag-Indo-1(London 1989)

Mg2+

O

N

O

N

O OMeOMe

OO

CC O

O

OO

C

C

O

O

O

O

(Tsien 1989)

Na+

Fura-2

R. Y. Tsien, J. Biol. Chem. 1985, 6, 3440

Internal charge transfer (ICT)

A D A D

BLU

E S

HIF

TB

LUE

SH

IFT

A D A DA DA D A DA D

BLU

E S

HIF

TB

LUE

SH

IFT

RE

D S

HIF

TR

ED

SH

IFT

D A D A

RE

D S

HIF

TR

ED

SH

IFT

D A D AD A D A

N

O

OO

O

O

NCCN

DCM-Crown

Valeur et al. J. Phys. Chem. 1989, 93, 3871

se il recettore è legato al gruppo elettron donatore

se il recettore è legato al gruppo elettron attrattore

Intrinsic chemosensor

Advantage: the direct interaction between the bound substrate and the fluorophore automatically leads to the modification of the emission properties. The transduction mechanism is somehow intrinsic to the chemosensor structure.

Design: the donor atoms for the complexation of the substrate are part of the fluorophore system, therefore the analyte binds to a receptor subsite which is an integrated part of the fluorophore aromatic system.

Weakness: rigidity of the design. They have to be designed around the substrate and any modification of the binding site may results in a change of the emission properties of the dye and vice versa.

INTRINSECO

On Off

CONIUGATO

AUTOASSEMBLATO +


Photoinduced electron transfer (PET)

LUMO

HO MO

excitedacceptor

donor

PE T

acceptorradical anion

donorradical cation

Back E TLUMO

HO MO

excitedacceptor

donor

PE T

acceptorradical anion

donorradical cation

Back E T

D

h h h’

D

e-

DD

h h h’

DDD

e-

PE T

LUMO

HO MO

fluorophore free receptor fluorophore

boundreceptor

recettore “libero” recettore “complessato”

Conjugate chemosensors: ET and PET

PET

LUMO

HOMO

fluorophorenitrogenlone pair fluorophore

free receptor

nitrogenlone pair

bound receptor

PETLUMO

HOMO

fluorophoreCu 2+

fluorophorez2

Cu 2+

x2 - y2

BeT

CH2

NH HN

OO

NH2H2N

2H+

CH2

N N

OO

H2NNH2

Cu2+

Cu2+, 2OH-

Active substrates

Fabbrizzi et al. Chem Eur. J. 1996, 2, 75.

Silent substrates

O O

N

O

O

O

N

N

N

N

De Silva, 1986 Czarnik Acc. Chem. Res., 1994, 27, 302-308

Zn2+K+

NH2

OCH3

H2N

OCH3

NH2

H3CO

NH2

OCH3

H2N

OCH3

NH3

H3CO

NH2

OCH3

HN

OCH3

NH3

H3CO

NH2

OCH3

HN

OCH3

NH2

H3CO

pK1 = 5.2 pK2 = 6.9 pK3 = 9.3

0

200

400

600

800

1000

4 5 6 7 8 9 10 11 12pH

I of f

luor

esce

nce

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

% o

f spe

cies

LHL

H2L

H3L

ATMCA = 1 M, ex = 368 nm, em = 415 nm

LH3L H2L H1L

HOMO

E

LUMO

excitedfluorophore

boundreceptor

hvFlu

HOMO

E

LUMO

excitedfluorophore

freereceptor

PET

HOMOHOMO

Conjugate chemosensors: ATMCA (pH)

NH2

OCH3

H2N

OCH3

NH3

H3CO

pH = 5NH

OCH3

NH

OCH3

NH

H3CO

Cu2+

Cu2+

[metal ion], M

0

20

40

60

80

100

0.00E+00 5.00E-07 1.00E-06 1.50E-06 2.00E-06 2.50E-06

I/I0 (

%, 4

15 n

m)

Cu(II)Co(II)Ni(II)Hg(II)Zn(II)Fe(II)Cd(II)Mn(II)Pb(II)Cu(II) + all

[ATMCA] = 1 M

Conjugate chemosensors: ATMCA (Cu2+)

logKapp = 8.0

0

100

200

300

400

500

600

0.00E+00 4.00E-07 8.00E-07 1.20E-06 1.60E-06 2.00E-06

[metal ion], M

I (41

5nm

)

Ni(II)Zn(II)Cu(II)Co(II)Cd(II)Fe(II)Hg(II)Pb(II)Mn(II)

pH = 7Zn2+NH

OCH3

NH

OCH3

NH

H3CO

Zn2+

NH2

OCH3

HN

OCH3

NH3

H3CO

[ATMCA] = 1 M

Conjugate chemosensors: ATMCA (Zn2+)

logKapp = 7.0

0

100

200

300

400

500

600

0 0.25 0.5 0.75 1 1.25 1.5 1.75 2

n

I

Zn(II)Zn(II) +Cu(II)Zn(II) +Co(II)Zn(II) +Hg(II)

0

0.2

0.4

0.6

0.8

1

1.2

0 0.25 0.5 0.75 1 1.25 1.5 1.75 2

n

I/I(m

ax)

Zn(II)

Zn(II) + Ni(II)Zn(II) + Fe(II)

Zn(II) + Mn(II)Zn(II) + Pb(II)

Zn(II) + Ni, Fe, Pb, Mn

[ATMCA] = 1 M

[ATMCA] = 1 M

Conjugate chemosensors: ATMCA (Zn2+)

Applicazione biomedica di sensori intrinseci

J.K. Tusa, H. He, JACS,

Formazione di eccimeri

M M*+ E*

OOO

O

O OOO

CO2Et

CO2Et

Na+ OOO

O

O OOO

OOEt

OEtO

excimer emission

monomer emission

calix[4]arene calix[4]arene

Jin et al. J. Chem. Soc., Chem. Commun., 1992, 499.

La complessazione del catione provoca una variazione conformazionale

Altri effetti dovuti alla variazione della conformazione

O O

O

OO

Ca2+

O O

O

OO

Ca2+

Finney et al. J. Am. Chem. Soc. 2001, 123, 1260.

FAM

TAMRA

K+

FAM

TAMRA

G

G

G

G

G

G

G

G

G

G

G

G

K+

K+

h

random coil

h

FRET

tetraplexstructure

O O-O

CO2-

HN

OOP O-OOGGGTTAGGGTTAGGGTTAGGG

P-OO

OHN

OH

O

O

CO2-

O N(CH3)2(H3C)2N

FAM(donor)

TAMRA(acceptor)

Takenaka et al. J. Am. Chem. Soc. 2002, 124, 14286.

increasing K+

concentration

planarizzazione del diarile

Conjugate chemosensors

Advantage: modularity. The two subunits (receptor and fluorophore) can be designed and optimized separately and then eventually connected.

Design: the receptor is electronically insulated from the -system of the fluorophore by a spacer.

Weakness: the overall design of the system must foresee the presence of some transduction mechanism, since the analyte and the signaling unit are no more in a direct contact. Moreover, the synthesis if often demanding

INTRINSECO

On Off

CONIUGATO

AUTOASSEMBLATO +


NH

OCH3

NH

OCH3

NH

H3CO

Zn2+

NH

OCH3

NH

OCH3

NH

H3CO

Zn2+

S

S

A organic substrate (anions) may bind to the Zn(II) ions forming a ternary complex.If the substrate is able to interact with the fluorophore this may result in thequenching of the fluorescence emission.

pH = 7

SELF-ASSEMBLED chemosensors: ATMCA·Zn(II) (organic anions)

0

0.04

0.08

0.12

0.16

0 10 20 30 40 50 60# of equivalents

Abs

orba

nce

(396

nm

)

GMPcytosine

0

0.2

0.4

0.6

0.8

1

0 10 20 30 40 50 60

# of equivalents

I/I0

GMPcytosine

Fluorescence UV-Visible

logKb = 4.1

logKb = 4.2

ATMCA = 50 M ATMCA = 50 M

Guanosine-5’monophosphate (GMP) Cytosine

N

N

NH2

OH

O

H

HHHHOH

OP-OO

O-

NH

N

N

O

NH2N

SELF-ASSEMBLED chemosensors: ATMCA·Zn(II) (nucleobases and nucleotides)

pKa = 12.2

pKa = 9.9 pKa = 9.4

pKa = 9.3

N

NN

N

NH2

R

NH

N

N

O

NH2N

R

N

N

NH2

O

R

HN

N

O

O

R

HN

N

O

O

R

O

H

HHHH

OH

OP-O

O

O-

R = H: Cytosine (no binding)R = A: CMP (no binding)

R = H: Thymine (3.6; 30%)R = A: TMP (4.5; 25%)

R = H: Uracyl (3.6; 31%)R = A: UMP (4.0; 30%)

R = H:Guanine (not soluble)R = A: GMP (4.2; 53%)

R = H: Adenine (not soluble)R = A: AMP (no binding)

A=

blu = logKbred = % of quenching

H2NNH2

NHOMe

OMeMeO

Zn2+N-

N

O

H3C

O

R

Binding and quenchingappear to be related to thepresence of an acidicamide (imide) proton andto stacking

N

N NH

NHHNZn2

+

Kimura et al. J. Am. Chem. Soc. 1994, 116, 3848.

SELF-ASSEMBLED chemosensors: ATMCA·Zn(II) (nucleobases and nucleotides)

O-

O

-O

O

Other dicarboxylic acids (malonic,succinic), monocarboxylic acids andamino acids do not bind to ATMCA.

H2NNH2

NH

OMe

OMeMeO

Zn2+O-

O

-O

O

The formation of a 5 atoms chelateappears to be crucial for binding andsignalling.

0

0.2

0.4

0.6

0.8

1

1.2

0 2 4 6 8# of equivalents

I/I0

logKb = 4.3

ATMCA = 50 MpH = 7.2

SELF-ASSEMBLED chemosensors: ATMCA·Zn(II) (carboxylic acids)

0

0.2

0.4

0.6

0.8

1

0 1 2 3

# of equivalents

I/I0

HN

N

O

O

H

COOH

[orotic acid] = 25 MpH = 7.2

H2N NH2NH

OMeMeO

OMe

Zn

N OO

O

O

NH

Orotic acid conjugates an acidicamide proton with theformation of a 5 atoms chelateand stacking.The result is a strong binding(logKb = 6.6) and total quenchof the fluorescence emission.

SELF-ASSEMBLED chemosensors: ATMCA·Zn(II) (vitamin B13)

Sistema autoassemblato mediante interazioni ione-ione: l’analita rimpiazza il rilevatore

chemosensing ensemble

NHNH

NNHN

NH

HNNH

N

OO O

CO2

CO2

HO

OO O

O

OO

NHNH

NNHN

NH

HNNH

N

OO O

CO2

CO2

HO

OO O

O

OO

Citrate Receptor Sensing EnsembleE.V. Anslyn, 1998

“Chemosensing ensemble”

Sistema autoassemblato mediante interazioni ione-metallo:stesso principio del “chemosensing ensemble”

NH

N

NHNH

Cu

NH

N

NHNH

Cu

para

meta

ortho

The approach is general and it has been applied to the detection of severalsubstrates (tartrate, gallic acid, heparin, phosphates, carbonate, amino acids andshort peptides).

Fabbrizzi et al. Angew. Chem. 2004, 43, 3847.

O

OO

OO

N

N

O O-

OO-

Figure 1 (A) Ligands and indicators used to construct sensor array. Absorbance spectra for (B) 1 [35 mM], Cu(OTf)2 [157 M], CCR [75 M], and Val [200 M]; (C) 3 [1.2 mM], Cu(OTf)2 [393 M], CAS [36 M], and Val [2.5 mM]; (D) N,N'-tetramethylethylenediamine [4.5 mM], Cu(OTf)2 [200 M], CAS [55 M], and Val [200 M]. (E) Colorimetric output for 1 [35 mM], Cu(OTf)2 [235 M], CAS [35 M], and amino acid [200 M]. All studies carried out in 1:1 MeOH:H2O, 50 mM HEPES buffer, pH = 7.8.

Pattern-Based Discrimination of Enantiomeric and Structurally Similar Amino Acids

I complessi con Cu(II) deileganti chirali 1-3 con icromofori interagisconocon ammino acidi (L e D)dando gradi diversi disostituzione e variazioni dicolore caratteristiche perciascun sistema

Taking different combinations of the ligands and indicators with Cu(OTf)2 (OTf = trifluoromethanesulfonate) and varying the concentrations of the species, we created a library of IDAs(Indicator Displacement Assay). Both enantiomers of the naturally occurring amino acids Leu, Val, Trp, and Phe, as well as the unnatural amino acid tert-leucine (Tle), were examined, giving a total of 10 analytes. For each analyte, absorbance spectra were recorded under a set of 21 different conditions.

Two-dimensional PCA (principal component analysis) plots for D and L amino acids prepared (A) from data for all 21 enantioselective indicator displacement assays (IDAs), (B) from data for 8 IDAs selective for D amino acids, and (C) from data for 13 IDAs selective for L configuration.

Pattern-Based Discrimination of Enantiomeric and Structurally Similar Amino Acids

Self-organized chemosensors: quantum dots


Analyte induced modulation of surface excitons recombination

Self-organization of binding sites

K. Konishi and T. Hiratani, Angew. Chem. Int. Ed., 2006, 45, 5191-5194

T. Jin, F. Fujii, H. Sakata, M. Tamura, and M. Kinjo, Chem. Commun., 2005, 4300-4302


Cyanide sensing by ET interruptionMaltose sensing by PET modulation

A. Touceda-Varela, E. I. Stevenson, J. A. Galve-Gasion, D. T. F. Dryden, and J. C. Mareque-Rivas, Chem. Commun., 2008, 1998-2000

M. G. Sandros, D. Gao, and D. E. Benson, J. Am. Chem. Soc., 2005, 127, 12198-12199


Chemosensing ensamble with a quencher, OFF-ON TNT detection

Chemosensing ensamble with a dye, FRET ratiometric sugar/dopamine detection

E. R. Goldman, I. L. Medintz, J. L. Whitley, A. Hayhurst, A. R. Clapp,H. T. Uyeda, J. R. Deschamps, M. E. Lassman, and H. Mattoussi, J.Am. Chem. Soc., 2005, 127, 6744-6751. R. Freeman, L. Bahshi, T. Finder, R. Gill, and I. Willner, Chem.

Commun., 2009, 764-766

Sensori autoassemblati

Vantaggi: preparazione, ottimizzazione e modificazione sono relativamente semplici

Punti deboli: trovare un meccanismo di trasduzione del segnale , diffocltà di progettazione del recettore

Struttura: recettore e unità attiva non solo non sono elettronicamente isolati ma addirittura non sono legati l’uno all’altro; si devono autoassemblare in soluzione

chemosensing ensemblechemosensing ensemble

template assisted chemosensor

templatetemplate templatetemplatetemplate

templateself-organization

Sensori organizzati da un opportuno agente templante

L’approccio si basa sull’autoassemblaggio (o l’autoorganizzazione) di unamolecola fluorescente e del recettore su di un opportuno agente templante così daformare un sistema organizzato. In questo sistema assemblato le due subunitànon interagiscono direttamente e la comunicazione tra substrato complessato emolecola fluorescente è garantita dalla loro vicinanza spaziale.

template template

self-organization

Agenti templanti:• Aggregati di tensioattivi• Monostrati• Superfici di vetro• Nanoparticelle

Self-Organized ChemosensorsIn Surfactant Micellar Aggregates

surfactant+

H2O+

+

+

+

+

+

+

+

+

+

highfluorescence

+

+

+

+

lowfluorescence

Cu(II)

ligand = C10GlyGLy

C10H21 NH HN

HO

O

O

C10H21 NH N

O

O

OCu

Cu(II)

- 2H+ 2+

PhNH SO3

fluorescent dye = ANS

ligand

fluorescent

selforganization

Angew. Chem. Int. Ed. 1999, 38, 3061-3064Langmuir 2001, 17, 7521-7528.

Cu2+ sensing and sensitivity tuning

0.0 1.0x10-4 2.0x10-4 3.0x10-40

20

40

60

80

100

I/I0

[Cu2+], M

[CTABr] = 0.94 mM

[CTABr] = 0.46 mM

[CTABr] = 0.23 mM

[ANS] = 0.5 mM[Hepes] = 0.01 mM, pH = 7exc = 375 nm, em = 500 nm[CTABr]/[C10GG] = 2

0.0 4.0x10-5 8.0x10-5 1.2x10-4 1.6x10-40

25

50

75

100 [C10GG] = 2.23 x10-4 M

[CTABr]/[L] = 5

[CTABr]/[L] = 2

I/I0

[Cu2+], M

[CTABr]/[L] = 10

10% Emission decrease at6.5×10-8 M Cu2+ concentration

Trito

n X

-100

Brij

35

DM

MA

PS

CTA

Br

ANS

1-NAFOSF

ACA

DANSA

0

20

40

60

80

100

II0-1/%

COOH

O P

OH

O

OH

SO2NH2

NH3C CH3

ACA

1-NAFOSF

DANSA

NHPh

SO3H

ANS

n-C12H25(OCH2CH2)23OH

Brij 35

(OCH2CH2)10OH

CH3

CH3

CH3

CH3

H3C

Triton X-100

n-C16H33 N

CH3

CH3

(CH2)3SO3

DMMAPS

n-C16H33 N

CH3

CH3

CH3

Br

CTABr

Fluorophores

Surfactants

Sensing in Micellar Aggregates: combinatorial screening

H2NO

HN O

HNO

H2NN

HN

NHO

C17H35

OH2N

NHO

C17H35

HN

SO

O

NA B

water subphase

air

B

B

B/A

B/A

Epifluorescence images of Langmuir monolayers of lipidsB, and A/B (90:10, molar ratio) in the absence and presence of copper ions (10-5 M) in the subphase

leblanc et al. J. Am. Chem. Soc. 2003, 125, 2680.

Utilizzo di monostrati come agenti templanti: sensore per Cu(II)

Self-Organized ChemosensorsIn Surfactant Micellar Aggregates

Advantages:• Prepared just by mixing components (no synthesis)

• Tuning of sensitivity just by variation of components ratio

• Modification of the system just by substitution of one component

Limitations:• Sensitivity to environmental conditions (temperature, ionic strength)

• Concentration limit (c.m.c.)

Self-Organized Chemosensors on SiO2 nanoparticlesSynthesis

SO

ONH

Si OMeOMe

OMeNH

Si OMeOMe

OMeON

NOO

O OO O OO OSiO2

Ludox

H2O/EtOH/AcOH60° C, 16 h

Chem. Commun. 2003, 3026-3027

NNH

O+ Cu2+, -H+

NN

O

Cu2+-

0

50

100

0 0.1 0.2

[Cu2+], mM

I/I0

%

= 0

= 0.15

= 0.40

= 0.58

= 0.91

][][][

LFL

Spectrofluorimetric titration of CSNs (0.03 mg/ml) with Cu(NO3)2 in 10% water/DMSO, HEPES buffer 0.01 M pH 7, 25 °C.

Self-Organized Chemosensors on SiO2 nanoparticlesCu2+ detection

10% Emission decrease at4×10-6 M Cu2+ concentration

[Cu2+]50% as a function of 2 molar fraction on the CSNs ([2] = 2 M, 10% water/DMSO, HEPES buffer 0.01 M pH 7, 25 °C

0

5

10

15

20

0 0.25 0.5 0.75 1

[Cu(

II)]50

%, m

M

O O

O OO

O OO O

h1

h2

SiO2 Nanoparticle

OOO O

O O OO O

SiO2 Nanoparticle

h1

h2

Self-Organized Chemosensors on SiO2 nanoparticlesSensitivity enhancement by cooperative binding

300 400 500 600

0.6

0.8

1.05 4687

I, ar

bitra

ry u

nits

, nm

5

HN Si OEt

OEt

OEtN

ON

O2NHN Si OEt

OEt

OEt

SO2

N

NH

Si OEt

OEt

OEt

OO

N Si OEt

OEt

OEtO

O

3 4

5 6

NH

Si OEt

OEt

OEt

O

O

7

NHO

O

O8

Si OEt

OEt

OEt

Self-Organized Chemosensors on SiO2 nanoparticlesComponents switching: signaling unit

0.0000 0.0005 0.00100

20

40

60

80

100 4a 5a 6a 7a 3a 8a

I/I0 (%

)

[Cu(II)], M

Conditions: DMSO/acqua 9:1, HEPES 0.01 M pH 7, 25 °C, exc=340 nm, em=520 nm.

0.00 0.05 0.10 0.15 0.20 0.25 0.300

20

40

60

80

100

I/I0 (

%)

[Cu(II)], mM0.000 0.0050

20

40

60

80

100

I/I0 (%

)

[Cu(II)], mM

NNH

O

Si OEt

OEt

OEt

1

NNH

O

HN Si OEt

OEt

OEt

2

10% Emission decrease at3.0×10-8 M Cu2+ concentration

Self-Organized Chemosensors on SiO2 nanoparticlesComponents switching: binding unit

J. Mat. Chem. 2004, 15, 2687-2696

0.0 0.2 0.4 0.6 0.820

40

60

80

100

I/Io %

(CCu(II)-CCu(II)CALC)/CDNS

O O

O OO

O OO O

SiO2 Nanoparticle

L:F=1:10.00 0.02 0.04 0.06 0.08 0.100

20

40

60

80

100

I/Io

%

(CCu(II)-CCu(II)CALC)/CDNS

O O

O OO

O OO O

SiO2 Nanoparticle

L:F=50:1

Self-Organized Chemosensors on SiO2 nanoparticlesSelectivity

SiO

OO

SiO

OO

fluoroforo

ligando

ione metallico

h1 h1

h2h2

QU

AR

TZ

Si

O

O

O

Si

O

O

O

Si

O

O

O

Si

O

O

O

QU

AR

TZ

Si

O

O

O

Si

O

O

O

Si

O

O

O

Si

O

O

O

SiO O

O

NH

HNR

SiO O

O

NH

HN

X

SiO O

O

NH

HNR

SiO O

O

NH

HNX

SiO O

O

NH

HNR

X or

O O

CO

N

=

R = H, COCH3, or CONHC6H13

N

SO2

N

SO2

GLASS

(H3CO)Si NH

NH2 SiO O

O

NH

H2N

SiO O

O

NH

H2N

SiO O

O

NH

H2N

SiO O

O

NH

H2N

SiO O

O

NH

H2N

Cl

1)

2) capping agent

sensitivity for Pb2+ in the 0.1 mM range

Crego-Calama and Reinhoudt Adv. Mater. 2001, 13, 1171

Superfici di vetro come agenti templanti: un sensore per ioni metallici

Crego-Calama, Reinhoudt and al. J. Am. Chem. Soc. 2004, 126, 7293

Superfici di vetro come agenti templanti: selezione combinatoria del sistema migliore

Sensori autorganizzati su/in nanoparticelle: PEBBLES

A. Burns, P. Sengupta, T. Zedayko, B. Baird, and U. Wiesner, Small, 2006, 2, 723-726

Core-shell silica nanoparticles for Pb2+ detection

0.0 5.0x10-5 1.0x10-40

25

50

75

100

I/I0 (

%)

[Pb2+], M

250 nm

SiEtO

OEt

OEtEtO

TEOS

NH3/H2O

EtOH, 25 °C8 h

SiO2NH3/H2O

EtOH, 25 °C16 h

TEOS,NH3/H2O

EtOH, reflux3 h

MPS, TEOS S

SH

S

HSSH

SHSH

HS

HS

SS

S

S

SH

SH

np0

np1

np2

np2

Langmuir 2007, 23, 8632-8636

S

SH

S

HSSH

SHSH

HS

HS

SS

S

S

SH

SH

Pb2+

Pb2+

Pb2+

Ratiometric sensing

SiEtO

OEt

OEtEtO

TEOS

NH3/H2O

EtOH, 25 °C16 h

SiO2NH3/H2O

EtOH, 25 °C16 h

TEOS,NH3/H2O

EtOH, reflux3 h

MPS, TEOS S

SH

S

HSSH

SHSH

HS

HS

SS

S

S

SH

SH

N

S OOHN

SiEtO OEtEtO

DNS

O

ONH

SiEtOEtO

OEt

MNC

300 400 500 6000

200

400

600

800

emis

sion

, a.u

.

nm

0.0 2.0x10-5 4.0x10-5 6.0x10-5 8.0x10-50.0

0.2

0.4

0.6

0.8

1.0

I 390/I 50

0

[Pb2+], M

S

SH

S

HSSH

SHSH

HS

HS

SS

S

S

SH

SH

Pb2+

Pb2+

Pb2+

Ratiometric sensing

300 400 500 6000

25

50

75

100

emis

sion

, a.u

.

wavelenght, nm0.0 1.5x10-5 3.0x10-5

0.2

0.4

0.6

0.8

I 390/I 50

0

[Pb2+], M

S

SH

S

HS

SH

SHSH

HS

HS

SS

S

S

SH

SH

Pb2+

Pb2+

Pb2+

TEOS

NH3/H2O

EtOH, 25 °C4 h

NH3/H2O

EtOH, 25 °C4 h

TEOS,NH3/H2O

EtOH, reflux3 h

MPS, TEOSS

SH

S

HS

SH

SHSH

HS

HS

SS

S

S

SH

SH

NH3/H2O

EtOH, 25 °C16 h

TEOS,

np3

a

b

c

FRET QI 87%Brightness 1000-fold

Active PEBBLES for O2 sensing

Single particle oxygen detection

C. F. Wu, B. Bull, K. Christensen, and J. McNeill, Angew. Chem. Int. Ed., 2009, 48, 2741-2745.

Other sensing schemes

Carboxylate detection

Eparin (polyanion) detectionP. Calero, E. Aznar, J. M. Lloris, M. D. Marcos, R. Martinez-Manez, J. V. Ros-Lis, J. Soto, and F. Sancenon, Chem. Commun., 2008, 1668-1670.

P. Calero, E. Aznar, J. M. Lloris, M. D. Marcos, R. Martinez-Manez, J. V. Ros-Lis, J. Soto, and F. Sancenon, Chem. Commun., 2008, 1668-1670.

NANO-HPLC

TEM micrograph of PLA-coated mesoporous MSN nanoparticles

Fluorescence increase by PLA-coated mesoporous MSN nanoparticles after addition of Dopamine (a), Tyrosine (b) and Glutamic acid (c)

a

b

c

V. S.-Y. Lin et al., JACS, 2004, 126, 1640-1641

Why zinc?: Zinc is only moderately abundant in nature, ranking 23rd of the elements. Zinc is, however, following iron, thesecond most abundant transition metal in the body. In total, the adult human body contains 2 –3 g zinc. The pronounced Lewisacid characteristics of the Zn2+ ion, its single redox state, and the flexibility of its coordination sphere with respect to geometryand number of ligands associated, combined with the kinetic lability of coordinated ligands, are responsible for its broad utilitywithin proteins. Thousands of proteins contain zinc. Zinc proteins can be divided into several groups according to the roleplayed by zinc. In the catalytic group (e.g.,carbonic anhydrase and carboxypeptidase A), zinc is a direct participant in thecatalytic function of the enzyme. In enzymes with structural zinc sites (e.g.,protein kinase C), one or more metal ions ensureappropriate folding for bioactivity. Enzymes in which zinc serves a co-catalytic function (e.g., superoxide dismutase), one orseveral zinc ions may be used for catalytic, regulatory, and structural functions. In addition, there are a large number oftranscription factors that utilize zinc, the so-called zinc fingers.While the total concentration of zinc in a cell is relatively high, the concentration of “free ”zinc, that is, the fraction of Zn2+ notstrongly bound to proteins, is extremely low and tightly controlled. Total cellular zinc can be determined by standardanalytical techniques such as AAS or ICP-MS, but the determination of the “free ”or “available ” Zn2+ concentrationshas proved difficult using classic techniques. This is because cell fractionation can readily lead to cross-contamination ofthe kinetically labile metal ion between intracellular sites. Thus, the knowledge gap between the structural chemistry of zinc andzinc homeostasis and action is, at least in part, due to the lack of techniques for tracking Zn2+ in biological systems. This led tothe emergence of zinc specific molecular sensors, which can make zinc “visible ”in tissue or even in live cells.

Spectroscopically silent zinc: The d10 electron configuration of the Zn2+ ion, the only zinc ion found in biological systems,has a number of practical implications for its detection. Zn2+ is colorless as it is devoid of d –d transitions. The Zn2+ ion is verystable and undergoes redox reactions only under extreme conditions, excluding the occurrence of ligand-to-metal charge-transfer bands in its complexes. These effects render UV-visible spectroscopy unsuitable for the detection of “free ”orcomplexed Zn2+. Zinc is also diamagnetic in all its compounds, prohibiting, for instance, EPR spectroscopy or magnetometricmeasurements.The d10 ion is not subject to ligand field stabilization effects, making it extremely flexible with respect to thecoordination geometries it can adopt in its complexes, and rendering it kinetically labile, allowing for rapid ligand exchangereactions. Finally, the major naturally occurring isotopes have zero nuclear spin, they are NMR silent.Much of what is known about the structure and function of Zn2+ containing proteins has been gleaned from X-ray crystalstructures, X-ray absorption data (EXAFS), and iso- morphous substitution experiments in which the Zn2+ was replaced bytraceable metal ions. None of these techniques are suitable for the tracking of Zn 2+ in cells and organisms. The use of the zincradioisotope 65Zn has allowed cell studies on bulk zinc uptake and egress, but this does not permit the direct observation of thetemporal and spatial distribution of zinc in live cells and questions of isotope equilibration with internal pools arise. Onetechnique to spectroscopically visualize zinc is the use of zinc-specific fluorescent molecular sensors.

Fluorescence chemosensors: the case of zinc

Desired optical properties: The ideal chemosensor for zinc is nonfluorescent in the free form and highlyfluorescent when coordinated to zinc; possibly, the response should be ratiometric. Moreover, the excitation wavelengthshould be as longer as possible to avoid UV-induced cell damage and to penetrate tissue better and with less scattering(giving rise to higher resolution imagesand), and to avoid UV-grade optics in the fluorescence microscopes used to observebiological samples.

Intrinsic chemosensors: the case of zinc

Determinazione della concentrazione di Zn(II) all’interno di cellule tumorali.

JACS 2004,126, 712-713.

N HN

HN

NH

NH

I > 300; ex 369 nm; em 535 nm

N

MeO

SO2

Me

HN N

O

SO2

Me

HN

EtO2C

MeN

MeO

SO2

Me

HNMe

TSQ ZINQUIN 2-Me-TSQ

ZINPYR-1

I > 3; ex 509 nm; em 525 nm


Microscopy images of mouse fibroblast cells by fluorescence in the presence of 10 mm 2-Me-TSQ.Nasir et al. J. Biol. Inorg. Chem. 1999, 4, 775.

Selectivity: Zinc is a borderline hard/soft metal with a variety of known coordination numbers, geometries, and donor atom sets. This makes the design of zinc-selective chelates somewhat difficult, but the number and concentration of competing metal ions in biological systems is limited, simplifying the task in practice.

In addition to zinc, the other divalent ions of Group 12 elicit a fluorescence response. Also, the soft-ion Pb2+ is found to bind. However, none of these toxic ions are expected to be present in any significant amount, excluding a false positive signal forzinc.The ions occurring in relatively large concentrations, such as Ca2+, Mg2+ (and Na+, K+) do not bind to cyclen, and therefore do not induce any fluorescence, even when present in a large molar excess.

Transition metals such as Mn2+, Fe2/3 +, and Cu2+ bind to many cyclen but they do not give a false positive fluorescence response as these paramagnetic ions quench fluorescence. In a refined and more relevant experiment, it is necessary to investigate how Zn2+ ions directly compete with varying concentrations of other transition-metal ions for the sensor bindingsite.


Affinity for zinc: a fluorescence titration of a given sensor with Zn2+ identifies the zinc concentration range in which the sensor can be used to measure relative concentrations of zinc. If the zinc concentration is too low, no enhanced fluo-rescence is measured because no significant binding takes place. In the upper limit range, the sensor is saturated andcannot give any information about relative concentration changes of zinc. Thus,every sensor is characterized by a useful working range of zinc concentrations.The ideal dissociation constant K d of the sensor for the analyte should be a value close enough to the projected concentration of the analyte to allow monitoring of changes in its concentration.

Binding kinetics: if the temporal resolution of changing zinc concentrations is desired, it is obligatory that the reversiblemetal binding event to the sensor is adequately fast. For instance, the binding of Zn2+ to the cyclen-based sensor 5 isvery slow (t1/2 =60 min). This is presumably due to the reorganization required to accommodate the metal in its convoluted binding site. Most sensors utilize non-macrocyclic polydentate chelates with fast binding kinetics. Rapidly binding sensors havebeen successfully used in time-resolved studies.

pH dependence: protons potentially compete with zinc for the lone pair(s) of the Lewis basic metal binding site. If the lone pair responsible for the PET process gets protonated, it becomes also less available for the quenching process, and fluorescence is switched on even in the absence of the metal ion. Hence the working pH range for any chemosensor needs to be determined to allow a judgment whether the sensor can operate within the pH range expected in the biological system studied.



Biodistribution properties : Ideally, the chemosensor is taken up by the cell or tissue, thus avoiding microinjection techniques. An indication whether endocytotic mechanisms or passive diffusion through the cell membrane is responsiblefor the uptake of the sensor can be derived by observing the temperature-dependence of its uptake. If incubation of thecells with the sensor at 4 °C results in cell uptake, it provides a strong indication for a passive diffusion mechanism, sinceendocytosis at this temperature is greatly inhibited. Once in the cell, the sensor may be excreted or metabolized, leading togradually diminishing fluorescence.

Sensor triacid 6 does not stain cells, whereas the triester 5 is taken up readily

ZINQUIN


Cell-impermeable zinc chemosensors: it is known that insulin and Zn2+ are co-stored in pancreatic -cells in secretory vesicles and are co-released by exocytosis.This process can be visualized by using the non-cell-permeable chemosensor FluoZin-3.The Figure shows the burst of fluorescence following the addition of glucose to pancreatic -cells. The time-lapse images following the burst show the fluorescence decrease due to diffusional dilution of the zinc concentration.


Single-wavelength excitation ratiometric zinc chemosensors: The signal derived from a fluorescence microscopy image of a cell stained with a zinc-specific chemosensor allows the determination of the presence of zinc. Relative emission increases can reasonably be correlated with increases of [Zn2 +] free but the fluorescence quantum yield of the sensor is in most cases solvent-dependent. Since the solvent properties of the local environments in which the sensors accumulate are not known,the absolute l emission measured cannot be correlated directly with the concentration of zinc. However, the measurement ofabsolute [Zn2+] free can be achieved by using a ratiometric sensor.

In a ratiometric sensor the binding of analyte the results in a shift of its max-emission, which may or may not be concomitant with an increase in l emission .This max-emission shift should be enough to distinguish the max-emission of the co-existing Zn 2 +-free and Zn 2 + -bound species, allowing the determination of their emission ratio.

Intrinsic chemosensors: the case of zincSingle-wavelength excitation ratiometric zinc chemosensors

Images of live COS cells stained with ZNP1 acetate. The pseudocolors depict the ratio of the fluorescence intensities at the twoemission wavelengths at 612 and 526 nm. The larger the ratio, the more zinc is present. In resting cells, little if any, “free endogenous zinc is present (A). Figure B shows the result of the addition of nitrosocyctein, an NO-delivery agent.The ratio in-creased, indicating the intracellular NO-triggered release of zinc.The cytosolic zinc was then chelated by TPEN, resulting in the complete loss of imageable zinc in the cells (C). TPEN = N ,N ,N ’N ’tetra(2-picolyl)ethylenediamine.

12-I12-II

Dual-wavelength excitation ratiometric zinc chemosensors

Phase contrast (A) and fluorescence (B, C) microscopy images of HeLa cells incubated with Coumazin-1 with the addition of ZnCl2 and sodium pyrithione. Fluorescence images were acquired with excitation at 400 –440 nm, band-pass of 475 nm (B) or with excitation at 460 –500 nm, band-pass of 510 –560 nm (C).


PEBBLES and Zn(II)

Chem. Eur. J 2007, 8, 2238-2245

SiEtO

OEt

OEtEtO

NH3/H2O

EtOH, 25 °C16 h

TEOS

TSQ

N

O

NHS

O

O

NH

HN

O

Si(OEt)3

O O

O

NH

(EtO)3Si

CUM

Zn2+

13 nm

Pebbles: Kopelman et al. Curr. Opin. Che. Biol. 2004, 8, 540-546

PEBBLES and Zn(II)

Chemical sensors (Chemosensors) “A chemosensor is molecule … lezione... · Chemical sensors...

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Transcript of Chemical sensors (Chemosensors) “A chemosensor is molecule … lezione... · Chemical sensors...