1

University of CreteDepartment of Chemistry

Laboratory of Analytical ChemistryIraklion, Crete, GREECE

Nanostuctures in BioSensors

Vicky Vamvakaki, N.A. Chaniotakis

Nanostuctures in BioSensors

URL: www.analytical_chemistry.uoc.gr

Nanomaterials have unique and novel physicochemical characteristics

Why Nano in Biosensors

High surface ratio

Novel optical properties

Increased catalytic activity

Enhanced electron transfer

Immobilization matrices

Transduction platforms

Mediators

NanoparticlesFunctionalization with inorganic and biological molecules

Nanoporous materialsImmobilization and stabilization of proteins and other biologicalmolecules

Schematic Diagram of Biosensors

ImmobilizationStabilization

MediationTransduction

TRANSDUCERBIORECOGNITION

ELEMENTDATA

ACQUISITIONANALYTESAMPLE

Basic Concepts in Biosensors Design

Problems in Biosensor DesignSensitivityStabilityReproducibility

Immobilization Matrices in BiosensorsBiocompatibilityStabilization of the biological moleculesSimplicity of immobilization procedurePossibility for direct transduction

2

Stabilization of Proteins in Confined SpacesEffect of confinement on the folding free energy as a function of the cage size

The radius of the protein in the native state (aN) was given by 3.73N1/3

Cage size (in units of 2aN) is given on a log scale.

Ν = 100Ν = 200

H.X. Zhou, K.A. Dill Biochemistry, 2001, 40 (38), 11289

Active Surface

Correlation between Protein size and Cage size

Maximum stabilization of proteins in spherical cages with diameter of 2 to 6times the diameter of the native protein

~20 -100 nm

~7 nm Glucose

Gluconic Acid

Enzyme

Enzyme withpolyelectrolyte

Materials

Porous Carbon

Silica Beads

Immobilization matrices in Biosensors

Nanotubes-Fullerenes

Liposomes

Nanocrystals

Porous Carbon

SEM images of the porous carbon

Micro pores < 80nmMeso pores 100-300nmVarious surface groups

S. Sotiropoulou, V. Gavalas, V. Vamvakaki, N.A. Chaniotakis Biosens. Bioelectron. 2003, 18, 211

3

Enzyme Stabilization into Porous CarbonEffect of the polyelectrolyte diethylaminoethyl-dextran on the stabilization of

Glucose Oxidase and Lactate Oxidase into porous carbon

Operational stability E (vs. Ag/AgCl) flow rate buffer

Gluocose sensor +800mV 1.0mL/min 10mM phosphate pH=7.5

Lactate sensor +600mV 0.52mL/min 10mM phosphate pH=7.0/1.0mM lactate

V. Gavalas, N.A. Chaniotakis, Anal. Chim. Acta 2000, 404, 67

Enzyme Stabilization into Porous Carbon

40

60

80

100

mai

ning

Act

ivity

m-AChE in porous carbon m-AChE free

60

80

100

120

140

mai

ning

Act

ivity

free m-AChE m-AChE in carbon powder

Leaching Continuous Operation

Mutant Acetylcholinesterase, +350 mV, 25 oC

0

20

40

% R

em

wash time48

hr19

hr6 h

r2 h

r0.5

hr

0 20 40 60 800

20

40

% R

em

time (hr)

S. Sotiropoulou, V. Vamvakaki, N.A. Chaniotakis, Biosens.Bioelectron. 2005, 20, 1674

Leaching ratem-AChE free: 1.8%/hrm-AChE in porous carbon: 0.7%/hr

AChE

Acetylcholine

Porous Carbon Pesticide Biosensor

Acetylcholine receptors

CH O P

O

OMeOMeC

Cl

Cl

Dichlorvos

O2N O P

O

OCH3

OCH 3

Paraoxon-methyl

50

60

70 dichlorvos paraoxon

35

40

45

50

dichlorvos paraoxon

Electrophorus electricus AChE Mutant (E69Y, Y71D)Drosophila melanogaster AChE

Inhibition Curves

Porous Carbon Pesticide Biosensor

8 10 12 14 16 18 20

0

10

20

30

40

50

% In

hibi

tion

-log[pesticide], M

6 8 10 12 14 16 18

0

5

10

15

20

25

30

% In

hibi

tion

-log[pesticide], M

S. Sotiropoulou, N.A. Chaniotakis, Biosens.Bioelectron. 2005, 20, 2347S. Sotiropoulou, N.A. Chaniotakis, Anal.Chim. Acta 2005, 530, 199

4

60

80

100

inin

g A

ctiv

ity

m-AChE in silica beads m-AChE free

100

120

140

160

180

200

inin

g A

ctiv

ity

free m-AChE m-AChE in silica beads

Porous Silica Beads

Leaching Continuous OperationPore diameter (10nm) ~ Enzyme diameter

0

20

40

% R

ema

48 hr

19 hr

2 hr

0.5 hr

6 hr

wash time

-10 0 10 20 30 40 50 60 70 800

20

40

60

80

% R

emai

time (hr)

S. Sotiropoulou, V. Vamvakaki, N.A. Chaniotakis, Biosens.Bioelectron. 2005, 20, 1674

Mutant Acetylcholinesterase, +350 mV, 25 oCLeaching ratem-AChE free: 4.6%/hrm-AChE in porous carbon: 0.4%/hr

Carbon Nanotubes

Transducer

Glucose

Gluconic acid

e-

EnzymeGlucose Oxidase

The carbon nanotubes were grown by the Chemical VaporDeposition method on a platinum platform , thus providingan array of MWNT, 15-20 microns long and with aninternal diameter of 150nm.

Carbon Nanotubes

SEM images of the Carbon Nanotubes

Initial Carbon Nanotube Array

Acid oxidation (HNO3/H2SO4)

Air oxidation(600 0C, 5min)

S. Sotiropoulou, N.A. Chaniotakis, Anal. Bioanal. Chem. 2003, 375, 103

30

40

50

60

µΑ)

1 0

1.5

2.0

Α)

1st day 2nd day

Carbon Nanotube Biosensor

Acid oxidation (HNO3/H2SO4)

0 1 2 3 4

0

10

20∆Ι (µ

[glucose] (mM)

0.0 0.5 1.0 1.5 2.0 2.5

0.0

0.5

1.0

∆Ι (

µ

[glucose] (mM)

Linear range: 0.25 - 2.5 MSensitivity: 93.9 ± 0.4 µA mM-1 cm-2

S. Sotiropoulou, N.A. Chaniotakis, Anal. Bioanal. Chem. 2003, 375, 103

5

Carbon Nanotube Biosensor

Air oxidation (600 0C, 5min)

2.2

2.4

2.6

2.8

3.0

3.2

µΑ)

0.4

0.5

0.6

0.7

(µΑ

)

0.0 0.5 1.0 1.5 2.01.2

1.4

1.6

1.8

2.0

∆Ι (

µ

[glucose] (mM)

0.0 0.5 1.0 1.5 2.0 2.5 3.0

0.1

0.2

0.3

∆Ι

[glucose] (mM)

1st day Ar atmosphere

Linear range: 0.05 - 0.5 MSensitivity: 15.6 ± 0.5 µA mM-1 cm-2

Mediation efficiency

Fullerenes

Fullerene C60multiple redox stateslow solubility in aqueous solutionsstable in many redox forms

Enzyme Glucose oxidase

Glucose

Gluconic acid

FAD

FADHMediator(ox)

Mediator(red)

+350 mV

e-

Fullerenes

V. Gavalas, N.A. Chaniotakis, Anal. Chim. Acta 2000, 409, 131

Calibration curve of the glucose biosensorcontaining 1.7µg C60/mg of electrodematerial. Measurements were performedin 10mM phosphate buffer, pH=7.5 underargon, at +350mV vs. Ag/AgCl.

Hydrodynamic voltammogram for theglucose biosensors constructed usingcarbon incubated for: 0 ( ), 4 ( ), 5 ( )cycles in the toluene-C60 solution

Liposomes

Lipids

300 ± 4 nm

enzymefluorescentindicatorporin substrate

Insertion of the porin OmpF in the

liposome membrane to allow substrate

entrance

Encapsulation ofAChE in liposomes

Encapsulation of the pH sensitive

fluorescent indicator, pyranine

The enzymatic reaction lowers the pH value which is correlated to substrate

concentration

AChEAcetylcholine + H2O choline + acetic acid

B. Chaize, M. Winterhalter, D. Fournier, BioTechniques 2003, 34, 1158

6

5 94

5,96

5,98

6,00

ce

5,9

6,0

6,1

nce

Calibration curve of the AChE/liposomebiosensor. The fluorescence intensity after10min reaction time was recorded for eachsubstrate concentration.

Fluorescence signal of the AChE/liposomebiosensor with time, for different ATChClconcentrations, 2.5, 5.0, 10.0 and 13.3 mM.

Liposome based Biosensor

2 4 6 8 10 12 145,84

5,86

5,88

5,90

5,92

5,94

Fluo

resc

enc

ATCh-Cl (mM)

0 5 10 15 20 25 30

5,6

5,7

5,8

5,9

t (min)

Fluo

resc

en

buffer 2.5 mM 5.0 mM 10.0 mM 13.3 mM

V. Vamvakaki, D. Fournier, N.A. Chaniotakis, Biosens.Bioelectron. 2005, In press

Linear range: 1.0 – 13.3 mMSensitivity: 8.2 x 10-3 Abs mM-1

activeenzyme

fluorescentindicatorporin substratepesticide inhibited

enzymeactive

enzymefluorescentindicatorporin substratepesticide inhibited

enzyme

Liposome based Pesticide Biosensor

6 7 8 9 10 11 12

102030405060708090

- Log [paraoxon] (M)

I (%

)

6 7 8 9 10 11 120

102030405060708090

I (%

)- Log [dichlorvos] (M)

V. Vamvakaki, N.A. Chaniotakis, Anal. Chim. Acta submitted

1.4 x 10-10 M 1.0 x 10-10 M

NGa

Gallium Nitride-Based SensorOn the surface of the GaN wurtzite crystal, each gallium atom has three complete

bonds to the underlying nitrogen atomic planeThese gallium atoms are relatively electropositive, due to the induced polarity of the

Ga to the N bondThus the gallium atoms of the GaN surface are expected to interact with anions of the

solutionIn such a case, the formation of a double layer at the GaN/solution interface can be

measured, and thus can be the basis for the development of a Chemical sensor

N G a

N G a N G a

N G a N G a

N G a N G a

G a N

G a N

G a N

G a N

G a N

G a N G a

N

N G a N G a

G a N G a

G a N G a

G a

N

N

N

Cl

Cl

Cl

ClC l

ClC l

ClC l

Electrometer

GaN Crystal

Indium contact(insulated)

Insulated Pt wire

Reference electrode

Y. Alifragis, G. Konstantinidis, A. Georgakilas, N.A. Chaniotakis, Electroanalysis 2005, 17, 527

Gallium Nitride-Based Sensor

-560-540-520-500-480-460-440-420-400-380

F-

Cl-

SO4-

solu

te P

oten

tial

N.A. Chaniotakis, Y. Alifragis, G. Konstantinidis, A. Georgakilas, Anal. Chem. 2004, 76, 5552

The sensitivity of the sensor is depended on the surface potential generated dueto the specific interaction with the active sites within the double layer

-6 -5 -4 -3 -2

-640-620-600-580 Cl

Br-

I-

Ab

Log[X-]

7

Gallium Nitride-Based SensorImpedance spectra of the GaN-Based sensor

as a function of different activities of KCl

500

750

1000

m) X

100

10-4M KCl

10-5M KCl

10-6M KCl

N.A. Chaniotakis, Y. Alifragis, G. Konstantinidis, A. Georgakilas, Anal. Chem. 2004, 76, 5552

There is a direct relationship between the anion activity and the capacitanceof the GaN-Solution interface

0 500 1000 1500 2000 2500

0

250

Z imag

(Oh

10-2M KCl

10-3M KCl

Zreal(Ohm) X 100

GaN as Transducer in DNA Biosensor Design

The experiments that have already carried out withGaN material prove that the electron defective Gaatoms of the GaN surface can coordinate withLewis acidic molecules and anions.

Consequently, negative charged organic orbiological molecules can be immobilized on theGaN surface by Lewis acid-base interactionsbetween the GaN surface and the negative chargedmolecules.

Immobilized ds DNA

GaN as Transducer in DNA Biosensor Design GaN as Transducer in DNA Biosensor Design

400

600

800

1000

1200

1400

'' (K

Ohm

)

Buffer ds DNA ss DNA

150

200

250

300

350

400

450

500

550

'' (K

Ohm

)

DNA DNA + ACTD 1.2nM DNA + ACTD 9.2nM

0 500 1000 1500 2000 2500 3000 3500 4000-200

0

200Z

Z ' (KOhm)

Immobilization of DNA molecules on the GaN surface

Detection of the DNA intercalator Actinomycin D

0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200

0

50

100

150

Z

Z ' (KOhm)

8

A quantum dot

Quantum Dots Quantum Dots - BiosensorsBy altering the particle size and the chemicalcomposition of the QDs the fluorescent emissionchanges

Bacteria

Direct Stainingof Bacteria and Virus

Quantum Dots - Biosensors

Organic or InorganicAnalyte

Conjugation,Silinization,

Functionalization

AcknowledgmentsThis work is being supported by the European Commission Programs “GANANO” and “SAFEGARD”, and the program “IRAKLITOS” of the Greek Ministry of Education.

Prof. N.A. ChaniotakisS S ti lS. SotiropoulouV. GavalasI. GherghiY. AlifragisProf. D. Fournier