University of Crete
Department of Chemistry
Laboratory of Analytical Chemistry
Iraklion, Crete, GREECE
Top Down Vs Bottom Up Approach in Bio-Sensors
Nanomaterials in the Design of
Chemical Sensors and Biosensors:
A bottom up Approach
Nikos A. Chaniotakis
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
The Controlling Parameters of Bio-Sensors
Selectivity
Disciplines Involved in the Design of Bio-Sensors
Detection Limit
Nanomaterials
Cost
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
CHEMISTRY
Organic
Physical
Inorganic
Macromolecular
Sensitivity
BIOLOGY
MATERIALS
Polymers
Nanoparticles
Semi-conductors
Reproducibility
DEVICES
DNA
Enzymes
Cells
Stability
Bio-Sensors
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
Schematic Diagram of Bio-Sensors
Nanomaterials in Bio-Sensors
Nanomaterials
Analyte
Electrode
Nanomaterials must have unique and novel physical
and/or chemical characteristics which can aid in the design
of bio-sensors with improved analytical characteristics:
Display
125
0
Signal Conditioning:
¾ High surface ratio
500
250
0
10
0
20
0
30
0
40
0
50
0
600
Potential,
P
t ti l Current,
C
t
Impedance, Light
¾ Novel electro-optical properties
¾ Increased catalytic activity
¾ Enhanced electron transfer
Signal Transduction
Analyte Recognizing System
Enzyme, Ionophore
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
1
Nanomaterials in Bio-Sensors
Operational Principles of Biosensors
The example of Glucose Oxidase
High surface ratio
Novel electro-optical properties
Increased catalytic activity
Enhanced electron transfer
Immobilization matrices
Stabilization matrices
Optical & electrochemical Mediators
Transduction platforms
Nano Materials
+800 mV
Enzyme
Glucose oxidase
H2 O2
FAD
Glucose
O2
FADH
Gluconic
acid
e-
Quantum Dots
9Nanomaterials
Quantum Dots?
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
Nanomaterials in Bio-Sensors
Stabilization in Nano Spaces
Materials
Immobilization and stabilization of proteins and other biological
molecules
-220
GaN Quantum Dots
Functionalization with inorganic and biological molecules
Potentiall (mV)
-200
-180
-160
-140
0
30
60
90
120
150
180
210
240
Time (days)
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
M. Vamvakaki, N.A. Chaniotakis, Anal. Chim. Acta 320 (1996) 53-61
Stabilization of Proteins in Confined Spaces
Protein and Cage Size
Effect of confinement on the folding free energy as a function of the cage size
Maximum stabilization of proteins in spherical cages with
diameter of 2 to 6 times the diameter of the native protein
Active Surface
Gluconic Acid
Ν = 100
Ν = 200
Enzyme
~7 nm
Glucose
~20 -100 nm
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.
H.X. Zhou, K.A. Dill Biochemistry, 2001, 40 (38), 11289
Enzyme with
polyelectrolyte
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
2
Enzyme Stabilization
Pesticide Biosensor
Stabilization of Glucose Oxidase into nanoporous carbon
Peripheral Site
Acetylcholine
W279
AChE
Acylation Site
W84
Acetylcholine
receptors
O
Cl
C
CH O P
Cl
O
OMe
OMe
O
O2 N
Dichlorvos
P
OCH 3
OCH 3
Paraoxon-methyl
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
V. Gavalas, N.A. Chaniotakis, Anal. Chim. Acta 2000, 404, 67
Porous Carbon Pesticide Biosensor
Nano Biosensors
Mutant (E69Y, Y71D) Drosophila melanogaster AChE
+350 mV 25 oC
120
free m-AChE
m-AChE in carbon nanopores
70
dichlorvos
paraoxon
60
% Inhibition
100
80
60
40
30
10
20
0
0
0
20
40
60
80
Lipids
50
20
40
8
10
12
14
16
18
20
-log[pesticide], M
time (hr)
fluorescent
indicator
porin
enzyme
Insertion of the porin
OmpF in the
liposome membrane
to allow substrate
entrance
Encapsulation of
AChE in
liposomes
Encapsulation of the
pH sensitive
fluorescent indicator,
pyranine
substrate
The enzymatic reaction lowers
the pH value which is
correlated to substrate
concentration
AChE
Acetylcholine + H2O
S. Sotiropoulou, N.A. Chaniotakis, Biosens.Bioelectron. 2005, 20, 2347
S. Sotiropoulou, N.A. Chaniotakis, Anal.Chim. Acta 2005, 530, 199
choline + acetic acid
B. Chaize, M. Winterhalter, D. Fournier, BioTechniques 2003, 34, 1158
Pesticide Biosensor
Fullerenes
Calibration Curve
Fullerene C60
90
Detection Limit:7.5 x 10-11 M
80
¾ multiple redox states
¾ low solubility in aqueous solutions
¾ stable in many redox forms
70
60
I (%)
% Remaining A
Activity
140
300 ± 4 nm
Calibration Curve
Continuous Operation
50
40
+350 mV
30
Enzyme
Glucose oxidase
Mediator(red)
20
FAD
Glucose
FADH
Gluconic
acid
10
0
6
7
8
9
10
11
12
-log[dichlorvos], M
V. Vamvakaki, N.A. Chaniotakis, Anal. Chim. Acta submitted
eMediator(ox)
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
3
Fullerenes
Fullerenes
+100mV
+350mV
Enzyme
Glucose Oxidase
Fullerene Mediator
eHydrodynamic voltammogram for the
glucose biosensors constructed using
carbon incubated for: 0 ( ), 4 ( ), 5 ( )
cycles in the toluene-C60 solution
Calibration curve of the glucose biosensor
containing 1.7µg C60/mg of electrode
material. Measurements were performed
in 10mM phosphate buffer, pH=7.5 under
argon, at +350mV vs. Ag/AgCl.
FAD
Glucose
FADH
Gluconic
acid
Flowchart of the processes involved in a light induced fullerene mediated
electrochemical biosensor. The operating potential has dropped to +100 mV.
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
V. Gavalas, N.A. Chaniotakis, Anal. Chim. Acta 2000, 409, 131
Fullerenes
Carbon Nanotubes
Glucose
-0.4
Pt Transducer
Gluconic acid
-0.2
∆Ι (µΑ
Α)
0.0
Light ON
0.2
Light OFF
e-
0.4
Enzyme
Glucose Oxidase
0.6
0.8
-1
0
1
2
3
4
The carbon nanotubes were grown by the CVD method on a platinum substrate, thus
providing an array of MWNT, 15-20 microns long and with an internal diameter of
150nm.
5
[Glucose], mM
S. Sotiropoulou, N.A. Chaniotakis, Anal. Bioanal. Chem. 2003, 375, 103
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
Carbon Nanotubes
Carbon Nanotube Biosensor
2.0
SEM images of the Carbon Nanotubes
Linear range: 0.05 - 2.5 M
Sensitivity: 93.9 ± 0.4 µA mM-1 cm-2
∆Ι (µΑ)
1.5
1.0
0.5
0.0
Initial Carbon
Nanotube Array
Acid oxidation
(HNO3/H2SO4)
Air oxidation
(600 0C, 5min)
S. Sotiropoulou, N.A. Chaniotakis, Anal. Bioanal. Chem. 2003, 375, 103
0.0
0.5
1.0
1.5
2.0
2.5
[glucose] (mM)
S. Sotiropoulou, N.A. Chaniotakis, Anal. Bioanal. Chem. 2003, 375, 103
4
Carbon Nanofiber Biosensor
Carbon Nanofiber Biosensor
Table 1. Carbon nanofiber physical characteristics
Nanofiber Grade
Diameter (nm)
2
N2 Surface Area (m /g)
LHT
HTE
GFE
70-150
80-150
80-150
43
80-100
> 50
Density (g/cm3)
> 1.95
1.98
2.17
Heat treatment (o C)
1000
1000
3000
Metal Content (wt. %)
< 0.50
< 0.50
< 0.01
Electrical Resistivity (Ohm/cm)
< 10-3
< 10-3
< 10-3
SEM image of HTE Nanofibers
mean diameter ~ 110 nm
length ~ tenths of nanometers
Carbon Nanofibers
Carbon Nanotubes
V. Vamvakaki, K. Tsagaraki, N.A. Chaniotakis, Anal. Chem. Is press
V. Vamvakaki, K. Tsagaraki, N.A. Chaniotakis, Anal. Chem. Is press
Carbon Nanofiber Sensor
Carbon Nanofiber BioSensor
Stability Study
-4
I (A
A)
5.0x10
GFE
HTE
LHT
NANOTUBES
GRAPHITE
1 50
1 40
% Remainiing Activity
-3
1.0x10
0.0
-4
-5.0x10
1 30
1 20
1 10
1 00
GFE
HTE
LHT
NA N OTUBES
GR AP HITE
90
80
-3
-1.0x10
70
-0.8 -0.6 -0.4 -0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
E (V)
0
20
40
60
80
100
t (hour s)
Reproducibility: RSD value < 1% (N = 3)
V. Vamvakaki, K. Tsagaraki, N.A. Chaniotakis, Anal. Chem. Is press
GaN Quantum Dots
By altering the particle size and the chemical
composition of the QDs the fluorescent emission
changes.
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
V. Vamvakaki, K. Tsagaraki, N.A. Chaniotakis, Anal. Chem. Is press
Quantum Dots
A quantum dot
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
5
Optical Properties of GaN quantum dots
Photoluminescence spectra
11000
GaN QDs
GaN QDs - KCl 1M
GaN QDs - KCl 2M
10000
9000
Depending on the KCl
concentration
• Blue shift
• Rise of intensity
Intensitty (a. u.)
8000
7000
6000
5000
4000
Conclusions-Future Directions
Nanomaterilas have unique properties that are
ideal for the development of
highly stable,
reproducible
reproducible,
and sensitive
3000
2000
chemical sensors and
biosensors
1000
400
450
500
550
600
650
700
750
Wavelength (nm)
The particle size and the
chemical composition altered
QDs fluorescent
emission changes
E-MRS Spring 2006. N. A. Chaniotakis University of Crete
Acknowledgments
9Sofia Sotiropoulou
9Vicky Vamvakaki
9Maria Fouskaki
9Jiannis Alifragis
9Antonis Volosirakis
9Kleri Karapidaki
Colaborations
9Microelectronics Group FORTH
9Prof. Ambacher and his group TUI
This work is being supported by the European Commission Programs “GANANO” and
“SAFEGARD”, “IRAKLITOS” and “ARCHIMIDIS” of the Greek Ministry of
Education.
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