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BIOSENSORS
Modern and future approaches to
medical diagnostics
James F. Rusling
Dept. of Chemistry
Dept. of Cell Biology, Univ. of CT Health
Center
Medical Diagnostics
Doctors increasingly rely on testing
Needs: rapid, cheap, and low tech
Done by technicians or patients
Some needs for in-vivo operation, with
feedback
Principle of Electrochemical Biosensors
substrate product
Enzyme
(label)
electrode
Figure 9
Equipment for developing electrochemical biosensors
potentiostat
insulator electrode
material
reference
Protein film
N2 counter
inlet
working electrode
E-t waveform
Cyclic
E, V
voltammetry
Electrochemical cell
time
A lipid-enzyme film
enzyme
Electrode
Cyclic voltammogram (CV) at 100 mV s-1 and 25 oC of Mycobacterium Tuberculosis KatG
catalase-peroxidase in a thin film of dimyristoylphospha tidylcholine on basal plane PG electrode,
in anaerobic pH 6.0 buffer.
2
Reversible
Reduction
Peaks for 1.5
Of FeIII
Direct electron 1
Transfer 0.5
I , A
-0.5
-1
Oxidation
-1.5
Of FeII
(not all proteins -2
0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8
do this) E, V vs SCE
A lipid-enzyme film
enzyme
Electrode
Catalytic enzyme electrochemistry
a basis for biosensor - glucose oxidase
I = f [glucose]
oxidation
Fc + glucose
+ enzyme
Mediator shuttles
Electrons between
Enzyme and electrode
Fc mediator
Fc Fc+ + e- (measured)
GOR + 2 Fc + --> GOox + 2 Fc
Dry coating of GO + Fc
es
Meter electrodes
Read glucose
Layer by layer
+ + + + + + + + + + + +
Protein
layer + + + + + + + + + + + +
Polycation soln.,
then wash
+ + + + + + + + + + + +
+ + + + + + + + + + + +
Polycation layers
Protein
layer
Figure 19
Detection of hydrogen peroxide
Conductive polymers efficiently wire
peroxidase enzymes to graphite
es
Xin Yu, G. A. Sotzing, F. Papadimitrakopoulos, J. F.
Rusling, Highly Efficient Wiring of Enzymes to Electrodes
by Ultrathin Conductive Polyion Underlayers: Enhanced
Catalytic Response to Hydrogen Peroxide, Anal. Chem.,
2003, 75, 4565-4571.
Horseradish Peroxidase (HRP)
100nm
50nm
+e- O2
PFe III - PFe II PFe II-O2
-e
2e-, 2H+
H2O2
PFeIV=O H2 O2 + PFeII
active oxidant
H2O2
PFeIII + H2 O + O2
-10
FeIII/FeII
0.2 0 -0.2 -0.4 -0.6 -0.8
E, V vs SCE
Rotating electrode amperometry at 0 V
HRP, 50 nmol H2O2 additions
1
reduction
span
with PAPSA
I, A
0.5
No span
without PAPSA
0.8
Span/HRP
I, A
PAPSA/HRP
0.6 Span/Mb
PAPSA/Mb
0.4
HRP
0.2 Mb
0
0 0.1 0.2 0.3 0.4 0.5 0.6
[H O ], M
2 2
Highly conductive,
flexible, strong,
patternable
Commercially Available
Single-Walled Carbon Nanotube
Forests: Antigen-Antibody Sensing
~1.4 nm diameter, high conductivity
SPAN or
Nafion
Ag Ag
Ab2
Ab1 Ab1
HPR
H
R
P
10
300 0.4 mM hydroquinone;
0.4 mM H O
I, A 8 140 2 2
45 controls
4
15
c d
2 7.5 a b 300
140
1.5 140
0
0
no SWNT bare PG
-2
0 500 1000 1500
t, s
SWNTs provide 10-20 fold signal enhancement; high surf.
area
Nanotubes aged in DMF fewer defects denser
forests
. Xin ,
Yu, Sang Nyon Kim , Fotios Papadimitr akopou los and James F. Rusli ng,
"Protein Imm unosen sor Using Single-Wall Carbon Nano tube Forests wit h Electrochemi cal
Detection of Enzy me Labels ", Molecular Biosystems, 2005, 1, 70-78.
Initial Target: Prostate Specific Antigen
~170 labels
per PSA
Using HRP-Ab2-nanotube
Amperometric response at 0.3 V and 3000 rpm for SWNT imm unosen sors incub ated with PSA
in 10 L und iluted newborn ca lf serum for 1.25 hr using the Ab 2-CNT-HRP bioconjuga te
Mediator + H 2O2
Xin Yu, Bernard Munge, Vyo mesh Patel, Gary Jens en, Ashw in Bhir de, Joseph D. Gong , Sang -
Nyon Kim, John Gillespie, J. Silvio Gutk ind, Fotios Papadimit rakopou los and James F. Rus ling,
"Carbon Nano tube Amplific ation Strategies fo r High ly Sens iti ve Immuno sens ing of Cancer
Biomarkers in Serum and Tis sue ", J. Am. Chem. Soc., 2006, 128, 11199-11205.
Accurate results obtained for cancer patient serum
Amperometric current at 0.3 V and 3000 rpm for human serum samples and PSA standards in
calf serum
Using conventional HRP-Ab2