ELECTROCHEMICAL

BIOSENSORS
Modern and future approaches to
medical diagnostics
James F. Rusling
Dept. of Chemistry, Univ CT, Storrs,
Dept. of Cell Biology, Univ. of CT Health
Center
F. A. Armstrong, H. A. Heering, and J. Hirst, Reactions of complex metalloproteins studies by
protein film voltammetry. J. Chem. Soc. Rev. 26, 169-179 (1997).
J. F. Rusling, Z. Zhang, Designing functional biomolecular films on electrodes. in J. F. Rusling,
Ed., Biomolecular Films, Marcel Dekker, N. Y., 2003, pp. 1-64.
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
electrode
substrate product
Figure 9
Enzyme
(label)
Apply voltage Measure current prop.
to concentration of substrate
Principle of Electrochemical Biosensors
E, V
time
E-t waveform
potentiostat
Electrochemical cell
counter
working electrode
N
2
inlet
Protein lm
Figure1
reference
insulator
electrode
material
Equipment for developing electrochemical biosensors
Cyclic
voltammetry
Electrode
enzyme
A lipid-enzyme lm
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
-0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8
I

,

A
E, V vs SCE
Cyclic voltammogram (CV) at 100 mV s
-1
and 25
o
C of Mycobacterium Tuberculosis KatG
catalase-peroxidase in a thin film of dimyristoylphosphatidylcholine on basal plane PG electrode,
in anaerobic pH 6.0 buffer.
Oxidation
Of Fe
II
Reduction
Of Fe
III
Reversible
Peaks for
Direct electron
Transfer
(not all proteins
do this)
Catalytic enzyme electrochemistry
a basis for biosensor - glucose oxidase
oxidation
Fc
mediator
Fc + glucose
+ enzyme
I = f [glucose]
A. Cass, G. Davis, G. D. Francis, H. O. A. Hill, W. J. Aston, I. J. Higgins, E. V. Plotkin, L. D. L.
Scott, A. P. F. Turner, Anal. Chem. 56, 667-671 (1984).
Mediator shuttles
Electrons between
Enzyme and electrode
Scheme 2
Glucose + GO(FAD) + 2 H
+
! gluconolactone + GO(FADH
2
) (1)
GO(FADH
2
) + 2 Fc
+
! GO(FAD) + 2 Fc + 2 H
+
(4)
Fc ! Fc
+
+ 2 e
-
(at electrode) (5)
Mechanism for catalytic oxidation of glucose
With Glucose oxidase (GO) and Fc mediator
Signal can also be measured by amperometry:
Hold const. E where oxidation occurs, measure I vs
time
Fc = ferrocenecarboxylate
Commercial Glucose Sensors
Biggest biosensor success story!
Diabetic patients monitor blood glucose
at home
First made by Medisense (early 1990s),
now 5 or more commercial test systems
Rapid analysis from single drop of blood
Enzyme-electrochemical device on a slide
Patient Diabetes Management
Insulin secretion by pancreas regulated
by blood glucose, 4.4 to 6.6 mM normal
In diabetes, regulation breaks down
Wide swings of glucose levels
Glucose tests tell patient what action to
take (e.g. administer insulin)
Most sensors use enzyme called glucose oxidase (GO)
Most sensors are constructed on electrodes, and use a
mediator to carry electrons from GO to electrode
Fc = mediator, ferrocene, an iron complex
These reactions occur in the sensor:
Fc Fc
+
+ e- (measured)
GO
R
+ 2 Fc
+
--> GO
ox
+ 2 Fc
GO
ox
+ glucose

--> GO
R
+ gluconolactone
Reach and Wilson, Anal. Chem. 64, 381A (1992)
G. Ramsay, Commercial Biosensors, J. Wiley, 1998.
Glucose biosensor test strips (~$0.50-1.00 ea.)
Meter
Read glucose
Dry coating of GO + Fc
Patient adds drop of blood,
then inserts slide into meter
Output:
Amperometry
Constant E
I
t
Patient reads glucose level on meter
(B.B. King
es
electrodes
http://www.bbking.com/)
Research on new glucose
sensors
Non-invasive biosensors - skin, saliva
Implantable glucose sensors to
accompany articial pancreas - feedback
control of insulin supply
Record is ~4 weeks for implantable
sensor in humans
Other biosensors
Cholesterol - based on cholesterol oxidase
Immunosensors - pathogenic bacteria,
disease detection (biomarkers)
Small molecules and ions in living things:
H
+
, K
+
, Na
+
, CO
2
, H
2
O
2
DNA hybridization and damage
Micro or nanoarrays, optical abs or uor.
Negative
surface
Polycation soln.,
then wash
+ + + + + + + + + + + +
soln. of negative protein
then wash
+ + + + + + + + + + + +
+ + + + + + + + + + + +
+ + + + + + + + + + + +
Repeat steps for desired
number of layers
Protein
layer
Polycation layers
Protein
layer
Polycation soln.,
then wash
Figure 19
Layer by layer
Film construction:
PSS layer
SPAN layer
Enzyme
layer
Detection of hydrogen peroxide
Conductive polymers efciently wire
peroxidase enzymes to graphite
Xin Yu, G. A. Sotzing,

F. Papadimitrakopoulos, J. F.
Rusling, Highly Efcient Wiring of Enzymes to Electrodes
by Ultrathin Conductive Polyion Underlayers: Enhanced
Catalytic Response to Hydrogen Peroxide, Anal. Chem.,
2003, 75, 4565-4571.
es
(sulfonated polyaniline)
Horseradish Peroxidase (HRP)
100nm
50nm
Tapping mode atomic force microscopy (AFM)
image of HRP film
O
2
H
2
O
2
PFe
III
PFe
II
PFe
II
-O
2
2e
-
, 2H
+
H
2
O
2
+ PFe
II
PFe
IV
=O
active oxidant
+e
-
-e
-
PFe
III
+ H
2
O + O
2
H
2
O
2
Possible reduced species in red
Electrochemical Response of Peroxidases
-10
0
10
20
30
40
50
60
-0.8 -0.6 -0.4 -0.2 0 0.2
I,A
E, V vs SCE
with SPAN
a
0
0.5
2
4
6
7.5
M H
2
0
2
Catalytic reduction of H
2
O
2
by peroxidase films
Catalytic cycles increase current
Fe
III
/Fe
II
reduction
0
0.5
1
0 100 200 300 400
I
,

A
t, s
with PAPSA
without PAPSA
Rotating electrode amperometry at 0 V
HRP, 50 nmol H
2
O
2
additions
span
No span
reduction
0
0.2
0.4
0.6
0.8
1
1.2
0 0.1 0.2 0.3 0.4 0.5 0.6
I
,

A
[H
2
O
2
], M
PAPSA/HRP
PAPSA/Mb
Mb
HRP
Rotating electrode amperometry at 0 V
Sensitivity much higher with conductive polymer (SPAN);
Electrically wires all the protein to electrode
Span/HRP
Span/Mb
Single walled (1.4 nm
o.d.) and multi-walled
Highly conductive,
exible, strong,
patternable
Commercially Available
Carbon Nanotubes
Single-Walled Carbon Nanotube
Forests: Antigen-Antibody Sensing
SPAN or
Naon
Chattopadhyay, Galeska, Papadimitrakopoulos, J. Am. Chem. Soc. 2001, 123, 9451.
End COOH groups allow chemical attachment to proteins (antibodies)
High conductivity to conduct signal (es) from enzyme label to meas. circuit
~1.4 nm diameter, high conductivity
AFM of SWNT forest with and without anti-HSA attached
SWNT forest on Si wafer
SWNT forest with anti-human serum
albumin (HSA) attached by amide links
Also linked enzymes to SWNT forests:X. Yu, D. Chattopadhyay, I. Galeska,
F. Papadimitrakopoulos, and J. F. Rusling, Peroxidase activity of enzymes bound to the ends
of single-wall carbon nanotubeforest electrodes, Electrochem. Commun., 2003, 5, 408-411.
Sandwich Electrochemical
Immunosensor Proteins
H2O2
Ab1
Ag
HRP
HRP
HRP HRP HRP
Ab1
Ag
H
R
P
HPR
Ab
2
Apply E measure I
SWNT forest
Conductive polymer
(SPAN)
protein
Amperometry Detection of Human Serum
albumin
-2
0
2
4
6
8
10
12
0 500 1000 1500
I , A
t, s
1.5
7.5
15
45

600 pmol/mL HSA
300
140
140
300
140
controls
a
0
b
c
d
no SWNT
bare PG
0.4 mM hydroquinone;
0.4 mM H
2
O
2
SWNTs provide 10-20 fold signal enhancement
Nanotubes aged in DMF ! fewer defects ! denser forests
. Xin Yu, Sang Nyon Kim, Fotios Papadimitrakopoulos
,
and James F. Rusling,
"Protein Immunosensor Using Single-Wall Carbon Nanotube Forests with Electrochemical
Detection of Enzyme Labels", Molecular Biosystems, 2005, 1, 70-78.
Initial Target: Prostate Specic Antigen
! PSA - Single chain
glycoprotein , MW 33 kDa
!Sensitive, specific
biomarker for detection of
prostate cancer up to 5 years
before clinical signs of disease
!Detection of PSA in serum:
clinical method for detection
of prostate cancer
!Led to less invasive
treatment protocols, avoid
surgery
Adapted From Brookhaven Protein Databank
Nanotube Strategies for PSA detection
~170 labels
per PSA
Washing with 2% BSA/0.05% Tween 20 to control non-specic binding
LOD - 4 pg/mL; 100-fold enhancement over HRP-Ab
2
Amperometric response at 0.3 V and 3000 rpm for SWNT immunosensors incubated with PSA
in 10 L undiluted newborn calf serum for 1.25 hr using the Ab
2
-CNT-HRP bioconjugate
Mediator + H
2
O
2
Using HRP-Ab
2
-nanotube
Xin Yu, Bernard Munge, Vyomesh Patel, Gary Jensen, Ashwin Bhirde, Joseph D. Gong, Sang-
Nyon Kim, John Gillespie, J. Silvio Gutkind, Fotios Papadimitrakopoulos and James F. Rusling,
"Carbon Nanotube Amplification Strategies for Highly Sensitive Immunosensing of Cancer
Biomarkers in Serum and Tissue", J. Am. Chem. Soc., 2006, 128, 11199-11205.
Amperometric current at 0.3 V and 3000 rpm for human serum samples and PSA standards in
calf serum
Accurate results obtained for cancer patient serum
Good correlation with ELISA!
Using conventional HRP-Ab
2
Future - arrays to detect many biomolecules at once
SWNT forest grown on
10 m Au Array elements
Prototype 8-electrode Array,
Univ. Edinburgh
Biosensors
Promising approach to medical diagnostics by
patients or in doctors ofces
Other important applications: cancer
biomarkers, DNA, peroxide, etc.
Method of choice for blood glucose in diabetics
Rapid diagnostics may lead to more timely and
effective treatment