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
Principle of Electrochemical Biosensors
substrate product

Enzyme
(label)
electrode

Apply voltage Measure current prop.

to concentration of substrate

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 dimyristoylphosphatidylcholine on basal plane PG electrode,
in anaerobic pH 6.0 buffer.

2
Reversible
Peaks for 1.5 Reduction
Direct electron 1
Of FeIII
Transfer 0.5
I , µA

-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
 Catalytic enzyme electrochemistry
a basis for biosensor - glucose oxidase
I = f [glucose]
oxidation
Fc + glucose
+ enzyme

Mediator shuttles
Electrons between
Fc Enzyme and electrode
mediator

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).
 Mechanism for catalytic oxidation of glucose
With Glucose oxidase (GO) and Fc mediator
Scheme 2

Glucose + GO(FAD) + 2 H+ ! gluconolactone + GO(FADH2) (1)

GO(FADH2) + 2 Fc+ ! GO(FAD) + 2 Fc + 2 H+ (4)

Fc ! Fc+ + 2 e- (at electrode) (5) Fc = ferrocenecarboxylate

Signal can also be measured by amperometry:

Hold const. E where oxidation occurs, measure I vs
time
 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)
GOR + 2 Fc + --> GOox + 2 Fc

GOox + glucose --> GOR + 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.)

Dry coating of GO + Fc

e’s
Meter electrodes
Read glucose

Patient adds drop of blood,

then inserts slide into meter
Patient reads glucose level on meter
I (B.B. King http://www.bbking.com/)
Output:
Amperometry t
Constant E
 Research on new glucose
sensors
• Non-invasive biosensors - skin, saliva
• Implantable glucose sensors to
accompany artificial 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+, CO2, H2O2
• DNA hybridization and damage
• Micro or nanoarrays, optical abs or fluor.
 Negative
surface
Polycation soln.,
then wash
+ + + + + + + + + + + +
Layer by layer
Film construction: soln. of negative protein
then wash

Protein
layer + + + + + + + + + + + +

Polycation soln.,
then wash
+ + + + + + + + + + + +

+ + + + + + + + + + + +

Repeat steps for desired

number of layers

Polycation layers
Protein
layer

Figure 19
 Detection of hydrogen peroxide
Conductive polymers efficiently wire
peroxidase enzymes to graphite

Enzyme PSS layer

layer
SPAN layer
(sulfonated polyaniline)

e’s
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

Tapping mode atomic force microscopy (AFM)

image of HRP film
Electrochemical Response of Peroxidases

+e- O2
PFe III - PFe II PFe II-O2
-e +
2e-, 2H
H2O2
II
•PFeIV=O H2O2 + PFe
active oxidant

H2O2

PFeIII + H2O + O2

Possible reduced species in red

Catalytic reduction of H2O2 by peroxidase films
Catalytic cycles increase current

60
µM H 0
2 2
50
a 7.5 reduction
40 6
I,µA 4
30
with SPAN
2
20
0.5
10
0
0
FeIII/FeII
-10
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
with PAPSA
span
I, µA

0.5

without PAPSA
No span
0

0 100 200 300 400

t, s
 Rotating electrode amperometry at 0 V
1.2

0.8
Span/HRP
I, µA

PAPSA/HRP
PAPSA/Mb
0.6 Span/Mb
0.4
HRP
0.2 Mb

0
0 0.1 0.2 0.3 0.4 0.5 0.6
[H O ], µM
2 2

Sensitivity much higher with conductive polymer (SPAN);

Electrically wires all the protein to electrode
 Carbon Nanotubes

• Single walled (1.4 nm

o.d.) and multi-walled

• Highly conductive,
flexible, strong,
patternable

• Commercially Available
 Single-Walled Carbon Nanotube
Forests: Antigen-Antibody Sensing
~1.4 nm diameter, high conductivity

SPAN or
Nafion

Chattopadhyay, Galeska, Papadimitrakopoulos, J. Am. Chem. Soc. 2001, 123, 9451.

End COOH groups allow chemical attachment to proteins (antibodies)
High conductivity to conduct signal (e’s) from enzyme label to meas. circuit
AFM of SWNT forest with and without anti-HSA attached

SWNT forest with anti-human serum

SWNT forest on Si wafer
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

protein HRP HRP

HRP
HRP HRP

Ag Ag

Ab
2 Ab1
Ab1

HPR

H
R
P

SWNT forest Conductive polymer

(SPAN)
Apply E measure I
 Amperometry Detection of Human Serum
12
albumin
600 pmol/mL HSA

10
300 0.4 mM hydroquinone;
0.4 mM H O
I , µA 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
• 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 Specific 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
 Using HRP-Ab2-nanotube
Amperometric response at –0.3 V and 3000 rpm for SWNT immunosensors incubated with PSA
in 10 µL undiluted newborn calf serum for 1.25 hr using the Ab2-CNT-HRP bioconjugate

Mediator + H2O2

Washing with 2% BSA/0.05% Tween 20 to control non-specific binding

LOD - 4 pg/mL; 100-fold enhancement over HRP-Ab2
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.
 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

Good correlation with ELISA!

 Future - arrays to detect many biomolecules at once
SWNT forest grown on Prototype 8-electrode Array,
10 µm Au Array elements Univ. Edinburgh
 Biosensors
• Promising approach to medical diagnostics by
patients or in doctors offices
• 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