1. BIOSENSORS
Modern and future approaches to medical
diagnostics
H.E.Braustein
“G.Wise” Life Science Institute
Dept. of Molecular Microbiology and Biotechnology
Tel Aviv University
2. 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
3. Principle of Electrochemical Biosensors
substrate product
electrode
Enzyme
Apply voltage Measure current prop.
to concentration of substrate
4. Equipment for developing electrochemical biosensors
E, V
E-t waveform
time
potentiostat
counter
Electrochemical cell
working electrode
N2
inlet
Protein film
reference
insulator electrode
material
Cyclic
voltammetry
5. C y c lic v o lt a m m o g ra m (C V ) a t 1 0 0 m V s-1 a n d 2 5 o C o f Mycobacterium Tuberculosis KatG
c a ta la se -p e ro x id a se in a th in film o f dimyristoylphosphatidylcholine o n b a s a l p la n e P G e le c tro d e ,
in a n a e ro b ic p H 6 .0 b u ffe r.
2
1.5
1
0.5
0
-0.5
-1
-1.5
-2
Reduction
Of FeIII
0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8
I , m A
E, V vs SCE
Oxidation
Of FeII
Reversible
Peaks for
Direct
electron
transfer
7. Catalytic enzyme electrochemistry
a basis for biosensor - glucose oxidase
oxidation
Fc + glucose
+ enzyme
Fc mediator
I = f [glucose]
Mediator shuttles
Electrons between
Enzyme and electrode
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).
8. Mechanism for catalytic oxidation of glucose
With Glucose oxidase (GO) and Fc mediator
Scheme 2
Glucose + GO(FAD) + 2 H+ Ÿ gluconolactone + GO(FADH2) (1)
GO(FADH2) + 2 Fc+ Ÿ GO(FAD) + 2 Fc + 2 H+ (4)
Fc Ÿ Fc+ + 2 e- (at electrode) (5)
Signal can also be measured by amperometry:
Hold const. E where oxidation occurs, measure I vs
time
Fc = ferrocenecarboxylate
9. 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
10. 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 how much
insulin to administer
11. • Most sensors use enzyme called glucose oxidase (GO)
• Most sensors are constructed on electrodes, and use a
mediator to carry electrons from enzyme to GO
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.
12. Glucose biosensor test strips (~$0.40-0.80 ea.)
Meter
Read glucose
Dry coating of GO + Fc
Patient adds drop of blood,
then inserts slide into meter
Output:
amperometry
I
t
Patient reads glucose level on meter
e’s
electrodes
13. Research on glucose sensors
• Non-invasive biosensors - skin, saliva
• Implantable glucose sensors to
accompany artificial pancreas - feedback
control of insulin supply
• Record is 3-4 weeks for implantable
sensor in humans
14. Other biosensors
• Cholesterol - based on cholesterol oxidase
• Antigen-antibody sensors - toxic
substances, pathogenic bacteria
• Small molecules and ions in living things:
H+, K+, Na+, CO2, H2O2
• DNA hybridization and damage
• Micro or nanoarrays, optical abs or
fluor.
15. Negative
surface
Polycation soln.,
then wash
+ + + + + + + + + + + +
soln. of negative protein
then wash
+ + + + + + + + + + + +
Polycation soln.,
then wash
+ + + + + + + + + + + +
+ + + + + + + + + + + +
Repeat steps for desired
number of layers
Protein
layer
Protein Polycation layers
layer
Figure 19
Layer by layer
Film construction:
16. Detection of hydrogen peroxide
Conductive polymers efficiently wire
peroxidase enzymes to graphite
PSS layer
SPAN layer
Enzyme
layer
e’s
(sulfonated polyaniline)
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.
18. Electrochemical Response of Peroxidases
O2
+ e-
- e-
PFeIII PFeII PFeII-O2
H2O2
2 e-, 2 H+
H2O2 + PF eII •PFeI V= O
active oxidant
PF eIII + H2O +O2
H2O2
Possible reduced species in red
19. Catalytic reduction of H2O2 by peroxidase films
Catalytic cycles increase current
60
50
40
30
20
10
0
-10
with SPAN
mM H
7.5
4
0.5
reduction
0.2 0 -0.2 -0.4 -0.6 -0.8
I,mA
E, V vs SCE
a
0
2
6
2
0
2
FeIII/FeII
20. Rotating electrode amperometry at 0 V
1
0.5
0
HRP, 50 nmol H2O2 additions
span
0 100 200 300 400
I, mA
t, s
with PAPSA
without PAPSA
No span
reduction
21. Rotating electrode amperometry at 0 V
1.2
1
0.8
0.6
0.4
0.2
0
Span/HRP
Span/Mb
0 0.1 0.2 0.3 0.4 0.5 0.6
I, mA
[H2O2 ], mM
PAPSA/HRP
PAPSA/Mb
Mb
HRP
Sensitivity much higher with conductive polymer (SPAN);
Electrically wires all the protein to electrode
22. Biosensors
• Promising approach to medical diagnostics by
patients or in doctors offices
• Other important applications: pathogens,
disease biomarkers, DNA, peroxide, etc.
• Method of choice for blood glucose in diabetics
• Rapid diagnostics may lead to more timely and
effective treatment
