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ELECTROCHEMICAL BIOSENSORS

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ELECTROCHEMICAL BIOSENSORS Modern and future approaches to medical diagnostics J. F. Rusling Dept. of Chemistry Dept. of Pharmacology, Univ. of CT Health Center – PowerPoint PPT presentation

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Title: ELECTROCHEMICAL BIOSENSORS


1
ELECTROCHEMICAL BIOSENSORS
  • Modern and future approaches to medical
    diagnostics
  • J. F. Rusling
  • Dept. of Chemistry
  • Dept. of Pharmacology, Univ. of CT Health Center

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
Enzyme (label)
electrode
Measure current prop. to concentration of
substrate
Apply voltage
Figure 9
4
Equipment for developing electrochemical
biosensors
potentiostat
electrode material
insulator
reference
Protein film
N2 inlet
counter
working electrode
E-t waveform
Cyclic voltammetry
E, V
Electrochemical cell
time
Figure1
5
A lipid-enzyme film
enzyme
Electrode
6
Reversible Peaks for Direct electron Transfer
(not all proteins do this)
Reduction Of FeIII
Oxidation Of FeII
7
A lipid-enzyme film
enzyme
Electrode
8
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
9
Mechanism for catalytic oxidation of glucose With
Glucose oxidase (GO) and Fc mediator
Fc ferrocenecarboxylate
Signal can also be measured by amperometry Hold
const. E where oxidation occurs, measure I vs time
10
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

11
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

12
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 --gt GOox 2 Fc
GOox glucose --gt GOR gluconolactone
Reach and Wilson, Anal. Chem. 64, 381A (1992) G.
Ramsay, Commercial Biosensors, J. Wiley, 1998.
13
Glucose biosensor test strips (0.50-1.00 ea.)
Dry coating of GO Fc
es
Meter Read glucose
electrodes
Patient adds drop of blood, then inserts slide
into meter
Patient reads glucose level on meter (B.B. King
Output Amperometry Constant E
I
http//www.bbking.com/)
t
14
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

15
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.

16
Layer by layer Film construction
17
Detection of hydrogen peroxide Conductive
polymers efficiently wire peroxidase enzymes to
graphite
PSS layer
Enzyme
layer
SPAN layer
(sulfonated polyaniline)
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.
18
Horseradish Peroxidase (HRP)
100nm
50nm
Tapping mode atomic force microscopy (AFM) image
of HRP film
19
(No Transcript)
20
Catalytic reduction of H2O2 by peroxidase
films Catalytic cycles increase current
reduction
FeIII/FeII
21
Rotating electrode amperometry at 0 V
HRP, 50 nmol H2O2 additions
reduction
span
No span
22
Rotating electrode amperometry at 0 V
Span/HRP
Span/Mb
Sensitivity much higher with conductive polymer
(SPAN) Electrically wires all the protein to
electrode
23
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 (es) from enzyme label to meas.
circuit
24
AFM of SWNT forest with and without anti-HSA
attached
SWNT forest with anti-human serum albumin (HSA)
attached by amide links
SWNT forest on Si wafer
Also linked enzymes to SWNT forestsX. 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.
25
Sandwich Electrochemical Immunosensor Proteins
protein
Ab2
HPR
Conductive polymer (SPAN)
SWNT forest
Apply E measure I
26
Amperometry Detection of Human Serum albumin
SWNTs provide 10-20 fold signal enhancement
Nanotubes aged in DMF ? fewer defects ? denser
forests
27
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
28
Nanotube Strategies for PSA detection
170 labels per PSA
29
Using HRP-Ab2-nanotube
Washing with 2 BSA/0.05 Tween 20 to control
non-specific binding LOD - 4 pg/mL 100-fold
enhancement over HRP-Ab2
30
Accurate results obtained for cancer patient serum
Using conventional HRP-Ab2
Good correlation with ELISA!
31
Future - arrays to detect many biomolecules at
once
SWNT forest grown on 10 mm Au Array elements
Prototype 8-electrode Array, Univ. Edinburgh
32
Biosensors
  • Promising approach to medical diagnostics by
    patients or in doctors offices
  • Other important applications pH, CO2, cancer
    biomarkers, DNA, peroxide, etc.
  • Method of choice for blood glucose in diabetics
  • Rapid diagnostics may lead to more timely and
    effective treatment
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