ELECTROCHEMICAL 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

electrode
Apply voltage

Enzyme (label)

Measure current prop. to concentration of substrate

Figure 9

Equipment for developing electrochemical biosensors

potentiostat
insulator electrode material

reference N2 inlet Protein film counter

working electrode
E-t waveform

E, V

Cyclic 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.
Reversible Peaks for Direct electron Transfer
I , A
2 1.5 1 0.5 0 -0.5 -1 -1.5

Reduction Of FeIII

Oxidation Of FeII
0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8

(not all proteins do this)

-2 0.8

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

A. Cass, G. Dav is, 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) GO(FADH2) + 2 Fc+ GO(FAD) + 2 Fc + 2 H+ Fc Fc+ + 2 e- (at electrode) (5) (1) (4)

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 how much insulin to administer; or what other action to take

 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.

Glucose biosensor test strips (~$0.50-1.00 ea.)

Dry coating of GO + Fc es
Meter Read glucose

electrodes

Output: I Amperometry Constant E

Patient adds drop of blood, then inserts slide into meter Patient reads glucose level on meter (B.B. King http://www.bbking.com/) t

Research on glucose sensors

Non-invasive biosensors - skin, saliva Implantable glucose sensors to accompany artificial pancreas - feedback control of insulin supply Typical use 3-4 weeks for implantable sensor in humans Failure involved fouling and inflammation

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 fluorescence

Negative surface

Layer by layer Film construction:

Protein layer

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

soln. of negative protein then wash + + + + + + + + + + + +

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

Repeat steps for desired number of layers

Protein layer

Polycation layers

Figure 19

Detection of hydrogen peroxide Conductive polymers efficiently wire

peroxidase enzymes to graphite

Enzyme layer

PSS 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.

Horseradish Peroxidase (HRP)

100nm

50nm

Tapping mode atomic force microscopy (AFM) image of HRP film

Electrochemical Response of Peroxidases

PFe III
+e-e
-

O2 PFe II PFe II-O 2

2e-, 2H+

H2O2
PFeIV=O active oxidant

H2 O2 + PFeII

H2O2
PFeIII + H2O + O2

Possible reduced species in red

Catalytic reduction of H2O2 by peroxidase films Catalytic cycles increase current

60 50 M H 0 2 2 7.5 6 4 with SPAN 2 0.5 0

reduction

I, A

40 30 20 10 0 -10 0.2

FeIII/FeII
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
I, A
with PAPSA

span

0.5
without PAPSA

No span

0 0 100 200 300 400

t, s

Rotating electrode amperometry at 0 V

1.2 1 0.8

I, A

Span/HRP PAPSA/HRP
0.6 0.4

PAPSA/M b

Span/Mb

HRP

0.2 0 0 0.1 0.2

2

Mb

0.3
2

0.4

0.5

0.6

[H O ], M

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 (es) from enzyme label to meas. circuit

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

protein
Ag

HRP

HRP

HRP

HRP

HRP

Ag

Ab2
Ab1

Ab1

HPR H R P

SWNT forest

Conductive polymer (SPAN)

Apply E

measure I

Amperometry Detection of Human Serum albumin 12

600 pmol/mL HSA

10
300

I, A

8 6 4
15 45

140

0.4 mM hydroquinone; 0.4 mM H O

2 2

controls c
140

2 0

d
300

7.5 1.5

a
140

b
0

no SWNT

bare PG

-2 0 500 1000 1500

SWNTs provide 10-20 fold signal enhancement; high surf. area Nanotubes aged in DMF fewer defects denser forests . Xin Yu, Sang Nyon Kim, Fotios Papadimitrakopoulos and James F. Rusling,
,

t, s

"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 L undiluted newborn calf serum for 1.25 hr using the Ab 2-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, V mesh Patel, Ga ry Jensen, Ashwin Bhirde, Joseph D. G yo ong , SangNyon Kim, John Gillespie, J. Sil vio Gutkind, Fotios Papadimit rakopou los and James F. Rusling, "Carbon Nano tube Amplification Strategies for Highly Sensitive Imm unosensing of Canc er 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!

PSA in prostate tissue samples indistinguishable by the best staining methods

cell lysates obtained after laser microdissection Using multiply labeled Ab2-nanotube-HRP 4 pg/mL DL (100 x better)

Future - arrays to detect many biomolecules at once Prototype 8-electrode Array, 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