bio sensors 3

8/6/2019 Bio Sensors 3

1/32

ELECTROCHEMICALBIOSENSORS

Modern and future approaches to

medical diagnosticsJames F. Rusling

Dept. of Chemistry, Univ CT, Storrs,

Dept. of Cell Biology, Univ. of CT Health

CenterF. A. Armstrong, H. A. Heering, and J. Hirst, Reactions of complex metalloproteins studies byprotein 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.


2/32

Medical Diagnostics

Doctors increasingly rely on testing

Needs: rapid, cheap, and low tech

Done by technicians or patients

Some needs for in-vivo operation, withfeedback


3/32

electrode

substrate product

Figure 9

Enzyme

(label)

Apply voltage Measure current prop.

to concentration of substrate

Principle of Electrochemical Biosensors


4/32

E, V

time

E-t waveform

potentiostat

Electrochemical cell

counter

working electrode

N2inlet

Protein film

reference

insulator electrode

material

Equipment for developing electrochemical biosensors

Cyclic

voltammetry


5/32

Electrode

enzyme

A lipid-enzyme film


6/32

-2

-1.5

-1

-0.5

0

0.5

1

1.5

2

-0.8-0.6-0.4-0.200.20.40.60.8

I,A

E, V vs SCE

Cyclic voltammogram (CV) at 100 mV s-1 and 25 oC ofMycobacterium Tuberculosis KatG

catalase-peroxidase in a thin film ofdimyristoylphosphatidylcholine on basal plane PG electrode,in anaerobic pH 6.0 buffer.

Oxidation

Of FeII

Reduction

Of FeIII

Reversible

Peaks for

Direct electron

Transfer

(not all proteins

do this)


7/32

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 shuttlesElectrons betweenEnzyme and electrode


8/32

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)

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


9/32

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


10/32

Patient Diabetes Management

Insulin secretion by pancreas regulatedby 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)


11/32

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.


12/32

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/)


13/32

Research on new glucose

sensors

Non-invasive biosensors - skin, saliva

Implantable glucose sensors toaccompany artificial pancreas - feedbackcontrol of insulin supply

Record is ~4 weeks for implantable

sensor in humans


14/32

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.


15/32

Negative

surfacePolycation soln.,

then wash

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

soln. of negative protein

then wash

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

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

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

Repeat steps for desired

number of layers

Protein

layer

Polycation layersProtein

layer

Polycation soln.,then wash

Figure 19

Layer by layerFilm construction:


16/32

PSS layer

SPAN layer

Enzyme

layer

Detection of hydrogen peroxide

Conductive polymers efficiently wire

peroxidase enzymes to graphite

Xin Yu, G. A. Sotzing,F. Papadimitrakopoulos, J. F.Rusling, Highly Efficient Wiring of Enzymes to Electrodesby Ultrathin Conductive Polyion Underlayers: EnhancedCatalytic Response to Hydrogen Peroxide,Anal. Chem.,

2003, 75, 4565-4571.

es

(sulfonated polyaniline)


17/32

Horseradish Peroxidase (HRP)

100nm

50nm

Tapping mode atomic force microscopy (AFM)

image of HRP film


18/32

O2

H2O2

PFeIII PFeII PFeII-O2

2e-, 2H

+

H2O2+PFeII

PFeIV=O

active oxidant

+e-

-e-

PFeIII +H2O+ O2

H2O2

Possible reduced species in red

Electrochemical Response of Peroxidases


19/32

-10

0

10

20

30

40

50

60

-0.8-0.6-0.4-0.200.2

I,A

E, V vs SCE

with SPAN

a

0

0.5

2

4

6

7.5

M H20

2

Catalytic reduction of H2O2 by peroxidase filmsCatalytic cycles increase current

FeIII/FeII

reduction


20/32

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 H2O2 additions

span

No span

reduction


21/32

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

[H2

O2 ], M

PAPSA/HRPPAPSA/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


22/32

Single walled (1.4 nmo.d.) and multi-walled

Highly conductive,flexible, strong,patternable

Commercially Available

Carbon Nanotubes


23/32

Single-Walled Carbon NanotubeForests: Antigen-Antibody Sensing

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

~1.4 nm diameter, high conductivity


24/32

AFM of SWNT forest with and without anti-HSA attached

SWNT forest on Si waferSWNT 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.


25/32

Sandwich Electrochemical

Immunosensor ProteinsH2O2

Ab1

Ag

HRPHRP

HRP HRPHRP

Ab1

Ag

H

R

P

HPR

Ab2

Apply E measure I

SWNT forest Conductive polymer(SPAN)

protein


26/32

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

140300

140

controls

a

0

bc d

no SWNT bare PG

0.4 mM hydroquinone;

0.4 mM H2O

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.


27/32

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


28/32

Nanotube Strategies for PSA detection

~170 labelsper PSA


29/32

Washing with 2% BSA/0.05% Tween 20 to control non-specific bindingLOD - 4 pg/mL; 100-fold enhancement over HRP-Ab2

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 Ab2-CNT-HRPbioconjugate

Mediator + H2O

2

Using HRP-Ab2-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.


30/32

Amperometric current at 0.3 V and 3000 rpm for human serum samples and PSA standards incalf serum

Accurate results obtained for cancer patient serum

Good correlation with ELISA!

Using conventional HRP-Ab2


31/32

Future - arrays to detect many biomolecules at once

SWNT forest grown on

10 m Au Array elementsPrototype 8-electrode Array,

Univ. Edinburgh


32/32

Biosensors Promising approach to medical diagnostics by

patients or in doctors offices

Other important applications: cancerbiomarkers, DNA, peroxide, etc.

Method of choice for blood glucose in diabetics

Rapid diagnostics may lead to more timely and

effective treatment

bio sensors 3

Documents