electrochemical biosensors l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
ELECTROCHEMICAL BIOSENSORS PowerPoint Presentation
Download Presentation
ELECTROCHEMICAL BIOSENSORS

Loading in 2 Seconds...

play fullscreen
1 / 34

ELECTROCHEMICAL BIOSENSORS - PowerPoint PPT Presentation


  • 420 Views
  • Uploaded on

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”

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

ELECTROCHEMICAL BIOSENSORS


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
electrochemical biosensors

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
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
slide3

Principle of Electrochemical Biosensors

substrate

product

Enzyme

(label)

electrode

Measure current prop.

to concentration of substrate

Apply voltage

Figure 9

slide4

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

slide5

A lipid-enzyme film

enzyme

Electrode

slide6

Reversible

Peaks for

Direct electron

Transfer

(not all proteins do this)

Reduction

Of FeIII

Oxidation

Of FeII

slide7

A lipid-enzyme film

enzyme

Electrode

slide8

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

slide9

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

slide10

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
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
slide12

• 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.

slide13

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

Dry coating of GO + Fc

e’s

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

research on glucose sensors
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
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
slide16

Layer by layer

Film construction:

slide17

Detection of hydrogen peroxide

Conductive polymers efficiently wire

peroxidase enzymes to graphite

PSS layer

Enzyme

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.

slide18

Horseradish Peroxidase (HRP)

100nm

50nm

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

slide20

Catalytic reduction of H2O2 by peroxidase films

Catalytic cycles increase current

reduction

FeIII/FeII

slide21

Rotating electrode amperometry at 0 V

HRP, 50 nmol H2O2 additions

reduction

span

No span

slide22

Rotating electrode amperometry at 0 V

Span/HRP

Span/Mb

Sensitivity much higher with conductive polymer (SPAN);

Electrically wires all the protein to electrode

slide23

Carbon Nanotubes

  • Single walled (1.4 nm o.d.) and multi-walled
  • Highly conductive, flexible, strong, patternable
  • Commercially Available
slide24

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

slide25

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 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.

slide26

H2O2

HRP

HRP

HRP

HRP

HRP

HRP

Ag

Ag

Ab1

Ab1

Sandwich Electrochemical Immunosensor Proteins

protein

Ab2

HPR

Conductive polymer

(SPAN)

SWNT forest

Apply E measure I

slide27

Amperometry Detection of Human Serum albumin

• SWNTs provide 10-20 fold signal enhancement; high surf. area

• Nanotubes aged in DMF  fewer defects  denser forests

slide28

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

slide30

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

slide31

Accurate results obtained for cancer patient serum

Using conventional HRP-Ab2

Good correlation with ELISA!

slide32

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)

slide33

Future - arrays to detect many biomolecules at once

Prototype 8-electrode Array, Univ. Edinburgh

biosensors
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