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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. Doctors increasingly rely on testing Needs: rapid, cheap, and “low tech”

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ELECTROCHEMICAL BIOSENSORS

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  1. ELECTROCHEMICAL BIOSENSORS Modern and future approaches to medical diagnostics James F. Rusling Dept. of Chemistry Dept. of Cell Biology, 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; or what other action to take

  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 + --> GOox + 2 Fc GOox + glucose --> 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 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

  14. 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

  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 fluorescence

  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) 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.

  18. Horseradish Peroxidase (HRP) 100nm 50nm Tapping mode atomic force microscopy (AFM) image of HRP film

  19. Catalytic reduction of H2O2 by peroxidase films Catalytic cycles increase current reduction FeIII/FeII

  20. Rotating electrode amperometry at 0 V HRP, 50 nmol H2O2 additions reduction span No span

  21. Rotating electrode amperometry at 0 V Span/HRP Span/Mb Sensitivity much higher with conductive polymer (SPAN); Electrically wires all the protein to electrode

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

  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 (e’s) 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 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. 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

  26. Amperometry Detection of Human Serum albumin • SWNTs provide 10-20 fold signal enhancement; high surf. area • 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. 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)

  32. Future - arrays to detect many biomolecules at once Prototype 8-electrode Array, Univ. Edinburgh

  33. 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

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