1. Amperometric Biosensors

2. Introduction Enzyme Catalyzed redox reactions The function of the enzyme is to generate or consume an electroactive species in a stoichiometric relationship with its substrate or target analyte Amperometric transducer allows for the electrometrical reactions to proceed at the electrode surface, thus giving rise to a current

3. Introduction - Amperometric Biosensor Schematic

4. Introduction S+C enzyme P + C’ S = Substrate C = Cofactor / Coreactant P = Product C’ = Cofactor Product

5. Introduction Three generations 1 st generation: the normal product of the reaction diffuses to the transducer and causes electrical response 2 nd generation: involves specific mediators between reaction and transducer to generate improved response

6. Introduction 3 rd generation: reaction itself causes the response

7. Amperometric Glucose Biosensor Developed by Updike and Hicks First reported use of enzyme electrode Glucose in blood-diagnosis of diabetes O 2 detection H 2 O 2 detection Enzyme mediation

8. Glucose Biosensor – O 2 Central – Pt cathode, surrounding Ag/AgCl anode, KCl electrolyte solution Teflon membrane Cathode (0.6V) O 2 + 4H + +4e - 2H 2 O Anode4Ag + 4Cl - 4AgCl + 4e - Glucose +O 2 glucoseoxidase Gluconic acid + H 2 O

9. Glucose Biosensor – O 2

10. Glucose Biosensor – H 2 O 2 Reverse polarity of electrodes for the O 2 set up Replace Teflon membrane with cellulose acetate membrane Cathode: 2Ag + 2Cl - 2AgCl + 2e - Pt Anode (0.6V): H 2 O 2 O 2 +2H + + 2e - Counter electrode: 4H + +O 2 2H 2 O – 4e -

11. Glucose Biosensor – H 2 O 2

12. Glucose Biosensor – redox mediators Replace O 2 with synthetic e- acceptor to shuttle e- from the flavin redox center of the enzyme to the electrode Measurements become insensitive to O 2 fluctuations, and can be carried out at lower potentials Enzyme mediators – dimethyl ferrocene, medola blue

13. Glucose Biosensor – Redox mediators Glucose + Gox(FAD)gluconic acid + Gox(FADH 2 ) Gox(FADH 2 )+2M ox Gox(FAD)+2M red +2H + 2Mred2M ox + 2e-

14. Common Enzyme electrodes Measured Species EnzymeDetected species LactateLactate dehydrgenase NADH LactateLactate oxidase H2O2H2O2 PhenolTyrosinaseQuinone CholesterolCholesterol oxidase O2O2

15. Some Commercial applications YSI : Glucose/Lactate Prostate specific antigen (PSA) NOVA Bioprofile analyzer (PO 2, Gluc, Gln, Glu, Lactate)

16. Other Applications Glucose: meat freshness, fermentation Rapid cell number monitor Monitor of herbicides in surface waters Amperometric and novel fluorescent DNA probes Rapid electrochemical sensor for Iron

17. References A.G. Elie,Principles of Potentiometric and Amperometric Biosensors,University of Virginia,(2002) P.V. Climent, M.L.M.Serralheiro, and M.J.F. Rebelo, Pure and Applied Chemistry 73, pp.1993-1999, 2001 http://www.lsbu.ac.uk.html