TOPICS IN (NANO) BIOTECHNOLOGY Immunosensors

1. PhD Course TOPICS IN (NANO) BIOTECHNOLOGY Immunosensors 30th June

2. Overview • Last week we looked at what is DNA and what is a gene. • We also looked at DNA replication and protein synthesis, and the path from the gene to protein • This week we will look at Recombinant DNA technology • We will also look at the amplification of DNA and finally at its sequencing

3. Immunosensors

4. What is an antibody?

5. How do we produce polyclonal and monoclonal antibodies? Polyclonal antibodies - larger quantities may be produced at a time - sometimes better selectivity and sensitivity due to recogintion of multiple epitopes - no guarantee of batch to batch reproducibility Monoclonal antibodies - long and expensive process - sometimes lower selectivity and sensitivity in comparison to Pabs observed - once cell line established constant reproducible supply of antibodies …. forever

6. substrate substrate substrate product product product Response Concentration Sandwich assay Useful for large molecules Robust assay - all reagents in excess Use with Pabs or different MAbs

7. substrate substrate product product Response Concentration Competition assay Useful for small molecules Reportedly less sensitive Concentrations of reagents critical

8. substrate substrate product product Response Concentration Displacement assay One step assay In practise difficulties to achieve - effect of non specific displacement Sub-optimum haptens met with some success

9. History of immunosensors • 1964 - Fluorescence polarisation labeled Ab and Ag • 1967 - First voltammetric immunosensor (Purdy et al) • 1976 - Use of FITC • 1972 - First PZ immunosensor (Shons et al) • 1975 - First potentiometric immunosensor (Janata) • 1976 - First report of EIA (Rubenstein et al) • 1976 - First amperometric immunosensor (Aizawa) • 1980 - First fluorescence immunoassay

10. Electrochemical transduction

11. Duan & Meyerhoff, 1994 • Gold coated microporous nylon membranes, serving as solid phase and working electrode • Ab immobilised via SAM of thioctic acid on gold side of membrane • Separation free sandwich assay - surface bound spatially resolved from excess conjugate in bulk • Substrate introduced through back side of porous membrane • Substrate diffuses rapidly through membrane first encountering ALP-Ab • Enzymatically generated product detected immediately via oxidation at gold electrode • Assay time of 30 minutes, measurement of 1 minute

12. Skladal et al, 1995

13. Bauer et al, 1996 • FIA system using bienzyme recycling for detection of 2,4-D • Clark-type electrode covered by membrane with PPO and PQQ-GDH • 350-fold amplification observed • 60 minute incubation with PP and zeptomole detection

14. Lu et al, 1997 • Electrically wired amperometric immunosensor • Demonstrated for detection of biotin • Redox polymer and antibody co-immobilised and competitive assay for biotin • Only surface bound biotin-HRP ‘wired’ • L.O.D. One order of magnitude better than ELISA

15. Rishpon & Ivnitski, 1997 • Separation free enzyme channelling immunosensor • Graphite pencil, Eapp = 0.0V • Poly(ethylene)imine film to discriminate surface bound and bulk HRP • Formats with I2, aminosalicylic acid • 10-30 minute assay

16. Keay & McNeil, 1998 • Separation free immunosensor based on enzyme channelling • Ab immobilised on Biodyne C membrane on SPEs • 15 minute assay time, L.O.D. 0.012mg/L (12 p.p.t)

17. Bi3+ Bi3+ Bi3+ Bi3+ Bi3+ 3é 3é Bi Bi Bi3+ Stripping Deposition Wang, Tian & Rogers, 1998 Potentiometric stripping analysis HSA used as model analyte Bismuth metal ion label 30 minute incubation HCl and Hg+ added to release metal label 10 minute deposition

18. Bäumner & Schmid, 1998 Ascorbic acid released SIGNAL • Pioneering work patented by Durst (1996) • Hapten tagged liposomes containing ascorbic acid • Competition, - unbound labeled hapten passes detergent loaded membrane - releases ascorbic acid for electrodetection

19. SmartSenseTM Ohmicron Co SmartSense Atrazine at p.p.b. Levels 15 minute assay time

20. Dequaire et al, 1999 • Sample and 2.4-D-ALP added to microwell-electrode format - 40 minute incubation • Microwell-electrode format supported on magnet holding block • Beads magnetically separated for 3 minutes and excess liquid removed • Phosphoric acid ester of [[(4-hydroxyphenyl)amino]-carbonyl]cobaltecium hexafluorophosphate used as substrate • Cationic phenol accumulated in Nafion film for 30 minutes • L.O.D. of 10ng/L (p.p.t) of 2.4-D

21. Campbell et al, 1999 • Ingenious assay - separation and reagentless immunosensor • Choline oxidase does not interact with wire - produces H2O2 to act as substrate for HRP • Washing not required as only surface bound HRP will be wired to electrode surface • ChOX and avidin immobilised on redox hydrogel followed by biotinylated specific antibody • An 18 minute assay - demonstrated with IgG

22. Kim et al, 2000 Attempted electrochemical detection of traditional immunochromatographic strips by measuring change in conductance upon aggregation of colloidal gold labels Direct detection - low sensitivity Used gold colloids coated with polyaniline Large improvement in sensitivity Demonstrated with HSA 6 minute assay time

23. Anti-analyte antibody Redox labeled analyte Analyte Size exclusion layer MWCO 20,000 Benkert et al, 2000 • SERI - size exclusion redox-labeled immunoassay • Analyte competes with redox-labeled analyte for antibody binding • Unbound redox-labeled passes therough the size exclusion layer and is indicated electrochemically • Demonstrated with creatinine - low L.O.D.

24. Deposition of IgG Consecutive assembly of PAH-FITC and PSS PS Microparticle Dye-labeled PS microparticle IgG conjugated dye-labeled PS microparticle Yang et al, 2001 Layer by layer (LbL) approach Applied to enyme and immunosensors Platform for fluorsecent immunosensors

25. Katz et al, 2001 • Sensing antibody using antigen monolayer electrode and anti idiotypic-HRP • Biocatalytic precipitation of insoluble product - forms an insulating layer on electrode surface, decreases interfacial electron transfer rate constant • Chronopotentiometry - measurement time of seconds, Faradaic impedance spectroscopy - 15 to 20 minutes

26. Competitive assay - immobilised antigen, labeled antibody • Originally used ALP label and p-APP substrate - 22 minute assay time, mainly due to substrate development • Using Os-amine mediator and HRP label assay time of 10 minutes Carbon working electrode Quasi counter – reference Ag/AgCl electrode Insulation Layer Area for sample application O’ Sullivan & Katakis, 2001

27. Market drivers • The market drivers for the biosensors market, in order of impact, are: • high demand, • expanding application areas • high levels of research & development • advancing technologies reducing production costs • increased customer awareness • legislation • integrating partnerships between academia and industry • innovative new product developments • strong economy

28. Biosensor Market Segments (Frost & Sullivan, 1998) Market predictions - 2001 to 2004 • Medical applications will continue to dominate • Overall best growth rate of 6.8% predicted for • environmental biosensors as applications will be • realised for site characterisation and clean-up • Growth attributed to development ofimmunosensors