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

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

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

8. Displacement assay substrate product substrate product Concentration Response 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, E app = 0.0V Poly(ethylene)imine film to discriminate surface bound and bulk HRP Formats with I 2, 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. Wang, Tian & Rogers, 1998 Bi3+ Bi Bi3+ 3é StrippingDeposition 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 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 Ascorbic acid released SIGNAL

19. SmartSense TM 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 H 2 O 2 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. Benkert et al, 2000 Anti-analyte antibody Redox labeled analyte Analyte Size exclusion layer MWCO 20,000 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. Yang et al, 2001 Layer by layer (LbL) approach Applied to enyme and immunosensors Platform for fluorsecent immunosensors Deposition of IgG PS MicroparticleDye-labeled PS microparticleIgG conjugated dye-labeled PS microparticle Consecutive assembly of PAH- FITC and PSS

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. O’ Sullivan & Katakis, 2001 Quasi counter – reference Ag/AgCl electrode Carbon working electrode Insulation Layer Area for sample application 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

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. Market predictions - 2001 to 2004 Biosensor Market Segments (Frost & Sullivan, 1998) 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 of immunosensors