Novel antibody-based biosensors for the detection of haptens, proteins and whole cells
Tully, Elizabeth (2007) Novel antibody-based biosensors for the detection of haptens, proteins and whole cells. PhD thesis, Dublin City University.
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The modification of basic sensor platforms, incorporating specially designed materials, enhances the capacity for the development of new and improved biosensors. Novel transduction strategies employ surface modification technologies as a key element in the detection of biologically important molecules. These modifications can be chemical, electrochemical or physical. Surface modification of polymer substrates for blochip applications, thin-film deposition of biocompatible surfaces for implants and electropolymerisation of electrodes for immunoassay development are the basis of much of the research described in this thesis. A number of immunosensing methods were investigated for the generahon of improved biosensors for the detection of small haptens, proteins and whole cells. These Included fluorescence, chemiluminescence and impedance-based detection systems. Microtitre plates, polymer chps, fluorescence-enhancement chips and screen-printed electrodes were all examined. The specific targets chosen were Internalin B@dB), an mnvasion-associated proteln of Listerla monocytogenes, parvovirus B19, the first human parvovirus and warfarin, the ninth most prescribed drug in the world.
A panel of antibodies and antibody fragments directed against InlB (previously produced) was used for the development of fluorescence and impedance-based assays. A recombinant form of the InlB protein was cloned, expressed ~n E.coli and purified by immobilised metal affinity chromatography (IMAC). An epitope mapping study of InlB was also performed, whereby the recombinant protein was portioned into three fragments to estabhsh the relative location of antibody-bindmg epitopes on its structure. The Listeria monocytogenes-derived proteins, peptides and anhbodies were characterised using plate-based methods and subsequently used for the development of immunoassays using novel fluorescent labels and an impedimettic sensor. The recomb~nant InlB-derived peptides were also cloned Into the PAC 4 vector for in vrvo biotinylation, expressed in E. coli and purif~ed using a monomeric streptavidin affinity column. The m vrvo hiotinylated fragments were characterised using SDS-PAGE, Western blotting, lmmunoassay and BiacoreTM (Chapter 3).
Quantum dots and phosphorescent porphyrins were used as labels in the generation of solidphase immunoassays. Fluorescence-based munoassays using functionalised dye-doped nanoparticles were also developed. These doped particles were conjugated to specific antibodies and proteins using various surface mohfication techniques. The optimisation of solid-phase fluorescence-based immunoassays, incorporating labelled antibody-nanoparticle conjugates, for the detection of hIgG was investigated. A number of plate-based assay formats for the detection of parvovirus B19 was also investigated, in conjunction with an industrial partner; Biotnn International Ltd. Enzyme, chemiluminescence and fluorescencebased detection strategies were employed. A series of immobilisation studies was performed to study biomolecule attachment onto cone platforms for detection. (Chapter 4)
Platform technologies using carbon screen-printed electrodes and ultra-thin polymer coatings for antibody-based biosensors were also examined. Antibodies directed against InlB were depos~tedo nto electrodes within conducting polyaniline (PANI) films to produce conductive affl~nty matrices with clearly defined binding characteristics. The binding of specific anhbodies to their target molecules was monitored via electrical impedance spectroscopy (EIS) , as the films function as labelfree reversible immuno-biosensors, when interrogated with a pulsed potential waveform (Chapter 5).
Finally, the thin-film deposition of biocompatible surfaces for implants was also studied. The adhesion of thin-films to 316L stainless steel substrates was investigated. These films were prepared by plasma-enhanced chemical vapour deposition (PECVD) of hexamethyldisiloxane W S O ) and oxygen mixtures. The film properties were found to be dependent on the Oxygen/HMDSO flow ratio and RF (radio frequency) power. Biocompatibility studies were camed out using rat aortic smooth muscle (RASM) cells. Cell proliferation, viability and toxicity were assessed using commercial kits and scanning electron microscopy (SEM) was performed on substrates post incubation in culture to monitor the biocompatibility effects of the films (Appendix).
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