The application of antibodies in optical and electrochemical transduction processes

Chapter 1 relates the background information of the structure of antibodies, the nature of their interaction with antigen and their production as polyclonal antibodies. Examples of antibodies which have been produced against luminescent molecules are given and it is illustrated how the specific interaction of antibodies with luminescent complexes can be used to gain information on the structure, function and rotational dynamics of antibodies. The principle features of immunoassays are outlined with a focus on amperometric immunosensing including some areas of active research within amperometric immunosensing. This serves as a general introduction to Chapter 4; the development of an amperometric immunosensor based on single wall carbon nanotubes. Chapter 2 begins with a review of the fundamental chemical, photochemical and electrochemical properties of Ru(II) and Os(II) polypyridyl complexes. It is shown how their photophysical properties can be modulated by the interaction of the complexes with biomolecules such as proteins, nucleic acids and antibodies. The synthesis and characterisation of [Os(bpy)2dcbpy] and some related Os(II) and Ru(II) complexes is described. The production and characterisation of a [Os(bpy)2dcbpy]-thyroglobulin conjugate which was used as the immunogen is described, as well as the purification and characterisation of the resulting polyclonal antibody. Competition ELISA served to confirm the cross-reactivity of the antibody with the Os and Ru complexes synthesised. Chapter 3 describes the effect of antibody binding on the spectrochemical properties of the complexes. Changes in the emission spectra and lifetimes were examined. Association constants were derived from emission titrations. The extent that the antibody binding site protects the complexes from excited state deactivation via interaction with solvent was investigated. The possibility of energy transfer from [Ru(bpy)2dcbpy] to [Os(bpy)2dcbpy] when both were bound to the same antibody was investigated, as were the effects of antibody binding to a self-assembled layer of [Os(bpy)2(p2 p)2]2+. Chapter 4 begins with an introduction to the structure and properties of CNTs and outlines their application thus far in biosensing. The assembly of oxidatively shortened SWNTs onto Nafion/iron oxide coated pyrolytic graphite electrodes is described and characterised by both AFM and resonance Raman spectroscopy. The immunosensing strategy investigated involved the adsorption of anti-biotin antibody to the carbon nanotube surface. The presence of HRP-labelled biotin was determined via the reduction of hydrogen peroxide in the presence of the soluble mediator hydroquinone. A short investigation is also presented on the ability of HRP-modified SWNT forest electrodes to detect H2O2 produced by a mutant catalase negative E. coli bacteria which was co-immobilised with the HRP. Recommendations for future work arising from this thesis are given in Chapter 5

Mediated amperometric immunosensing using single walled carbon nanotube forests

A prototype amperometric immunosensor was evaluated based on the adsorption of antibodies onto perpendicularly oriented assemblies of single wall carbon nanotubes called SWNT forests. The forests were self-assembled from oxidatively shortened SWNTs onto Nafion/iron oxide coated pyrolytic graphite electrodes. The nanotube forests were characterized using atomic force microscopy and resonance Raman spectroscopy. Anti-biotin antibody strongly adsorbed to the SWNT forests. In the presence of a soluble mediator, the detection limit for horseradish peroxidase (HRP) labeled biotin was 2.5 pmol ml[-1] (2.5 nM). Unlabelled biotin was detected in a competitive approach with a detection limit of 16 nmol ml[-1] (16 μM) and a relative standard deviation of 12%. The immunosensor showed low non-specific adsorption of biotin-HRP (approx. 0.1%) when blocked with bovine serum albumin. This immunosensing approach using high surface area, patternable, conductive SWNT assemblies may eventually prove useful for nano-biosensing arrays

Production and characterization of a polyclonal antibody fornext term Os(II) and Ru(II) polypyridyl complexes

The characterization of a polyclonal antibody produced via immunization with an [Os(bpy)2dcbpy] hapten is described. Bpy is 2,2′-bipyridine and dcbpy is 2,2′-bipyridine-4,4′-dicarboxylic acid. The cross-reactivity of the antibody for the Ru(II) analogue of the hapten was also investigated. Large increases in the emission and luminescent lifetime of a series of Os and Ru complexes were observed on binding of the antibody. Association equilibrium constants were derived from luminescence titration data and were found to be 5.6 × 108 and 5.0 × 108 M−1 for [Os(bpy)2dcbpy] and [Ru(bpy)2dcbpy], respectively. Spectroscopic changes were likely due to the exclusion of H2O from the complex/antibody binding cleft and blocking of vibrational relaxation pathways of the Os/Ru excited state. D2O/H2O experiments confirmed that the antibody protected approx. 82% of [Os(bpy)2dcbpy] and 80% of [Ru(bpy)2dcbpy] from excited state deactivation by the aqueous solvent