Optical modulation of high-affinity biomolecules function via photochromic dyes : a step towards an artificial control of biological activity

摘要

Prior to this study, there has yet to be a clear demonstration of an artificial control of antibody affinity via photochromic dyes. The research described in this thesis sets out to address this by investigating photochromic dyes and their subsequent applications with high affinity biomolecules - primarily to photomodulate the functions of biomolecules. The main avenue of investigation explored the conjugation of photochromic dyes (spiropyrans) to proteins (an enzyme and five different antibodies), to achieve reversible photomodulation of protein function for possible applications in biosensor technology (such as the development of reagentless bio¬reversible sensing systems). A secondary aim involved the investigation of the feasibility of antibody-antigen binding in the presence of ionic liquids. Ionic liquids have recently experienced growing interest as replacements for traditional organic solvents in a number of industrial applications. The practicability of spiropyrans in ionic liquids was also investigated (with the future possibility of photomodulated antibody-antigen interactions in ionic liquids to deliver a variety of improved analytical performances).The synthesis and photoswitching properties of an appropriate range of spiropyran dyes are reported. The spiropyran dyes are synthesised to possess a carboxyl group to aid carbodiimide mediated conjugation to lysine amino groups of proteins. The photochromic behaviour of the spiropyran dyes in various solvents, temperature and pH ranges were observed. Conjugation of carboxylated spiropyran dyes to an enzyme: horseradish peroxidise, was initially observed to aid development of experimental protocol for the target study group i.e. antibodies. Photomodulation of the modified horseradish peroxidase was found to demonstrate ~ 60 % decline in enzyme activity, an effect which was reversible as a result of the photoswitching capabilities of the attached spiropyran dyes. The five different antibodies; anti Atrazine, anti GroEL, anti Phytanic Acid, anti FITC and anti Staphylococcus aureus were modified with spiropyran dyes as with horseradish peroxidase. Reversible antibody affinity photomodulation was observed via their reaction in an ELISA which yielded a decline of ~ 15 %, ~ 40 %, ~ 50 %, ~ 55 % and ~ 65 % in binding signal respectively. A fatigue assessment was conducted on the photoswitching capabilities of both the conjugated and the unconjugated spiropyran dyes. This was expressed as ten photoswitching cycle experiments, the first evenly spaced over ten days and a second over ten weeks. The initial results suggested dye degradation increased with consecutive photoswitching cycles of the conjugated spiropyran dyes. It was observed that the level of degradation for the unconjugated spiropyran dyes was independent to the timing interval between photoswitching cycles, suggesting storage stability of the compound. However the level of degradation for the conjugated spiropyran dyes was dependent to the timing interval between photoswitching cycles, suggesting storage instability.A subsequent study involved the demonstration of the feasibility of antibody-antigen binding in ionic liquids for the first time. Various combination ratios of ionic liquids with aqueous phosphate buffered saline were employed. Initial experimentation of antibody-antigen binding showed that use of solutions with an ionic liquid content of 50 % and below, produced identical results to that of the standard aqueous phosphate buffered saline. At 95 % ionic liquid content, a lower level of binding activity was observed. The possibility of a photomodulated antibody-antigen interactions in ionic liquids did not produce a significant result on this occasion with the observation of spiropyran dyes failure to photoswitch in solutions with as low as 10 % ionic liquid content.In summary, although the development of a reagentless bio-reversible sensing system continues beyond the period of this PhD thesis, significant progress has been made with regards to photochromic antibodies as possible candidates for further studies and applications, also the establishment of antibody-antigen binding in various ionic liquids can serve as ways to further enhance the applicability of such reactions under different environmental conditions.