Detection and analysis of proteins in the solid phase using extrinsic and intrinsic fluorescence
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Over the past two decades a body of evidence concerning residual biological contamination on cleaned surgical instruments has accumulated. This is substantiated by the number of yearly surgery cancellations due to visible residue on instruments in surgical packs and incidences of iatrogenic Creutzfeldt-Jakob disease (iCJD). It is therefore imperative to develop a method of protein detection for use in clinical sterile services departments (SSDs) for monitoring of decontamination quality. This Thesis describes the development and use of an epifluorescence surface scanner (EFScan) technology in the assessment of proteinaceous residue on surgical instruments, by detecting protein pre-labelled with fluorescein isothiocyanate (FITC), and exploratory studies on the feasibility of label-free detection, using intrinsic protein fluorescence. Measurements using FITC labelling showed that residual protein on the order of micrograms can be found on new, single-use instruments (i.e. prior to use). This is comparable to the amount of residual protein found on retired, reusable instruments. To confirm the suitability of fluorescence techniques in the detection and quantification of proteinaceous residue, a blind, pilot study was carried out in conjunction with groups from Queen Mary University and the University of Southampton. Each University used a different labelling and detection method, and results showed good agreement between these methods. This showed that fluorescence is a suitable technique for the detection and quantification of proteinaceous contamination on surgical instruments. The next step in the project focussed on detection of contamination via intrinsic protein fluorescence from tryptophan residues, with a view to elimination of the labelling step. Intrinsic fluorescence of proteins in solution is widely characterised; however, fluorescence characteristics of solid or surface-bound protein have been little studied. Therefore, the characterisation of solid protein fluorescence and the emission characteristics of protein adsorbed onto stainless steel was undertaken. Analysis of the commonly used protein standard bovine serum albumin (BSA) showed that the two tryptophan residues it contains are highly susceptible to photo-oxidation in the solid state, resulting in conversion to the fluorescent photoproducts n-formylkynurenine (NFK) and kynurenine. Therefore, BSA is not suitable for use as a standard in the development of intrinsic fluorescence detection of surface-bound protein. The 72-tryptophan protein fibrinogen, as well as a series of other multi-tryptophan proteins, were assessed and it was found that photo-oxidation of the tryptophan residues did not occur on the irradiation timescale of 1 hour utilized. Therefore, it was concluded that lysozyme or gamma-globulins, a prominent group of serum proteins, would be more suitable candidates as a standard in subsequent research into the intrinsic detection and quantification of proteinaceous contamination. A third study explored the potential use of fluorescence in the early diagnosis of cataract. This involved the fluorescence characterisation of healthy porcine lenses and the use of UV irradiation of the lens to attempt to create cataract in vitro. There was found to be a large variation in fluorescence characteristics from lens to lens, suggesting that fluorophore concentrations can vary significantly. This implies that identification of a suitable standard for the early detection of cataract may be problematic. Attempts to create cataract resulted in the photo-oxidation pathway which had been observed in BSA, and although NFK and kynurenine play a role in cataractogenesis, the accumulation of these photoproducts is not analogous to a natural cataract. It was found that these products could be destroyed by irradiation of the lens at appropriate photo-bleaching wavelengths. However, this also destroyed intrinsic, protective fluorophores within the lens, suggesting that a light-based method of cataract treatment may not be achievable.