Several factors affect the viability of biosensor design. This thesis presents the development of a computer-based model that will enable the sources and effects of noise and variations in concentrations within an evanescent field immunosensor to be analysed. The model was developed as a series of modules, each representing one aspect of the sensor, which when linked provide a simulation of the whole sensor. A complete solution of the complex biochemical reactions involved in the immunoassay module was achieved using a Markov chain approach. More traditional methods of solving sets of equations, such as optimisation, genetic algorithms and simulated annealing, all failed to produce satisfactory results. Two alternative assays, a sandwich and a competitive assay, are presented. The light module details the modelling of the coupling into a planar monomode waveguide and calculation of fluorescence excited by the resulting evanescent field using standard electromagnetic formulae. However, both beam divergence and scattering from the immobilised antibody layer were incorporated into the model. Two alternative coupling techniques were modelled, prism coupling and coupling through a "resonant mirror" multilayer. The detection system modelled the amplification of the fluorescence by a photomultiplier tube. The resulting model represents the most rigorous modelling undertaken in this area and the potential applications and benefits of such a model were detailed. Analysis of noise within the sensor allowed the impact of variation in the physical parameters defining the sensor to be determined and compared. The model was used to compare different protocols and confirmed that the sandwich assay produced the more sensitive device. A study of the kinetic response of the assay determined that measurements could be performed at half the time taken to reach equilibrium without significant loss of sensitivity. An analysis of the effect of scattering at the waveguide surface showed this to be significant noise factor. An initial study of the impact of the humectant layer illustrated that this is an issue that merits further consideration.

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The conventional biochemical diagnosis of disease using isolated blood biomarkers must be revisited by clinicians, replacing it with a multi-biomarker, personalised profile of human health. A label-free nanoparticle array technology, Liscar, has been developed. It is capable of performing rapid, multi-biomarker assays from complex biological samples which, if employed to assay the Complement cascade of the innate immune system, has the potential for a novel systemic profile of patient health. An assay for IgG has been developed on the Liscar platform with a detection limit of 380 ± 100 ng/mL IgG in model sera. Furthermore, addition of a chaotropic agent to the complex sample is shown to improve the accuracy of the IgG assay. Competitive binding between nonspecific interfering proteins and specific target analytes (IgG) at the sensor surface is studied, and a quantitative mathematical model is developed to analyse the data, yielding evidence for the active displacement of albumin by IgG-antigen binding. With a sensitive, accurate multi-biomarker detection platform, a systemic profile of patient health may be possible by examination of the Complement cascade. The Complement system can be activated by a variety of immunological challenges, causing large numbers of activation biomarkers to be produced quickly. Assays for three activation markers, C3d, TCC and Bb are developed, with detection limits of 0.864 ACS Units, 2.32 ng/ml and 54.7 ng/ml respectively. Complement activation was tested in a prospective cohort study of 45 patients undergoing major abdominal surgery. Patient recovery was monitored from admission to ~60 hours postoperatively by Complement activation and consumption using C3d, TCC, Bb, C3 and C4 as biomarkers. A response profile was obtained for the entire cohort for C3 and C4 assays by normalising with respect to individual analyte levels on admission, against which individual responses are compared. 22% of patients in the study suffered postoperative complications, and 73% showed Complement activation by increased levels of C3d, as expected from the initial trauma of surgery. Expansion of the trial is needed to establish clinical significance and utility, especially in relation to the presymptomatic diagnosis of disease.

Cardiac surgery with cardiopulmonary bypass (CPB) can trigger systemic inflammatory response syndrome (SIRS) in patients. Pro-inflammatory cytokines lL-6 and IL-8 are reported as particular markers in blood of the inflammatory •espouse to CPB. It would be useful to measure the inflammatory response in the surgical theatre in real/near real time to improve the effectiveness and timings for interventions which may counteract any undesirable inflammatory response. Currently, Enzyme Linked Immunosorbent Assays (ELISAs) and similar systems are used in the central laboratory to measure inflammatory markers however they do not provide results within the appropriate timescale for rapid intervention. Consequently other approaches to assay provision at point of care must be considered. This thesis is concerned with the design, fabrication and characterisation of a prototype, electrochemical, lateral flow system opening a new approach to rapid, point of care (POC), quantitative monitoring of inflammatory markers.

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A demand exists for a fast, sensitive, reliable and economical test for pyogenic sepsis that provides a “real time” bed-side assessment. Detection of significant intra-abdominal sepsis can be particularly problematic in the ICU setting and in patients with multi-system organ failure. Lysozyme, first reported by Fleming (1922), is a bacteriolytic enzyme released during phagocytosis. Previous studies have shown significant correlation between lysozyme levels and the presence of intra-abdominal abscess in both animals and humans. A method which determines and quantifies lysozyme as part of an assessment of an acutely ill patient in whom major sepsis is suspected; would significantly aid diagnosis and prescription of the most effective form of treatment. To date measurement of lysozyme has been by turbidometry, with consequent poor sensitivity and reliability. Other methods of assay include fluorescence, radial immunodiffussion and enzyme linked immunosorbent assay (ELISA). This study reports a modified ELISA technique which provides a cheap, sensitive and reliable method of lysozyme determination, producing results in <100 minutes. The ELISA has been tested with ~200 clinical samples provided by the patients at the Great Western Hospital, Swindon. Two ELIFA techniques were also developed for lysozyme and E.coli detection. These techniques also provide a cheap and rapid alternative to the more traditional immunoassays. Results from the ELIFA and mini-ELIFA were obtained qualitatively after only 10 minutes. An SPR detection technique was also devised. The BIAcore 3000 was used to create a biosensor for serum lysozyme using an artificial receptor in the form of an aptamer. This system was tested with clinical serum samples, is reusable and took <80 minutes to immobilise a ligand on a blank sensor and analyse a serum sample for lysozyme. Although further research and development is required on the mini-ELIFA and lysozyme biosensor, the ELISA detection system may prove a useful tool in the diagnosis of sepsis in critically ill patients.