Development of a multiplexing biosensor platform using SERS particle immunoassay technology

A thesis submitted to the University of Bedfordshire in partial fulfilment of the requirements of the degree of Doctor of PhilosophyThe purpose of this study is to demonstrate the ability of surface enhanced Raman scattering (SERS) active particles to enable multiplexed immunoassays in a lateral flow format for point of care (POC) testing. The SERS particles used for this study are chemically active glass coated gold particles, containing tracer molecules which in principle can be chosen to provide Raman Spectra with unique features allowing multiple tracers to be simultaneously measured and distinguished without interference between each other. Lateral flow immunoassay technology is the important part of this study and can be conveniently packaged for the use of other than highly skilled technicians outside of the laboratory. A well-known (single channel - simplex) device for the pregnancy test is a typical example of the lateral flow assay. Similar formats have been/are being developed by others for a range of POC applications – but most diagnostic applications require simultaneous determination of a range of biomarkers and multiplexed assays are difficult to achieve without significant interference between the individual assays. This is where SERS particles may provide some advantages over existing techniques. Cardiac markers are the growing market for point of care technology therefore biomarkers of cardiac injury (Troponin, myoglobin and CRP) have been chosen as a model. The object of the study is to establish the proof of concept multiplexing assay using these chosen biomarkers. Thus, initially all different particles were characterised in single and mixture form. Also development of conjugate chemistry between antibodies for each analyte that have been purchased from commercial sources and SERS particles were analysed using different conditions like buffer, pH and antibody loading concentration to get the optimum intensity. The selected SERS particles and their conjugates were tested for size, aggregation and immune quality using a range of techniques: ultraviolet-visible (UV/Vis) absorption spectroscopy, dynamic light scattering (DLS) and lateral flow assay. These characterisations methodologies gave the understanding of optimum conditions of the each conjugates and individual’s behaviour in mixture conditions as well. After the characterisation all conjugates were tested singularly on the lateral flow assay using buffers and serum. The results of this single analyte immunoassay explained the individual’s bioactivity on the lateral flow strip. Further in study, multiplex assay have been demonstrated in serum. These outcomes have described each candidate characteristic in a mixture form on the lateral flow strip. In order to get the optimum Raman intensity from multiplex assay, the detection and capture antibodies loading concentrations were tuned in the assay. Later on different combinations (high, medium and low concentrations) of all three analytes were analysed and has found some interferences in multiplex assay. To investigate these issues various aspect were considered. First of all, different possibilities of non-specific interactions between the co-analytes and antibodies were tested. In addition, steric hindrance and optical interference investigations were performed via several assays and analysis using Scanning electron microscopy. The outcomes have confirmed related optical interferences. Therefore other assay (wound biomarkers) established to eliminate the interferences. In summary, the works reported here have built and test the equipment and necessary reagents for individual assays before moving on the more complicated task. In addition, the entire study has given a deep knowledge of multiplex assay on a single test line including the investigation of the issues for selected cardiac biomarkers and their applications in the future