Synthesis and characterisation of Raman-tagged microspheres for immunoassay

The swift, unambiguous identification of disease markers in low concentrations is fundamental to early detection and good prognosis. Immunoassays, which are analytical disease diagnostic tests, provide quantitative recognition based on antibody-antigen specificity. The cornucopia of available antibodies has led to the development of assays for almost any perceivable antigen, making them a pillar of modern diagnostics. The existence of two antibodies to one antigen allows the immobilisation of capture antibodies, which bind an antigen, onto which a detection antibody can be bound forming a sandwich structure. These assays rely on radio, fluorescent or enzymatic labelled detection antibody for readout. These suffer from several limitations, such as high limits of detection and difficult multiplexation, requiring the design of arrays or complex microfluidics in order to achieve spatial separation for multiplex analysis. Raman spectroscopy has the capacity for high level multiplex detection. Utilisation of the narrow vibrational transitions has provided an inimitable method for sensitive and multiplex diagnostics. Aiming to establish a platform for a specific and multiplex immunoassay, multi-layered microsphere sensors were developed which exploit the plasmonic properties of silver metal nanoparticle films to enhance locally the Raman scattering of surface bound molecules in surface enhanced Raman spectroscopy (SERS), thus overcoming the weak scattering efficiency of normal Raman. These Raman encoded microspheres were enveloped in a protective silica layer, and grafted with amine surface chemistry to immobilise monoclonal antibodies. Scanning electron microscopy, zeta potential analysis and Raman and UV-vis spectroscopy were used to characterise the metal films and subsequent protective shells. In particular, highly reproducible SERS substrates were developed as well as a broad protective strategy allowing for the exploitation of widely varying chemical moieties for excellent spectral separation. These sensors became microscopic immunoassay barcode substrates onto which a sandwich immunoassay could be performed employing fluorescent secondary labels for localisation and quantification while the sensor provided recognition. Raman micro-spectroscopy and laser scanning confocal microscopy verified the sensors as applicable substrates for sensitive, highly flexible and multiplex immunoassay. The convenience of the prepared substrates lend themselves to a unique dip’n’dry methodology where high surface area can allow for rapid detection of disease antigens. These were used for targeting of a unique panel of antigens for pancreatic cancer, and establishing attomolar detection limits with tumour necrosis factor alpha. These sensors, therefore, are an excellent candidate for qualitative and quantitative immunoassay