Upconversion nanoparticle based optical biosensor for the detection of molecular biomarkers

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Developing sensitive, specific and user-friendly biosensors in order to detect and quantify disease-related molecular biomarkers, is highly significant for early stage diagnosis in the clinic. Current optical biosensors have limitations due to high auto-fluorescent background noise, low detection sensitivity, and are labour intensive. This thesis explores the surface properties of upconversion nanoparticles (UCNPs), along with identification of a suitable surface modification ligand, which ultimately results in the development of a set of optical biosensors for use in the detection and quantification of both nucleic acid and protein molecular biomarkers for diagnostic purposes. This project has employed UCNPs as a new generation optical nanomaterial for biosensing applications, which have the distinct advantage of eliminating background interference arising from auto-fluorescence. This favourable advantage comes from the physicochemical properties of the UCNPs as they stepwise absorb near-infrared photons and emit anti-Stokes visible luminescence within a narrow spectral bandwidth. This project further explores the use of UCNPs in luminescence resonance energy transfer (LRET) to simplify biomolecular assays, as their distance-dependent energy transfer properties can be exploited to avoid tedious washing steps, resulting in simplicity by saving both labour and analytical time. To realize this, an enzyme-assisted signal amplification technique has been applied to further improve the detection sensitivity of trace amounts of nucleic acids. The research program reported herein involves three core projects with three key techniques successfully implemented: the first project contains a systematic and comparative study on the colloidal stability and biocompatibility of hydrophilic UCNPs capped with four ligands. In this project, a newly designed polymer ligand have been identified to form highly water stable and biocompatible UCNPs (Chapter 2); the second project reports an Exonuclease III-assisted upconversion resonance energy transfer in a wash-free suspension DNA assay. Using this approach, an ultra-high sensitivity assay with a detection limit of 15 pM of DNA has been achieved. This assay has achieved one order of magnitude higher sensitivity compared to conventional LRET DNA assay systems using UCNPs (Chapter 3); the third project introduces UCNP based resonance energy transfer for immunoassay of glypican-1 (GPC-1), as a molecular biomarker for prostate cancer diagnosis. In this system, a rational core-shell structure of UCNPs was designed to increase the on/off ratio of detection, and a bispecific antibody was used to improve immune-affinity and simplicity. As a result, a labour-saving, specific and rapid optical biosensor to detect the prostate cancer-relevant GPC-1 biomarker has been demonstrated (Chapter 4). Several advances in biosensor development have been achieved and reported in this thesis, and further developments are still required towards real world applications. By the end of this thesis, the simplicity of detecting devices and exploitation in point-of-care application are further discussed, which present an array of new opportunities for biomarker assays (conclusions and perspectives Chapter 5).
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