Novel customisable phases for micro solid-phase extraction and automated biological sample preparation

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Protein biomarkers play an important role in clinical settings as they serve as measurable indicators for normal or abnormal biological processes. They aid in accelerated disease identification, diagnosis, prognosis, and response to treatments. Recently, mass spectrometry (MS) assays have been introduced as an alternate to the conventionally used immunoassays. MS provides accurate and precise quantification due to its high sensitivity and specificity. However, major challenges lie with sample preparation involving lengthy workflows, limited automation, and challenges related to highly complex biological samples where several techniques are applied. This thesis aims to improve sample preparation techniques to provide an accurate, rapid, and automated method for protein biomarker quantification using micro-solid-phase extraction (µSPE) technology through the development of a micro-immobilised-enzyme reactor (IMER) and a µSPE immunoaffinity cartridge to alleviate sample preparation bottlenecks caused by conventional ~18-hour digestions. A novel and customisable material was prepared for bio-ligand immobilisation. A hybrid inorganic-organic material using silica modified with carboxymethylated dextran (CMD) was prepared. This material was packed into µSPE cartridges and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) coupling was exploited to immobilise enzymes and antibodies in-situ using a programmable syringe driver allowing for the development of an automated workflow. Reproducible trypsin digestion was observed using high performance liquid chromatography (HPLC) through the cleavage of N-α-Benzoyl-L-arginine ethyl ester (BAEE). Three model proteins (bovine serum albumin, cytochrome c and thyroglobulin) and a human serum sample were analysed, and compared to conventional in-solution digestion by employing liquid chromatography orbitrap mass spectrometry (LC-OT-MS) and liquid chromatography quadrupole time of flight mass spectrometry (LC-QToF-MS). The IMER facilitated protein digestion within 10 minutes at room temperature and overall, observed lower sequence coverages and number of identified proteins compared to the conventional in solution digestion method at 37 °C. In the same manner as trypsin, anti-BSA was immobilised and BSA isolation was confirmed using size exclusion chromatography hyphenated to triple quadrupole inductively coupled plasma mass spectrometry (SEC-ICP-MS/MS). Whilst immobilisation of anti-BSA was achieved, challenges with low level protein detection were observed. The immunoaffinity and trypsin µSPE cartridge were combined into an automated workflow for protein pre-concentration and digestion. Using BSA as the model protein, BSA standards and BSA spiked into human serum samples were subjected to the workflow. BSA extraction and digestion was achieved, however, the complex human serum matrix negatively impacted the BSA isolation compared to neat standards. Further investigation and optimisation of the workflow must be performed.
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