ENABLING RAPID DIAGNOSTICS FROM LARGE-VOLUME COMPLEX SAMPLES WITH AUTOMATED BIOMARKER ENRICHMENT STRATEGIES

Abstract

Rapid diagnostic testing is critical for enabling timely and targeted clinical decision-making, yet many clinical biomarkers are dilute and embedded in inhibitor-rich biological sample matrices, complicating their detection. These challenges encompass the central bottleneck for near-patient molecular diagnostics: recovering and enriching dilute biomarkers while minimizing user steps and dependence on central-laboratory infrastructure. To address these challenges, this work presents diagnostic strategies that combine magnetic bead capture with thermally responsive alkane partitions (TRAPs) to enable automated purification, enrichment and detection of dilute biomarkers in complex sample matrices.In this dissertation, three automated assay workflows are presented across distinct sample types and targets. First, a sample-to-answer assay for circulating microRNA in whole blood integrates TRAPs, a novel displacement-ligation mechanism, and loop-mediated isothermal amplification for detection in a portable, hands-free device. Second, the Automated Magnetically and Thermally Induced Conical Separator (Auto-MATICS) is introduced to recover and concentrate magnetic beads from up to 15 mL samples into a small output volume, providing large enrichment factors. This platform enables detection of dilute, fragmented Borrelia burgdorferi genomic DNA in synthetic urine. Third, the Auto-MATICS is adapted for bloodstream infection workflows by pairing it with Fc-containing mannose binding lectin (FcMBL)-functionalized magnetic beads to capture and enrich low-concentration bacteria from 8 mL whole blood within ~4.5 hours. This workflow offers a path to substantially shorten the diagnostic turnaround time for bloodstream infection diagnostics. Collectively, these contributions advance automated sample preparation as a practical route to rapid diagnostics that are closer to the site of patient care.