Encoded hydrogel microparticles for high-throughput molecular diagnostics and personalized medicine

Abstract

The ability to accurately detect and quantify biological molecules in complex mixtures is crucial in basic research as well as in clinical settings. Advancements in genetic analysis, molecular diagnostics, and patient-tailored medicine require robust detection technologies that can obtain high-density information from a range of physiological samples in a rapid and cost-effective manner. Compared to conventional microarrays and methods based on polymerase chain reaction (PCR), suspension (particle-based) arrays offer several advantages in the multiplexed detection of biomolecules, including higher rates of sample processing, reduced consumption of sample and reagent, and rapid probe-set modification for customizable assays. This thesis expands the utility of a novel hydrogel-based microparticle array through (1) the creation of a microfluidic, flow-through fluorescence scanner for high-throughput particle analysis, (2) the development of a suite of techniques for the highly sensitive and specific detection of microRNA (miRNA) biomarkers, and (3) the investigation of new methods for directly measuring biomolecules at the single-cell level. Graphically-encoded hydrogel microparticles synthesized from non-fouling, bioinert poly(ethylene glycol) (PEG) and functionalized with biomolecule probes offer great promise in the development of high-performance, multiplexed bioassays. To extend this platform to applications in high-throughput analysis, particle design was optimized to ensure mechanical stability in high-velocity flow systems, and a single-color microfluidic scanner was constructed for the rapid fluorescence interrogation of each particle's spatially-segregated "code" and "probe" regions. The detection advantages of three-dimensional, probe-laden hydrogel scaffolds and the operational efficiencies of suspension array technology were then leveraged for the rapid multiplexed expression profiling of miRNA. The graphical encoding method and ligationbased labeling scheme implemented here allowed for scalable multiplexing with a simple workflow and an unprecedented combination of sensitivity, flexibility, and throughput. Through the rolling circle amplification of a labeling oligonucleotide, it was possible to further enhance the system's sensitivity and resolve single-molecule miRNA binding events on particle surfaces, enabling the first direct detection of low-abundance miRNA in human serum without the need for RNA extraction or target amplification. Finally, by arraying cells and gel particles in polydimethylsiloxane (PDMS) microwells, it was possible to dramatically improve the particles' target capture efficiency and thereby move closer to a regime in which miRNAs and other biological molecules may be directly detected without target amplification from single cells.