Hydrogel-based microfluidic assays for multiplexed medical diagnostics

Abstract

There is high demand for next-generation biomolecule detection platforms arising from clinical need for more robust point-of-care diagnosis systems, fundamental research need to characterize complex biological processes, and the emergence of personalized medicine. These detection systems should accordingly provide multiplexed, sensitive, and highly specific quantification of biomarkers from several types of biological fluids. Hydrogels, which are loosely crosslinked networks of polymer chains, are particularly favorable substrates for robust detection of clinical analytes due to their non-fouling properties, ability to immobilize high concentrations of probe molecules, and solution-like environment which often leads to lower free energies of target-probe binding relative to solid surfaces. In this thesis, a novel array of microfluidic gel-based diagnostic tools is created for interrogation of proteins and microRNAs (miRNAs) from a range of biologically complex samples. To this end, we developed two distinct systems that are each applicable in different clinical contexts and both make use of microfluidic photolithographic approaches: (1) well-mixed hydrogel particle arrays for high-throughput and multiplexed quantification of proteins from cell culture, serum, and tissue lysates, and (2) channel-immobilized hydrogel posts for high-sensitive and multiplexed detection of microRNAs in an entirely on-chip format. Graphically-encoded polyethylene glycol (PEG) hydrogel microparticles bearing distinct coding regions and probe regions have previously demonstrated superior detection properties relative to surface-based bead arrays or microarrays for detection of short nucleic acids such as microRNAs. They have also been interfaced with a custom microfluidic highthroughput scanner for real-time post-assay analysis. Now, this platform is extended to multiplexed endogenous protein detection using both aptamer and antibodies to capture and label targets. Through our development and optimization, gel particles could be suspended into several types of unprocessed biological media without fouling or losing detection capability and demonstrated sensitivities that are comparable to commercial standards without needing amplification. In particular, aptamer-based protein detection using gel particles was found to provide up to three orders of magnitude better sensitivity than competing platforms using the hydrated and flexible gel particles also without using signal amplification. The chemical and thermodynamically favorable properties of the gel scaffold were also exploited to engineer an entirely on-chip assay that could have applications in clinical settings. Probe-containing porosity-tuned PEG posts were immobilized into microfluidic channels using a spatial encoding scheme. We developed a novel enzymatic amplification scheme that made use of the unique properties of a laminar two-phase flow around a gel post in a channel to completely isolate the gel posts within a fluorinated oil phase without needing surfactants. These confined gel chambers could then be used to dramatically concentrate products from an enzymatic amplification reaction leading to large (up to 57-fold) increase in sensitivity relative to direct labeling. The platform was integrated with an existing scheme for multiplexed miRNA detection from low amounts (10 - 50 ng) of total RNA inputs to enable entirely on-chip detection that shows great promise for eventual clinical integration to measure these biomarkers for disease diagnosis. Together, the advances reported here have significantly furthered both gel particle arrays and immobilized hydrogel posts as platforms that could eventually be integrated into clinical and research settings for robust biomolecule quantification.