(416f) High-Throughput Flow Alignment and Scanning of Barcoded Hydrogel Microparticles

The ability to accurately detect and quantify biological molecules in a complex mixture is crucial in both basic research and clinical settings. Advancements in the fields of genomics and proteomics require robust technologies that can obtain high-density information from biological samples in a rapid and cost-effective manner. Suspension (particle-based) arrays offer several advantages over conventional planar arrays in the multiplexed detection and quantification of biomolecules, including the use of smaller sample volumes, more favorable probe-target binding kinetics, and rapid probe-set modification. We have previously described a microfluidic-based process (stop-flow lithography, SFL) for rapidly generating geometrically and chemically complex hydrogel microparticles (10,000 per hour) that can be used for the sensitive, multiplexed detection of DNA and RNA oligomers.

We now present a microfluidic system for the rapid flow alignment of these multifunctional hydrogel microparticles, which are designed to bear one or several biomolecule probe regions, as well as a graphical code to identify the embedded probes. Using high-speed imaging, we have developed and optimized a flow-through system that allows for a high particle throughput, ensures proper particle alignment for decoding and target quantification, and can be reliably operated continuously without clogging. A tapered channel flanked by side focusing streams is used to orient the flexible, tablet-shaped particles into a single-file, well-ordered flow in the center of the channel. The effects of channel geometry, particle geometry, particle composition, particle loading density, and barcode design are explored to determine the best combination for eventual use in biological assays. Particles in the optimized system move at velocities of ~50 cm/s and with throughputs of ~40 particles/s. Simple physical models and COMSOL Multiphysics simulations have been used to investigate flow behavior in the device.

The ability to align particles in the channel with high precision enables photomultiplier tube (PMT)-based detection with a simple one-dimensional line scan that integrates particle fluorescence intensity along a thin laser excitation beam established perpendicular to the flow direction. The resulting high-resolution profile is then used to determine the identity of a passing particle, ascertain the probe(s) it carries, and measure the extent of the binding events on the various probe and negative control regions. We present our latest advances in the development of this microfluidic scanning system, with particular emphasis on the instrumentation employed.