Microfluidic Bead Suspension Hopper

Abstract

Many high-throughput analytical platforms, from next-generation DNA sequencing to drug discovery, rely on beads as carriers of molecular diversity. Microfluidic systems are ideally suited to handle and analyze such bead libraries with high precision and at minute volume scales; however, the challenge of introducing bead suspensions into devices before they sediment usually confounds microfluidic handling and analysis. We developed a bead suspension hopper that exploits sedimentation to load beads into a microfluidic droplet generator. A suspension hopper continuously delivered synthesis resin beads (17 μm diameter, 112,000 over 2.67 h) functionalized with a photolabile linker and pepstatin A into picoliter-scale droplets of an HIV-1 protease activity assay to model ultraminiaturized compound screening. Likewise, trypsinogen template DNA-coated magnetic beads (2.8 μm diameter, 176,000 over 5.5 h) were loaded into droplets of an in vitro transcription/translation system to model a protein evolution experiment. The suspension hopper should effectively remove any barriers to using suspensions as sample inputs, paving the way for microfluidic automation to replace robotic library distribution.

hνSABR: Photochemical Dose–Response Bead Screening in Droplets

“…Histogram analysis confirmed the stochastic nature of bead distribution. 16 Histograms of 3 min windows of data were integrated to determine the total number of droplets in each population (empty, 1-bead, etc.) and the average beads/droplet for each time point in order to compare the observed bead distribution with the expected Poisson distribution (Figure 5B).…”

“…The calibration channel was rinsed with water, dried, and filled with trimethylsiloxy-terminated PDMS. Beads displaying a fluorescein-labeled compound attached to resin with an o -nitrobenzyl photolabile linker (PC-FAM beads) were distributed into droplets (20 μg/mL phycoerythrin in thrombin cleavage buffer, AQ1) via a suspension hopper 16 and directed through the UV illumination region for photochemical cleavage. Photochemically cleaved compound fluorescence was detected at turn 4 in the incubation channel (1 kHz).…”

“…We previously disclosed the suspension hopper, a key microfluidic circuit component that enables introduction and distribution of hundreds of thousands of synthesis resin beads into picoliter-scale droplets. 16 Applying these findings to large OBOC libraries, however, would require reproducible and predictive compound liberation from library beads under high-throughput flow conditions to initiate screening. Here, we present devices fabricated using two-tone soft lithographic patterning with avobenzone-doped PDMS that feature a well-defined droplet illumination region and enable miniaturized droplet-scale, dose-dependent functional screens of OBOC libraries.…”

Discovery in Droplets

“…Alternatively, using a capped cartridge “suspension hopper” introduces one-bead-one-compound (OBOC) combinatorial library beads into a circuit for encapsulation into droplets. 29 The polyvalent synthesis resin particles inherently deliver multiple library member copies to a single picoliter-scale droplet, an event that the hopper can reproduce over 200,000 bead distributions. Once encapsulated, photolytic cleavage of compound from the bead surface provides sufficiently high concentrations (> 30 μ M) for screening in a UV dose-dependent fashion.…”

“…Academic researchers particularly have access to very few large, plate-based libraries from which to sip, prompting exploration of new library synthesis, delivery and distribution strategies. One example of such a scalable methodology for distributing > 10 5 chemical library members could entail the use of synthesis beads, wherein single droplets house single beads, 29 and libraries are the products of split-and-pool combinatorial synthesis.…”