Alexander K. Price

Use of microchip-based hydrodynamic focusing to measure the deformation-induced release of ATP from erythrocytes

In order to understand the role that erythrocytes play in conditions such as pulmonary hypertension, in vitro mimics of the microcirculation are needed. This paper describes the use of microchip-based hydrodynamic focusing to develop a mimic that allows both mechanical deformation of erythrocytes and quantification of the adenosine triphosphate (ATP) that is subsequently released in response to this deformation. In this mimic, two sheathing streams of a luciferin/luciferase mixture are used to focus and deform a central fluid flow of an erythrocyte sample. The focusing width is changed by simply manipulating the sheath flow rate. This allows a variety of cross-sectional areas to be studied using single point chemiluminescent detection. It was shown that increasing the sheath flow rate does result in elevated levels of ATP release. For example, one sample of rabbit erythrocytes released 0.80 (+/- 0.13) microM ATP when focused to a cross-section of 3480 microm(2), while focusing the same sample to a smaller cross-section (1160 microm(2)) led to a release of 6.43 (+/- 0.40) microM ATP. In addition, two different inhibitors, diamide and glibenclamide, were used to ensure a lack of cell lysis. This approach can be used to examine a wide range of deformation forces in a high throughput fashion and will be of interest to researchers studying the mechanisms leading to vasodilation in the microvasculature.

Discovery in Droplets

In the summer of 2009, Daniel Chiu’s prescient review in Analytical Chemistry described droplet microfluidics, an emerging concept and malleable analytical tool.1 The article primarily discussed the potential of single droplets to aid in the study of immensely complex systems, such as single cells and organelles. But droplets, by way of miniaturization and parallelization, were also clearly poised to survey much broader swathes of chemical and biological space in discovery-oriented high-throughput experimentation. Picoliter-scale analysis coupled with microfluidic integration could very realistically deliver million-fold improvements in throughput, potentially revolutionizing diverse applications from protein engineering to drug lead identification. The technology needed to tackle these difficult problems was still just emerging, but 2009 featured a dramatic expansion in microfluidic componentry for generating and manipulating large quantities of droplets (e.g., incubation, picoinjection, sorting, etc.). Today they collectively form the microfluidic circuit engineer’s standard palette of parts. Microfluidic circuit component integration, once largely concerned with moving bulk fluid between reactions and separation-based analysis channels, has entered a digital renaissance. Single devices now generate, handle, and analyze sample collections that vastly eclipse the capabilities of even the most sophisticated robotic automation. This review highlights recent (primarily 2013-2015) themes in technology development that continue to build the foundation of droplet-based discovery platforms, and new challenges in droplet-scale information storage and retrieval that have coalesced around these new platforms.

Generation of Nonbiased Hydrodynamic Injections on Microfluidic Devices Using Integrated Dielectric Elastomer Actuators

Sample introduction is a crucial, yet often overlooked step in chemical analysis. Its importance is clearly portrayed in the case of electrokinetic injection for electrophoretic separations, where sampling bias favors the introduction of the fastest moving analytes in a mixture. To this end, a poly(dimethylsiloxane) (PDMS)-based microfluidic device that incorporates miniaturized and fully integrated dielectric elastomer actuators (IDEAs) in order to perform sample injection for electrophoresis is reported. These electromechanical actuators produce hydrodynamic fluid pulses within the channel network without the need for any modifications to the channel design and without the use of large, off-chip equipment. Separations of Fluorescein thiocarbamyl-labeled amino acids reveal that IDEA-derived injections have a more stable chemical composition than electrokinetic injections, with peak area relative standard deviations (RSDs) less than 1.1% over 30 injections at six different volumes. Moreover, the efficiency and resolution of separations with IDEA-derived injections are not significantly different from electrokinetic injections under similar separation conditions. The reproducibility of peak heights and peak areas over the course of 64 consecutive injections reveal that the actuation mechanism is very stable with peak area RSDs less than 1.8%. These results, coupled with facile fabrication and operation of IDEA devices, suggest that widespread adaptation of this technology could be very advantageous for many types of miniaturized analysis systems.

Microfluidic Bead Suspension Hopper

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

With the potential for each droplet to act as a unique reaction vessel, droplet microfluidics is a powerful tool for high-throughput discovery. Any attempt at compound screening miniaturization must address the significant scaling inefficiencies associated with library handling and distribution. Eschewing microplate-based compound collections for one-bead-one-compound (OBOC) combinatorial libraries, we have developed hνSABR (Light-Induced and -Graduated High-Throughput Screening After Bead Release), a microfluidic architecture that integrates a suspension hopper for compound library bead introduction, droplet generation, microfabricated waveguides to deliver UV light to the droplet flow for photochemical compound dosing, incubation, and laser-induced fluorescence for assay readout. Avobenzone-doped PDMS (0.6% w/w) patterning confines UV exposure to the desired illumination region, generating intradroplet compound concentrations (>10 μM) that are reproducible between devices. Beads displaying photochemically cleavable pepstatin A were distributed into droplets and exposed with five different UV intensities to demonstrate dose-response screening in an HIV-1 protease activity assay. This microfluidic architecture introduces a new analytical approach for OBOC library screening, and represents a key component of a next-generation distributed small molecule discovery platform.

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing.