Brian M. Paegel

High throughput DNA sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor

High throughput DNA sequencing has been performed by using a microfabricated 96-channel radial capillary array electrophoresis (μCAE) microchannel plate detected by a 4-color rotary confocal fluorescence scanner. The microchannel plate features a novel injector for uniform sieving matrix loading as well as high resolution, tapered turns that provide an effective separation length of 15.9 cm on a compact 150-mm diameter wafer. Expanded common buffer chambers for the cathode, anode, and waste reservoirs are used to simplify electrode addressing and to counteract buffering capacity depletion arising from the high electrophoretic current. DNA sequencing data from 95 successful lanes out of 96 lanes run in parallel were batch-processed with basefinder, producing an average read length of 430 bp (phred q ≥ 20). Phred quality values were found to exceed 40 (0.01% probability of incorrectly calling a base) for over 80% of the read length. The μCAE system demonstrated here produces sequencing data at a rate of 1.7 kbp/min, a 5-fold increase over current commercial capillary array electrophoresis technology. Additionally, this system permits lower reagent volumes and lower sample concentrations, and it presents numerous possibilities for integrated sample preparation and handling. The unique capabilities of μCAE technology should make it the next generation, high performance DNA sequencing platform.

Stepwise synthesis of giant unilamellar vesicles on a microfluidic assembly line.

Among the molecular milieu of the cell, the membrane bilayer stands out as a complex and elusive synthetic target. We report a microfluidic assembly line that produces uniform cellular compartments from droplet, lipid, and oil/water interface starting materials. Droplets form in a lipid-containing oil flow and travel to a junction where the confluence of oil and extracellular aqueous media establishes a flow-patterned interface that is both stable and reproducible. A triangular post mediates phase transfer bilayer assembly by deflecting droplets from oil, through the interface, and into the extracellular aqueous phase to yield a continuous stream of unilamellar phospholipid vesicles with uniform and tunable size. The size of the droplet precursor dictates vesicle size, encapsulation of small-molecule cargo is highly efficient, and the single bilayer promotes functional insertion of a bacterial transmembrane pore.

High-performance genetic analysis using microfabricated capillary array electrophoresis microplates

This review focuses on some recent advances in realizing microfabricated capillary array electrophoresis (νCAE). In particular, the development of a novel rotary scanning confocal fluorescence detector has facilitated the high‐speed collection of sequencing and genotyping data from radially formatted νCAE devices. The concomitant development of a convenient energy‐transfer cassette labeling chemistry allows sensitive multicolor labeling of any DNA genotyping or sequencing analyte. High‐performance hereditary haemochromatosis and short tandem repeat genotyping assays are demonstrated on these devices along with rapid mitochondrial DNA sequence polymorphism analysis. Progress in supporting technology such as robotic fluid dispensing and batched data analysis is also presented. The ultimate goal is to develop a parallel analysis platform capable of integrated sample preparation and automated electrophoretic analysis with a throughput 10–100 times that of current technology.

Layer-by-layer cell membrane assembly

Eukaryotic subcellular membrane systems, such as the nuclear envelope or endoplasmic reticulum, present a rich array of architecturally and compositionally complex supramolecular targets that are yet inaccessible. Here we describe layer-by-layer phospholipid membrane assembly on microfluidic droplets, a route to structures with defined compositional asymmetry and lamellarity. Starting with phospholipid-stabilized water-in-oil droplets trapped in a static droplet array, lipid monolayer deposition proceeds as oil/water phase boundaries pass over the droplets. Unilamellar vesicles assembled layer-by-layer support functional insertion of both purified and in situ expressed membrane proteins. Synthesis and chemical probing of asymmetric unilamellar and double bilayer vesicles demonstrate the programmability of both membrane lamellarity and lipid leaflet composition during assembly. The immobilized vesicle arrays are a pragmatic experimental platform for biophysical studies of membranes and their associated proteins, particularly complexes that assemble and function in multilamellar contexts in vivo.

Microfluidic Compartmentalized Directed Evolution

Directed evolution studies often make use of water-in-oil compartments, which conventionally are prepared by bulk emulsification, a crude process that generates nonuniform droplets and can damage biochemical reagents. A microfluidic emulsification circuit was devised that generates uniform water-in-oil droplets (21.9 +/- 0.8 microm radius) with high throughput (10(7)-10(8) droplets per hour). The circuit contains a radial array of aqueous flow nozzles that intersect a surrounding oil flow channel. This device was used to evolve RNA enzymes with RNA ligase activity, selecting enzymes that could resist inhibition by neomycin. Each molecule in the population had the opportunity to undergo 10(8)-fold selective amplification within its respective compartment. Then the progeny RNAs were harvested and used to seed new compartments. During five rounds of this procedure, the enzymes acquired mutations that conferred resistance to neomycin and caused some enzymes to become dependent on neomycin for optimal activity.

Darwinian Evolution on a Chip

Computer control of Darwinian evolution has been demonstrated by propagating a population of RNA enzymes in a microfluidic device. The RNA population was challenged to catalyze the ligation of an oligonucleotide substrate under conditions of progressively lower substrate concentrations. A microchip-based serial dilution circuit automated an exponential growth phase followed by a 10-fold dilution, which was repeated for 500 log-growth iterations. Evolution was observed in real time as the population adapted and achieved progressively faster growth rates over time. The final evolved enzyme contained a set of 11 mutations that conferred a 90-fold improvement in substrate utilization, coinciding with the applied selective pressure. This system reduces evolution to a microfluidic algorithm, allowing the experimenter to observe and manipulate adaptation.

DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis

The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the “structure elucidation problem”: the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS’s utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 104 molecules/bead and sequencing allowed for elucidation of each compound’s synthetic history. We applied DESPS to the combinatorial synthesis of a 75 645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward, and may prompt the chemistry community to revisit the synthesis of more complex and diverse libraries.

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.

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.