Publications by authors named "John Heyman"

High-throughput screening and enrichment of antibody-producing cells have many important applications. Herein, we present a droplet microfluidic approach for high-throughput screening and sorting of antibody-secreting cells using a Förster resonance electron transfer (FRET)-based assay. The FRET signal is mediated by the specific binding of the secreted antibody to two fluorescently labeled probes supplied within a droplet.

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Fluorosurfactants have expanded the landscape of high-value biochemical assays in microfluidic droplets, but little is known about how the spatial geometries and polarity of the head group contribute to the performance of fluorosurfactants. To decouple this, we design, synthesize, and characterize two linear and two dendritic glycerol- or tris-based surfactants with a common perfluoropolyether tail. To reveal the influence of spatial geometry, we choose inter-droplet cargo transport as a stringent test case.

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Monoclonal antibodies are powerful tools for scientific research and are the basis of numerous therapeutics. However, traditional approaches to generate monoclonal antibodies against a desired target, such as hybridoma-based techniques and display library methods, are laborious and suffer from fusion inefficiency and display bias, respectively. Here we present a platform, featuring droplet microfluidics and a bead-based binding assay, to rapidly identify and verify antigen-binding antibody sequences from primary cells.

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Quantification of cell-secreted molecules, e.g., cytokines, is fundamental to the characterization of immune responses.

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Multiple sclerosis is a chronic inflammatory disease of the CNS. Astrocytes contribute to the pathogenesis of multiple sclerosis, but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations.

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Here, we demonstrate use of a Mg-dependent, site-specific DNA enzyme (DNAzyme) to cleave oligos from polyacrylamide gel beads, which is suitable for use in drop-based assays. We show that cleavage efficiency is improved by use of a tandem-repeat cleavage site. We further demonstrate that DNAzyme-released oligos function as primers in reverse transcription of cell-released mRNA.

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We report an additive-free method to lyse bacteria and extract nucleic acids and protein using a traveling surface acoustic wave (TSAW) coupled to a microfluidic device. We characterize the effects of the TSAW on E. coli by measuring the viability of cells exposed to the sound waves and find that about 90% are dead.

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Fluorosurfactant-stabilized microfluidic droplets are widely used as pico- to nanoliter volume reactors in chemistry and biology. However, current surfactants cannot completely prevent inter-droplet transfer of small organic molecules encapsulated or produced inside the droplets. In addition, the microdroplets typically coalesce at temperatures higher than 80 °C.

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The selection of improved producers among the huge number of variants in mutant libraries is a key issue in filamentous fungi of industrial biotechnology. Here, we developed a droplet-based microfluidic high-throughput screening platform for selection of high-cellulase producers from filamentous fungus Trichoderma reesei. The screening system used a fluorogenic assay to measure amount of cellulase and its activity.

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Article Synopsis
  • A new method has been developed for detecting specific bacterial strains using a DNAzyme and a molecular beacon, making the process simpler.
  • This improved technique eliminates a previously time-consuming preparation step while still keeping high sensitivity for detecting bacteria.
  • The method aims to enhance the efficiency of bacterial detection in various applications.
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Active manipulation of droplets is crucial in droplet microfluidics. However, droplet polydispersity decreases the accuracy of active manipulation. We develop a microfluidic "droplet filter" that accurately separates droplets by size.

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Single-span membrane proteins (ssMPs) represent approximately one-half of all membrane proteins and play important roles in cellular communications. However, like all membrane proteins, ssMPs are prone to misfolding and aggregation because of the hydrophobicity of transmembrane helices, making them difficult to study using common aqueous solution-based approaches. Detergents and membrane mimetics can solubilize membrane proteins but do not always result in proper folding and functionality.

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Quantitative protein analysis of single cells is rarely achieved due to technical difficulties of detecting minute amounts of proteins present in one cell. We develop a mix-and-read assay for drop-based label-free protein analysis of single cells. This high-throughput method quantifies absolute, rather than relative, amounts of proteins and does not involve antibody labeling or mass spectrometry.

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Simultaneous encapsulation of multiple active substances in a single carrier is essential for therapeutic applications of synergistic combinations of drugs. However, traditional carrier systems often lack efficient encapsulation and release of incorporated substances, particularly when combinations of drugs must be released in concentrations of a prescribed ratio. We present a novel biodegradable core-shell carrier system fabricated in a one-step, solvent-free process on a microfluidic chip; a hydrophilic active (doxorubicin hydrochloride) is encapsulated in the aqueous core, while a hydrophobic active (paclitaxel) is encapsulated in the solid shell.

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We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells.

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Micrometer-sized hydrogel particles that contain living cells can be fabricated with exquisite control through the use of droplet-based microfluidics and bioinert polymers such as polyethyleneglycol (PEG) and hyperbranched polyglycerol (hPG). However, in existing techniques, the microgel gelation is often achieved through harmful reactions with free radicals. This is detrimental for the viability of the encapsulated cells.

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Droplet microfluidics offers significant advantages for performing high-throughput screens and sensitive assays. Droplets allow sample volumes to be significantly reduced, leading to concomitant reductions in cost. Manipulation and measurement at kilohertz speeds enable up to 10(8) samples to be screened in one day.

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We report the preparation of polyglycerol particles on different length scales by extending the size of hyperbranched polyglycerols (3 nm) to nanogels (32 nm) and microgels (140 and 220 μm). We use miniemulsion templating for the preparation of nanogels and microfluidic templating for the preparation of microgels, which we obtain through a free-radical polymerization of hyperbranched polyglycerol decaacrylate and polyethylene glycol-diacrylate. The use of mild polymerization conditions allows yeast cells to be encapsulated into the resultant microgels with cell viabilities of approximately 30%.

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Protein localization data are a valuable information resource helpful in elucidating eukaryotic protein function. Here, we report the first proteome-scale analysis of protein localization within any eukaryote. Using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis, we have epitope-tagged 60% of the Saccharomyces cerevisiae proteome.

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