Generating electric fields in PDMS microfluidic devices with salt water electrodes

Sciambi, Adam; Abate, Adam R.

doi:10.1039/c4lc00078a

Cited by 91 publications

(82 citation statements)

References 25 publications

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“…1(b)]. These four channels connected at the high voltage amplifier deflected droplets of interest into the collection channels by electrophoresis2728. Our method was adapted from agarose gel liquid electrodes19.…”

Section: Resultsmentioning

confidence: 99%

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution

Girault¹,

Kim²,

Arakawa³

et al. 2017

Sci Rep

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A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.

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Section: Resultsmentioning

confidence: 99%

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution

Girault¹,

Kim²,

Arakawa³

et al. 2017

Sci Rep

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“…The resulting microchannels obtained in polydimethylsiloxane (PDMS) were plasma bonded on a thin cured PDMS layer (5 g PDMS in a Petri dish; Ø, 9 cm), flushed with 2% (vol/vol) Trichloro(1H,1H,2H,2H-perfluorooctyl)silane in HFE-7500, put in a 65°C oven for 30 min, and the PDMS-PDMS devices then bonded to a microscope glass slide. Electrodes were made by filling channels with salt solution (5 M NaCl) as a practical alternative to low-melting point metals (54). Optical fibers (SMA Fiber Patch Cable, 50 microns, 0.22 NA; Thorlabs) were stripped (so that only the cladding was left) and manually inserted into the chip under a microscope.…”

Section: Methodsmentioning

confidence: 99%

Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS)

Gielen

Hours

Emond

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

236

253

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Ultrahigh-throughput screening, in which members of enzyme libraries compartmentalized in water-in-oil emulsion droplets are assayed, has emerged as a powerful format for directed evolution and functional metagenomics but is currently limited to fluorescence readouts. Here we describe a highly efficient microfluidic absorbance-activated droplet sorter (AADS) that extends the range of assays amenable to this approach. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to 300 droplets per second (i.e., >1 million droplets per hour). To validate this device, we implemented a miniaturized coupled assay for NAD+-dependent amino acid dehydrogenases. The detection limit (10 μM in a coupled assay producing a formazan dye) enables accurate kinetic readouts sensitive enough to detect a minimum of 1,300 turnovers per enzyme molecule, expressed in a single cell, and released by lysis within a droplet. Sorting experiments showed that the AADS successfully enriched active variants up to 2,800-fold from an overwhelming majority of inactive ones at ∼100 Hz. To demonstrate the utility of this module for protein engineering, two rounds of directed evolution were performed to improve the activity of phenylalanine dehydrogenase toward its native substrate. Fourteen hits showed increased activity (improved >4.5-fold in lysate; kcat increased >2.7-fold), soluble protein expression levels (up 60%), and thermostability (Tm, 12 °C higher). The AADS module makes the most widely used optical detection format amenable to screens of unprecedented size, paving the way for the implementation of chromogenic assays in droplet microfluidics workflows.

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“…By default, all droplets flow through the sorting junction to OUT. If a droplet is determined to contain an active compound, a salt water electrode (4 M NaCl, orange) 47 delivers a high-voltage AC pulse to the sorting junction, generating an electric field that deflects the desired droplet toward HIT output (Figure 1C). 32 …”

Section: Resultsmentioning

confidence: 99%

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

2017

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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.

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Generating electric fields in PDMS microfluidic devices with salt water electrodes

Abstract: Droplet merging and sorting in microfluidic devices usually rely on electric fields generated by solid metal electrodes. We show that simpler and more reliable salt water electrodes, despite their lower conductivity, can perform the same droplet manipulations at the same voltages.

Cited by 91 publications

References 25 publications

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution

Ultrahigh-throughput–directed enzyme evolution by absorbance-activated droplet sorting (AADS)

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

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