picoinjection-of-microfluidic-drops-without-metal-electrodes

Picoinjection of Microfluidic Drops Without Metal Electrodes

Existing methods for picoinjecting reagents into microfluidic drops require metal electrodes integrated into the microfluidic chip. The integration of ...

Unversity of California, San Francisco

The ability to add reagents to drops in a sequential fashion is necessary for numerous applications of microfluidics in biology. An important method for accomplishing this is picoinjection, a technique in which reagents are injected into aqueous drops using an electric field. While picoinjection has been shown to allow the precise addition of reagents to drops, its compatibility with biological reactions is yet to be thoroughly demonstrated. Here, we investigate the compatibility of picoinjection with digital RT-PCR Taqman assays, reactions that incorporate nucleic acids, enzymes, and other common biological reagents. We find that picoinjection is compatible with this assay and enables the detection of RNA transcripts at rates comparable to workflows not incorporating picoinjection. We also find that picoinjection results in negligible transfer of material between drops and that the drops faithfully retain their compartmentalization.

Picoinjection enables digital detection of RNA with droplet rt-PCR.

The behaviors of complex biological systems are often dictated by the properties of their heterogeneous and sometimes rare cellular constituents. Correspondingly, the analysis of individual cells from a heterogeneous population can reveal information not obtainable by ensemble measurements. Reverse-transcriptase polymerase chain reaction (RT-PCR) is a widely used method that enables transcriptional profiling and sequencing analysis on bulk populations of cells. Major barriers to successfully implementing this technique for mammalian single-cell studies are the labor, cost, and low-throughput associated with current approaches. In this report, we describe a novel droplet-based microfluidic system for performing ~50000 single-cell RT-PCR reactions in a single experiment while consuming a minimal amount of reagent. Using cell type-specific staining and TaqMan RT-PCR probes, we demonstrate the identification of specific cells from a mixed human cell population. The throughput, robust detection rate and specificity of this method makes it well-suited for characterizing large, heterogeneous populations of cells at the transcriptional level.

Ultrahigh-throughput Mammalian single-cell reverse-transcriptase polymerase chain reaction in microfluidic drops.

We present a method to add reagent to microfluidic droplets by enveloping them as a double emulsions in reagent-filled droplets and then rupturing them with an electric field. When the double emulsions rupture, they release their contents into the enveloping droplets, ensuring mixing with reagent while limiting cross-droplet contamination.

Adding reagent to droplets with controlled rupture of encapsulated double emulsions.

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.

Article	Year
Picoinjection enables digital detection of RNA with droplet rt-PCR. PloS one\| PubMed ID: 23658657	2013
Ultrahigh-throughput Mammalian single-cell reverse-transcriptase polymerase chain reaction in microfluidic drops. Analytical chemistry\| PubMed ID: 23885761	2013
Adding reagent to droplets with controlled rupture of encapsulated double emulsions. Biomicrofluidics\| PubMed ID: 24404045	2013
Generating electric fields in PDMS microfluidic devices with salt water electrodes. Lab on a chip\| PubMed ID: 24671446	2014

Adam Sciambi

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