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Recent advances in transcriptomic analysis at single-cell resolution reveal cell-to-cell heterogeneity in a biological sample with unprecedented resolution. Partitioning single cells in individual micro-droplets and harvesting each cell's mRNA molecules for next-generation sequencing has proven to be an effective method for profiling transcriptomes from a large number of cells at high throughput. However, the assays to recover the full transcriptomes are time-consuming in sample preparation and require expensive reagents and sequencing cost. Many biomedical applications, such as pathogen detection, prefer highly sensitive, reliable and low-cost detection of selected genes. Here, we present a droplet-based microfluidic platform that permits seamless on-chip droplet sorting and merging, which enables completing multi-step reaction assays within a short time. By sequentially adding lysis buffers and reactant mixtures to micro-droplet reactors, we developed a novel workflow of single-cell reverse transcription loop-mediated-isothermal amplification (scRT-LAMP) to quantify specific mRNA expression levels in different cell types within one hour. Including single cell encapsulation, sorting, lysing, reactant addition, and quantitative mRNA detection, the fully on-chip workflow provides a rapid, robust, and high-throughput experimental approach for a wide variety of biomedical studies.
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Microfluidic on-demand droplet generation, storage, retrieval, and merging for single-cell pairing
A multifunctional microfluidic platform combining on-demand aqueous-phase droplet generation, multi-droplet storage, and controlled merging of droplets selected from a storage library in a single integrated microfluidic device is described. A unique aspect of the technology is a microfluidic trap design comprising a droplet trap chamber and lateral bypass channels integrated with a microvalve that supports the capture and merger of multiple droplets over a wide range of individual droplet sizes. A storage unit comprising an array of microfluidic traps operates in a first-in first-out manner, allowing droplets stored within the library to be analyzed before sequentially delivering selected droplets to a downstream merging zone, while shunting other droplets to waste. Performance of the microfluidic trap is investigated for variations in bypass/chamber hydrodynamic resistance ratio, micro-chamber geometry, trapped droplet volume, and overall flow rate. The integrated microfluidic platform is then utilized to demonstrate the operational steps necessary for cell-based assays requiring the isolation of defined cell populations with single cell resolution, including encapsulation of individual cells within an aqueous-phase droplet carrier, screening or incubation of the immobilized cell-encapsulated droplets, and generation of controlled combinations of individual cells through the sequential droplet merging process. Beyond its utility for cell analysis, the presented platform represents a versatile approach to robust droplet generation, storage, and merging for use in a wide range of droplet-based microfluidics applications.
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- Award ID(s):
- 1844299
- PAR ID:
- 10201837
- Date Published:
- Journal Name:
- Lab on a Chip
- Volume:
- 19
- Issue:
- 3
- ISSN:
- 1473-0197
- Page Range / eLocation ID:
- 493 to 502
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/c8lc01178h
