Abstract
Wider application of single-cell analysis has been limited by the lack of an easy-to-use and low-cost strategy for single-cell isolation that can be directly coupled to single-cell sequencing and single-cell cultivation, especially for small-size microbes. Herein, a facile droplet microfluidic platform was developed to dispense individual microbial cells into conventional standard containers for downstream analysis. Functional parts for cell encapsulation, droplet inspection and sorting, as well as a chip-to-tube capillary interface were integrated on one single chip with simple architecture, and control of the droplet sorting was achieved by a low-cost solenoid microvalve. Using microalgal and yeast cells as models, single-cell isolation success rate of over 90% and single-cell cultivation success rate of 80% were demonstrated. We further showed that the individual cells isolated can be used in high-quality DNA and RNA analyses at both gene-specific and whole-genome levels (i.e. real-time quantitative PCR and genome sequencing). The simplicity and reliability of the method should improve accessibility of single-cell analysis and facilitate its wider application in microbiology researches.
Highlights
Wider application of single-cell analysis has been limited by the lack of an easy-to-use and low-cost strategy for single-cell isolation that can be directly coupled to single-cell sequencing and singlecell cultivation, especially for small-size microbes
The droplet-based microfluidic chip consists of four functional units (Fig. 1a): (i) cell encapsulation in water-in-oil droplet by “T-junction” configuration, (ii) droplet deceleration by a branch-channel structure and inspection under microscope, (iii) single-cell droplet sorting by solenoid valve suction, and (iv) exporting of single-cell droplet into a tube via an embedded capillary interface
Cells were pumped into the chip through inlet hole 1, while oil through inlet holes 2 and 3; droplet sorting was realized by the valve through inlet hole 4; wastes were exported through outlet hole 5, and the selected single-cell droplets were exported through the capillary interface
Summary
Wider application of single-cell analysis has been limited by the lack of an easy-to-use and low-cost strategy for single-cell isolation that can be directly coupled to single-cell sequencing and singlecell cultivation, especially for small-size microbes. Serial dilution[4] and micro-pipetting[5] methods were used in early single-cell studies with the advantages of being cheap and easy to perform, they usually suffer greatly from being imprecise, hard to validate and prone to DNA contamination. More automated methods such as optical/magnetic tweezers[6] Raman-activated cell sorting (RACS)[7] and fluorescence-activated cell sorting (FACS)[8] require expensive instruments that are equipped with laser beam, force clamp or fluorescence flow cytometer, which limits their wider applications. Single-cell trapping systems based on on-chip valves and microchambers www.nature.com/scientificreports/
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