A Fluorescence-Activated Single-Droplet Dispenser for High Accuracy Single-Droplet and Single-Cell Sorting and Dispensing

Abstract

The ability to sort and dispense droplets accurately is essential to droplet-based single-cell analysis. Here we describe a fluorescence-activated single-droplet dispenser (FASD) that is analogous to a conventional fluorescence-activated cell sorter, but sorts droplets containing single cells within an oil emulsion. The FASD system uses cytometric detection and electrohydrodynamic actuation-based single-droplet manipulation, allowing droplet isolation and dispensing with high efficiency and accuracy. The system is compatible with multi-well plates and can be integrated with existing microfluidic devices and large-scale screening systems. By enabling sorting based on single-cell reactions such as PCR, this platform will help expand the basis of cell sorting from mainly protein biomarkers to nucleic acid sequences and secreted biomolecules.

Capillary-Mediated Single-Cell Dispenser

“…The challenge is to generate and manipulate droplets at a volume that minimizes the effect of evaporation while efficiently separating single cells. We addressed this challenge previously 29 by constructing a dropon-demand single-cell dispenser (Scheme 1) inspired by electrohydrodynamic jet (e-jet) printing. 32,33 Here, we switch to aqueous-only droplets to improve assay compatibility and optimize the performance of the single-cell dispenser with a combination of simulations and experimentation.…”

“…We previously built a fluorescence-activated single-droplet dispenser to isolate onchip-generated double-emulsion (water-in-oil) droplets containing single cells based on fluorescence signals and dispense the droplets with nanoliter precision into multiwell plates for analysis. 29 Here, we extend this system by switching to allaqueous droplet generation (no oil emulsion) to improve assay compatibility and by systematically optimizing critical parameters including capillary tip diameter, flow rate, and applied voltage using computational simulations and experimentation to achieve accurate drop generation and efficient single-cell manipulation, sorting, and dispensing. We demonstrate an application of the system by sorting and dispensing single cells from rare cell subpopulations into multiwell plates for singlecell PCR analysis.…”

Acoustofluidic Droplet Sorter Based on Single Phase Focused Transducers

“…Compared to the regular transducer design, the SPFT could focus more energy to the focal point, allowing it to generate a much stronger standing SAW field with the same input power. In contrast to the recently released sorting strategy, [ 33,65–68 ] the SPFT based sorter is able to conduct precise cell‐laden droplets under a low input pulse, and still maintains a high sorting purity of up to 99.2%. Overall, the SPFT based droplet sorter is able to continuously perform accurate droplet sorting with a throughput as high as 1187 events per second.…”

Advances in single‐cell sequencing and its application to musculoskeletal system research

“…The initial two methods for single‐cell separation, continuous dilution and micromanipulation, both had the disadvantages of low throughput and cell susceptibility to mechanical damage. More recent cell separation methods have included magnetic activated cell sorting (MACS), 47 fluorescence activated cell sorting (FACS) 48 and microfluidics. 49 Notably, microfluidics involves fewer step and has the advantages of high analytical sensitivity and specificity as well as high throughput.…”