Development of a facile droplet-based single-cell isolation platform for cultivation and genomic analysis in microorganisms

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.

Figure 1. The droplet microfluidic platform for single-cell dispensing.

(a) Schematics of the platform and single-cell isolation process including (i) cell encapsulation, (ii) droplet deceleration, (iii) sorting of single-cell droplets, and (iv) export of single-cell droplets into tubes. (b) Photo of the integrated microfluidic platform including the chip, syringe pumps, a microscope, a NI controlling board, a solenoid valve, and cell collecting tubes.

Figure 2. The process of single-cell droplet isolation under microscope.

(a) Droplet generation at the “T junction” of the microchannels. (b) Droplet deceleration at the inspection zone. (c) On-demand droplet sorting by the solenoid valve. (d) Export of single-cell droplets through the dispensing channel. Photos were taken with time intervals 0-0.92-1.2 s, 0-0.7-1.4 s, 0-0.2-0.4 s and 0-0.24-0.44 s for (a), (b), (c), (d) respectively. Each photo showed roughly the whole microscope field of this step respectively.

Figure 3. Evaluation of the single-cell droplet isolation.

(a) Each sorted droplet was dispensed on a glass slide and the number of cells in each droplet was counted under microscope. Single cells were marked with red circles; droplets without cells were marked with blue-dotted boxes. Size and shape of droplets varied due to their expansion on the glass slide. One of the triplicates was shown. (b) The ratio of single-cell droplets (gray block) compared with the theoretical ratio of single-cell droplets when formed at the “T-junction” of the microchannels (λ = 0.3, black triangle) and the upper limit of single-cell droplets (λ = 1, black circle). (c) Result of the single-cell cultivation experiment. S: single-cell samples; N: blank droplet. (d) Microscopic images of P. rhodozyma cells from one of the tubes in (c).

Figure 4. Results of the single-cell gene-specific analyses.

(a) Amplification curves of single-cell droplet samples and standard DNA. The x axis represents PCR cycle number and the y axis represents fluorescence intensity. Only samples with C_T value < 60 were shown. (b) Melting curves of these samples. The x axis represents melting temperatures and the y axis represents the −ΔF/ΔT (change in fluorescence/change in temperature). Only samples with C_T value < 60 were shown. (c) Linear fitting of log transformed DNA concentrations vs. C_T values using standard DNA samples (dots). The single-cell droplet samples (squares) were plotted on the fitting curve by C_T values. (d) Bioanalyzer electropherograms of a representative cDNA sample, showing the size distribution of cDNA molecules. (e) 1.2% agarose gel electrophoresis of the partial C. reinhardtii 18S rRNA gene PCR products. L: DNA ladder; 1–10: ten single-cell droplet samples. Samples 6 and 9 failed to show positive amplicon in either RT-PCR or 18S rRNA gene targeted PCR.

Figure 5. Results of single-cell genomic DNA amplification and sequencing.

(a) 0.8% agarose gel electrophoresis of MDA products. L: DNA ladder; 1–3: three representative single-cell droplet samples; B: blank droplets. (b) The Circleator figure of reads aligned to the S. cerevisiae S288c genome. From outside to inside: coordinate labels of the S288c genome; forward and reverse strand genes of the S288c genome; percent GC content of the sequencing reads of the single-cell sample (shown in red); read coverage of the single-cell sample (shown in blue). The chromosome name, NCBI accession and size of the chromosome were specified in the center. Only the Chromosome III of the yeast was shown here, as an example (additional details in the Supplementary Information).