A programmable droplet-based microfluidic reaction array applied to multiparameter single cell analysis
Kaston Leung, Hans Zahn, Tim Leaver, and Carl Hansen
Centre for High-Throughput Biology, University of British Columbia
Microfluidic systems have been shown to provide numerous advantages for biological analysis including automation, enhanced reaction efficiency in nanoliter volumes, and cost-effective analysis of limited samples. Yet the application of microfluidics to biological research remains restricted to laboratories focused on device development. This is due to conventional design methodology which “hardwires” devices for specific fluid handling steps, necessitating a custom design and fabrication cycle for each application or change in assay protocol. Such design iterations require expensive equipment and specialized training for microfabrication, which are inaccessible to the typical biologist. A general, flexible, and user-programmable microfluidic platform would remove this barrier to widespread adoption of microfluidics by the general scientific community.
Here, we present a droplet-based programmable microfluidic reaction array, which combines the flow control of microvalves with the compartmentalization of droplets to enable programmable formulation of an array of nanoliter-scale reactions. Using integrated peristaltic pumping, user-defined reagent volumes are dispensed into droplets in an oil phase, which are directed to any one of 95 individually addressable storage chambers designed to merge all incoming droplets. The consecutive merging of user-defined sequences of droplets of different reagents allows for the assembly of any multistep reaction with fully programmable composition, timing, and volume. In addition, an integrated cell sorter, which allows for phenotypic sorting of single cells, enables execution of any single cell assay protocol. An integrated elution nozzle and a robotic interface permit automated elution of reaction products from individual chambers directly into standard microfuge tubes for further off-chip analysis.
We first evaluated the formulation accuracy of our device by generating a series of fluorescent dye concentrations, which were found to match target values (R2=0.999). We then demonstrated quantitative nucleic acid amplification with single molecule sensitivity and used this to quantify cross-contamination during loading and elution of individual chambers, which was found to be below 1 part in 1500 and 1 part in 480,000 respectively. We then demonstrated the utility of the device for single cell multiparameter analysis by phenotypically sorting single bacteria from a mixed suspension into chambers for monoclonal culture and two-strain co-culture, for genotyping by PCR assays, and for single-cell whole genome amplification (WGA). Next-generation sequencing of WGA product determined that our method obtained 99% genome coverage from a single Escherichia coli cell, suggesting excellent potential for the genetic analysis of uncultivated single microbes. We have applied these single-cell genetic analysis methods to a wide range of sample types including single microbes isolated from an oral biofilm and marine sediment, and single primary breast cancer cells. This device provides a general platform that can be programmed by end-users for numerous applications. In particular, we have demonstrated its broad utility for single cell genomics.
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