SCO-pH: Microfluidic dynamic phenotyping platform for high-throughput screening of single cell acidification.

Studies on the dynamics of single cell phenotyping have been hampered by the lack of quantitative high-throughput metabolism assays. Extracellular acidification, a prominent phenotype, yields significant insights into cellular metabolism, including tumorigenicity. Here, we develop a versatile microfluidic system for single cell optical pH analysis (SCO-pH), which compartmentalizes single cells in 140-pL droplets and immobilizes approximately 40,000 droplets in a two-dimensional array for temporal extracellular pH analysis. SCO-pH distinguishes cells undergoing hyperglycolysis induced by oligomycin A from untreated cells by monitoring their extracellular acidification. To facilitate pH sensing in each droplet, we encapsulate a cell-impermeable pH probe whose fluorescence intensities are quantified. Using this approach, we can differentiate hyperglycolytic cells and concurrently observe single cell heterogeneity in extracellular acidification dynamics. This high-throughput system will be useful in applications that require dynamic phenotyping of single cells with significant heterogeneity.

Static array of droplets and on-demand recovery for biological assays.

Microfluidics has revolutionized several research areas by providing compact yet powerful microanalytical devices that in many cases outperform conventional systems. Among different microfluidics technologies, droplet microfluidics has emerged as a powerful platform to enable analyses of biological samples and phenomena because of its simplicity and versatility. Droplet microfluidics enables high-throughput encapsulation, manipulation, and analysis of single cells while drastically reducing the cost and time required by conventional technologies. For many of these microanalysis systems, manipulation of individual droplets is extremely important as it enables multiplexed high dimensional phenotyping of the targets, going beyond surface phenotyping. One of the key manipulation steps that needs to be implemented with high precision is enabling long-term observation of droplets and recovery of a subset of these droplets for further analysis. This Perspective highlights the recent advances and provides an outlook on future developments that will enable highly complex analyses of biological samples.

Photoactivated Selective Release of Droplets from Microwell Arrays.

Droplet microfluidics has enabled a significant reduction in reaction volume and analysis time, which in turn has led to transformative advances in high-capacity screening and assays. By arranging droplets into a static array, it is possible to monitor dynamic events that occur within these microchambers over an extended period of time, facilitating the identification of rare events and cell types. In many instances, it is highly desirable to recover a small number of droplets that contain unique analytes or cells for further analyses; however, few techniques allow for selective recovery of droplets from such an array without using a complex network of physical valves, which also require a large number of control units external to the microfluidic device. In this report, we present photoactivated selective release of droplets from a static microwell array enabled by a photoresponsive polymer layer integrated into the microfluidic device. This photoresponsive layer is placed in between a microwell array that traps a large number of droplets and a PDMS slab with or without a top flow channel that can be used for recovery. By using focused light, the photoresponsive layer can either be punctured for release-up recovery or induced to create a bubble by local heating to selectively push-down release droplets. We show that the photoresponsive layer is optically transparent within the visible spectrum and thus does not interfere with optical observation of droplets. The type of photoacoustic dye and the physical properties of the photoresponsive layer can be engineered to induce either puncture of the photoresponsive layer or pushing of droplets out of the microwell arrays with low thermal impact on the droplets. We believe that the photoresponsive layer will have a broad impact in the field of soft lithography-based microfluidic devices for various applications including photoresponsive valves as well as high-throughput single-cell sequencing.