High-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Synthesis of biocompatible surfactants and identification of gas-permeable storage systems allowed human cells, and even a multicellular organism (C. elegans), to survive and proliferate within the microcompartments for several days. Microcompartments containing single cells could be reinjected into a microfluidic device after incubation to measure expression of a reporter gene. This should open the way for high-throughput, cell-based screening that can use >1000-fold smaller assay volumes and has approximately 500x higher throughput than conventional microtiter plate assays.

Drops of water-in-fluorocarbon emulsions have great potential for compartmentalizing both in vitro and in vivo biological systems; however, surfactants to stabilize such emulsions are scarce. Here we present a novel class of fluorosurfactants that we synthesize by coupling oligomeric perfluorinated polyethers (PFPE) with polyethyleneglycol (PEG). We demonstrate that these block copolymer surfactants stabilize water-in-fluorocarbon oil emulsions during all necessary steps of a drop-based experiment including drop formation, incubation, and reinjection into a second microfluidic device. Furthermore, we show that aqueous drops stabilized with these surfactants can be used for in vitro translation (IVT), as well as encapsulation and incubation of single cells. The compatability of this emulsion system with both biological systems and polydimethylsiloxane (PDMS) microfluidic devices makes these surfactants ideal for a broad range of high-throughput, drop-based applications.

Nonlinearity in finite-Reynolds-number flow results in particle migration transverse to fluid streamlines, producing the well-known “tubular pinch effect” in cylindrical pipes. Here we investigate these nonlinear effects in highly confined systems where the particle size approaches the channel dimensions. Experimental and numerical results reveal distinctive dynamics, including complex scaling of lift forces with channel and particle geometry. The unique behavior described in this Letter has broad implications for confined particulate flows.

Encapsulation of cells within picoliter-size monodisperse drops provides new means to perform quantitative biological studies on a single-cell basis for large cell populations. Variability in the number of cells per drop due to stochastic cell loading is a major barrier to these techniques. We overcome this limitation by evenly spacing cells as they travel within a high aspect-ratio microchannel; cells enter the drop generator with the frequency of drop formation.While drop-based microfluidics 1,2 promises breakthrough applications in biotechnology such as directed evolution, 3 tissue printing 4 and bead-based PCR in emulsions, 5 it also facilitates many quantitative studies of biology at the most fundamental level, that of single cells. Because each cell can be made to reside within its own picoliter-volume drop, chemically isolated from all other drops, cell-secreted molecules rapidly achieve detectable concentrations in the confined fluid surrounding the encapsulated cell. Similarly, uptake of trace chemicals specific to individual cells can be probed due to their depletion within the confined extracellular fluid. Moreover, highly monodisperse drops of water in an inert and immiscible carrier fluid can be formed at rates of several kHz using microfluidic techniques, 6 and these drops can be pairwise combined, 7 split in two, 8 and selected based on the contents of individual drops. 9,10 Despite their great potential, studies of single cells in drops suffer from an intrinsic limitation; because the process of loading cells into drops is purely random, the distribution is dictated by Poisson statistics. Thus, the probability of a drop containing k cells is λ k exp(−λ) / k!, where λ is the average number of cells per drop, so the ratio of drops containing one cell to those containing two is 2/λ. This means that to minimize the number of drops that contain more than a single cell requires very low average loading densities, meaning that most drops actually contain no cells whatsoever. This constraint significantly reduces the number of usable drops; for example, only 15.6 % of all drops will contain one cell if no more than one in ten of the occupied drops can be allowed to hold two or more cells.Recent work describes a method to passively sort drops containing single cells from smaller empty drops after each cell triggers its own encapsulation upon entering a narrow aqueous jet formed in a flow-focusing device. 11 This clever mechanism can also be used to sort cells based on their size since, for this system, drops are always slightly larger than the cell they contain; however, to overcome the inherent limitations of stochastic encapsulation of cells within controlled-size (monodisperse) drops, one (and only one) cell should be present whenever a drop is generated. This can be achieved manually for each drop, 12 or passively, and with a much higher throughput, by organizing cells in the direction of flow so that they enter the microfluidic nozzle with the same frequency at which drops form. * These autho...

Inertial focusing in a pressure-driven flow refers to the positioning of particles transverse to the mean flow direction that occurs as a consequence of a finite particle Reynolds number. In channels with rectangular cross-sections, and for a range of channel aspect ratios and particle confinement, experimental results are presented to show that both the location and the number of focusing positions depend on the number of particles per unit length along the channel. This axial number density is a function of both the channel cross-section and the particle volume fraction. These results are rationalized using simulations of the particle-laden flow to show the manner in which hydrodynamic interactions set the preferred locations in these confined flows. A criterion is presented for the occurrence of a stepwise transition from one to two or more trains of particles. At small but finite particle Reynolds numbers, particles in a well-established pipe flow migrate across streamlines to specific positions in the channel or tube cross-section. 1-8 Such focusing of particles is a consequence of inertia and was first observed in channels with a circular cross-section, where the particles migrate to an annular region approximately three-fifths of the radius from the center. 1 In this geometry, the underlying mechanisms have been studied extensively, and particle focusing is understood to arise from the force balance between a wall effect that pushes the particles toward the center of the channel and a shear-gradientinduced migration that pushes particles toward the boundary. 9-12 More recently, inertial focusing has been observed in channels with square and rectangular crosssections, where the particle size often approaches the dimensions of the channel cross-section. [6][7][8]13,14 Because this phenomenon localizes the particles to specific positions, it has been used for separation, filtration, and improved encapsulation efficiencies, and has the potential for incorporation into microfluidic lab-on-a-chip technologies. [6][7][8]15 While the focusing positions and particle ordering in these confined, rectangular cross-sections have been described [6][7][8]14 and the lift force on particles in square cross-section channels has been investigated, 13 questions remain. In particular, consequences of the combination of confining geometries, inertia, and particle concentration have not been characterized.Given the intense interest in the application of microfluidic approaches for manipulating particles and cells, there is a need to develop prediction methods based on understanding the basis of this inertially modulated ordering. To address this need, in this letter, we study the effects of particle concentration and channel geometry on inertial focusing in microfluidic channels with rectangular cross-sections. We find that both the location and the number of focusing positions depend on the number of particles per unit length along the channel, which is a function of both the channel crosssection and particle volume fraction. F...