Digital Polymerase Chain Reaction (dPCR) is a novel method for the absolute quantification of target nucleic acids. Quantification by dPCR hinges on the fact that the random distribution of molecules in many partitions follows a Poisson distribution. Each partition acts as an individual PCR microreactor and partitions containing amplified target sequences are detected by fluorescence. The proportion of PCR-positive partitions suffices to determine the concentration of the target sequence without a need for calibration. Advances in microfluidics enabled the current revolution of digital quantification by providing efficient partitioning methods. In this review, we compare the fundamental concepts behind the quantification of nucleic acids by dPCR and quantitative real-time PCR (qPCR). We detail the underlying statistics of dPCR and explain how it defines its precision and performance metrics. We review the different microfluidic digital PCR formats, present their underlying physical principles, and analyze the technological evolution of dPCR platforms. We present the novel multiplexing strategies enabled by dPCR and examine how isothermal amplification could be an alternative to PCR in digital assays. Finally, we determine whether the theoretical advantages of dPCR over qPCR hold true by perusing studies that directly compare assays implemented with both methods.

The evolution of carbon dioxide bubbles dissolving in water is experimentally examined using long microchannels. We study the coupling between bubble hydrodynamics and dissolution in confined geometries. The gas impregnation process in liquid produces significant flow rearrangements. Depending on the initial volumetric liquid fraction, three operating regimes are identified, namely saturating, coalescing, and dissolving. The morphological and dynamical transition from segmented to dilute bubbly flows is investigated. Tracking individual bubbles along the flow direction is used to calculate the temporal evolution of the liquid volumetric fraction and the average flow velocity near reference bubbles over long distances. This method allows us to empirically establish the functional relationship between bubble size and velocity. Finally, we examine the implication of this relationship during the coalescing flow regime, which limits the efficiency of the dissolution process.

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We present a novel method for conducting true single-cell encapsulation at very high efficiency for the manipulation of precious samples. Our unique strategy is based on the sequential capture and original encapsulation of single-cells into a series of hydrodynamic traps. We identified two distinct modes of encapsulation and we established their associated design rules. We improved the trapping scheme to reach a near perfect capture efficiency and make it compatible with the encapsulation process. Finally, we developed the complete device operation that permits highly efficient single-cell encapsulation and droplet retrieval. This platform provides the foundation to a fully integrated multiparameter platform that will impact the analysis of tissues at single-cell resolution.

We examine the initial dynamical behavior of dissolving microbubbles composed of carbon dioxide gas in highly viscous silicone oils over a range of flow rates and pressure conditions. Microfluidic periodic trains of CO(2) bubbles are used to probe the interrelation between bubble dissolution and high-viscosity multiphase flows in microgeometries. We investigate bubble morphology from low to large capillary numbers and calculate the effective mass diffusion flux across the interface by tracking and monitoring individual bubbles during shrinkage. The initial flux is characterized using a dissolution coefficient that reveals the influence of the oil molecular weight on the dissolution process. Our findings show the possibility to control and exploit the interplay between capillary and mass transfer phenomena with highly viscous fluids in small-scale systems.

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The negative tone photoresist SU-8 permits the creation of micrometer-scale structures by optical lithography. It is also the most used photoresist in soft lithography for the fast-prototyping of microfluidic devices. Despite its importance, the effect of capillary forces on SU-8 multi-layering onto topographical features has not been thoroughly studied. In particular, the profile of the added layer has not been examined in detail. The overlaying process exhibits a set of distinct behaviors, or regimes, depending on the relative thickness of the overlay and the underlying rectangular pattern. We demonstrate how capillary effects control the profile of multi-layer microchannels in a predictable manner. We derive a simple static model to describe the evolution of the overlay as a function of dimensionless geometric parameters. Our study provides a critical understanding of the parameters that govern multi-layer spin coating.