Microfluidic step emulsification passively produces highly monodisperse droplets and can be easily parallelized for high throughput emulsion production. The two main techniques used for step emulsification are: i) edge-based droplet generation (EDGE), where droplets are formed in a single, very wide and shallow nozzle, and ii) microchannel emulsification (MCE), where droplets are formed in many separated narrow nozzles. These techniques differ in modes of droplet formation that influence the throughput and monodispersity of produced emulsions. Here we report a systematic study of novel grooved step emulsifying geometries, a hybrid of MCE and EDGE architectures. We introduce partitions of different heights to a wide (EDGE-like) slit to establish optimal geometries for high-throughput droplet production. We demonstrate that the volume and monodispersity of the produced emulsion can be tuned solely by changing the height of these partitions. We show that the spacing of the partitions influences the size of the produced droplets, but not the population monodispersity. We also determine the moment of transition between two distinct droplet generation modes as a function of the geometrical parameters of the nozzle. The optimized grooved geometry appears to combine the advantages of both MCE and EDGE, i.e. spatial localization of droplet forming units (DFUs), high-throughput formation of tightly monodisperse droplets from parallel DFUs, and low sensitivity to variation in the flow rate of the dispersed phase. As a proof-of-concept we show grooved devices that for a 260-fold increase of flow rate produce droplets with volume increased by just 75%, as compared to 91% increase in volume over a 180-fold increase of flow rate of the dispersed phase in MCE devices. We also present the optimum microfluidic device geometry that almost doubles the throughput of an MCE device in the generation of nanoliter droplets.

In microfluidic step emulsification, the size of droplets generated in the dripping regime is predominantly determined by the nozzle's height and only weakly depends on the applied flow rates or liquid properties. While the generation of monodisperse emulsions at high throughput using step emulsifiers has been well established, the generation of double emulsions, i.e., liquid core–shell structures, is still challenging. Here, we demonstrate a novel double-step emulsification method for the direct generation of multi-core double-emulsions and provide a predictive model for the number of cores. While the mechanism of the formation of the core droplets or empty shell droplets follows the well-established scenario of simple step emulsification, the formation of double-emulsion droplets is strongly affected by the presence of the cores. Passing of the cores through the narrowing neck of the shell postpones shell pinch-off. In particular, we demonstrate that our system can be used for the generation of arbitrary large, tightly packed droplet clusters consisting of a controllable number of droplets. Finally, we discuss the options of upscaling the system for high-throughput generation of tailored double emulsions.

We demonstrate detection and quantification of bacterial load with a novel microfluidic one-pot wash-free fluorescence in situ hybridization (FISH) assay in droplets. The method offers minimal manual workload by only requiring mixing of the sample with reagents and loading it into a microfluidic cartridge. By centrifugal microfluidic step emulsification, our method partitioned the sample into 210 pL (73 µm in diameter) droplets for bacterial encapsulation followed by in situ permeabilization, hybridization, and signal detection. Employing locked nucleic acid (LNA)/DNA molecular beacons (LNA/DNA MBs) and NaCl-urea based hybridization buffer, the assay was characterized with Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis. The assay performed with single-cell sensitivity, a 4-log dynamic range from a lower limit of quantification (LLOQ) at ~3 × 10³ bacteria/mL to an upper limit of quantification (ULOQ) at ~3 × 10⁷ bacteria/mL, anda linearity R² = 0.976. The total time-to-results for detection and quantification was around 1.5 hours.

We present a centrifugal microfluidic cartridge for the eight-fold parallel generation of monodisperse water-in-oil droplets using standard laboratory equipment. The key element is interfacing centrifugal microfluidics with its design based on polar coordinates to the linear structures of standard high-throughput laboratory automation. Centrifugal step emulsification is used to simultaneously generate droplets from eight samples directly into standard 200 μl PCR 8-tube strips. To ensure minimal manual liquid handling, the design of the inlets allows the user to load the samples and the oil via a standard multichannel pipette. Simulation-based design of the cartridge ensures that the performance is consistent in each droplet generation unit despite the varying radial positions that originate from the interface to the linear oriented PCR 8-tube strip and from the integration of linear oriented inlet holes for the multichannel pipettes. Within 10 minutes, sample volumes of 50 μl per droplet generation unit are emulsified at a fixed rotation speed of 960 rpm into 1.47 × 10⁵ monodisperse droplets with a mean diameter of 86 μm. The overall coefficient of variation (CV) of the droplet diameter was below 4%. Feasibility is demonstrated by an exemplary digital droplet polymerase chain reaction (ddPCR) assay which showed high linearity (R² ≥ 0.999) across all of the eight tubes of the strip.

A centrifugal microfluidic disk with four-scale partitioning (60–0.48 nL) enables a compact layout and broad dynamic range, while integrated vapor-based pressurization (∼70 kPa) preserves partitions during thermal cycling without external hardware.