Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.

Directed differentiation of human pluripotent stem cells to kidney organoids brings the prospect of drug screening, disease modelling and the generation of tissue for renal replacement. Currently, these applications are hampered by organoid variability, nephron immaturity, low throughput and limited scale. Here we apply extrusion-based 3D cellular bioprinting to deliver rapid and high throughput generation of kidney organoids with highly reproducible cell number and viability. We demonstrate that manual organoid generation can be replaced by 6- or 96-well organoid bioprinting and evaluate relative toxicity of aminoglycosides as a proof of concept for drug testing. In addition, 3D bioprinting enabled precise manipulation of biophysical properties including organoid size, cell number and conformation, with modification of organoid conformation substantially increasing nephron yield per starting cell number. This facilitated the manufacture of uniformly patterned kidney tissue sheets with functional proximal tubular segments. Hence, automated extrusion-based bioprinting for kidney organoid production deliver improvements in throughput, quality control, scale and structure, facilitating in vitro and in vivo applications of stem cell-derived human kidney tissue.

Electrophoretic separations carried out in capillary tubes offer the possibilities of rapid and automated analyses of small volumes of complex mixtures with unprecedented resolution and sensitivity. Some emerging developments of this new instrumentation are reviewed.

We present a tunable three-dimensional (3D) self-assembled droplet packing method to achieve high-density micro-reactor arrays for greater imaging efficiency and higher-throughput chemical and biological assays. We demonstrate the capability of this platform's high-density imaging method by performing single molecule quantification using digital polymerase chain reaction, or digital PCR, in multiple self-assembled colloid-like crystal lattice configurations. By controlling chamber height to droplet diameter ratios we predictively control three-dimensional packing configurations with varying degrees of droplet overlap to increase droplet density and imaging sensor area coverage efficiency. Fluorescence imaging of the densely packed 3D reactor arrays, up to three layers high, demonstrates high throughput quantitative analysis of single-molecule reactions. Now a greater number of microreactors can be observed and studied in a single picture frame without the need for confocal imaging, slide scanners, or complicated image processing techniques. Compared to 2D designs, tunable 3D reactor arrays yield up to a threefold increase in density and use 100% of the sensor's imaging area to enable simultaneous imaging a larger number of reactions without sacrificing digital quantification performance. This novel approach provides an important advancement for ultra-high-density reactor arrays.

The design of a simple, on-column connector for microcolumn separations Is described. The connector, In the form of a cross or tee, Is fabricated from the fused silica capillary tubing, Itself, and has sufficiently low dead volume to be compatible with the tubing dimensions normally associated with microcolumn separation techniques. As a demonstration of one possible application, a mixture of amino acids Is separated by capillary zone electrophoresis (CZE), the amino acids are derlvatlzed on-column with o-phthaldialdehyde (OPA) Introduced from a cross connector, and the highly fluorescent adducts are detected downstream by laser-induced fluorescence. Zone broadening by the connector Is determined to be approximately 10% for CZE separations performed In 75 pm l.d. capillary tubes. Detector response Is found to be linear over more than 3 orders of magnitude with minimum limits of detection In the subfemtomole range.