Tunable 3D droplet self-assembly for ultra-high-density digital micro-reactor arrays

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.