Influence of microfabrication on digital PCR performance in bead-based microwell array assays

Digital PCR (dPCR) is a highly sensitive analytical technique used to quantify DNA targets. Detection sensitivity can be further enhanced by capturing target sequences onto beads for preconcentration and sample cleanup prior to analysis in microfluidic microwell arrays. However, robust digital analysis requires individual beads to be interrogated within individual wells. Fabricating microwells with dimensions ≤ 3 μm is challenging, and the high surface area-to-volume ratio of the wells leaves PCR susceptible to inhibition stemming from materials used during device processing. This report describes the development of a microfabrication procedure to create ultralow-volume wells (100 fL) for bead-based dPCR and characterize the effects of microprocessing materials on assay performance. Standard microfabrication protocols used for creating microelectronics resulted in devices with nanoscopic debris originating from photoresists used during processing. A model dPCR assay was developed to characterize the effects of this debris, which revealed variable PCR inhibition. Debris within microwells attenuated digital and analog assay signals to a greater extent than debris on the device surface. Spatial heterogeneity of debris across devices was quantified to characterize regional PCR inhibition and intra- and inter-device variability. Ultimately, a fabrication procedure was developed to create pristine microfluidic arrays using dual processes to remove positive resist and forgoing use of negative resist entirely, which enabled robust amplification with digital signals matching theoretical predictions. Results from this work catalog the unique performance artifacts from device microfabrication and provide a guide for future studies seeking to conduct robust, high-sensitivity bead-based dPCR assays.