Permeability-Engineered\nCompartmentation System-Enabled\nDigital PCR (PECS-dPCR): A Digital Platform toward Multistep Biomolecular\nAssays

Droplet-based\ndigital PCR has emerged as a powerful platform\nfor\nnucleic acid–based detection. However, the formation of droplet\ncompartments and the subsequent amplification process in oil present\nsignificant drawbacks: instability under harsh thermal conditions,\nhigh background fluorescent noise inside droplets, and major difficulty\nin supporting multistep assays. Alternatively, droplets made of a\nhydrogel, or other advanced materials, have been adopted and demonstrate\npromising improvement over conventional droplet-based platforms. In\nthis context, we present permeability-engineered compartmentation\nsystem-enabled digital PCR (PECS-dPCR), a novel digital platform that\nfacilitates multistep biomolecular assays with thermal stability,\nminimized background noise, and long-term preservation capability.\nWe achieve compartmentalization by forming a core–shell structure\nusing the aqueous two-phase system (ATPS). The hydrogel shell provides\nexceptional mechanical strength and thermal stability to these compartments.\nThe permeability of the shell can be fine-tuned to retain larger DNA\ntargets while sieving out smaller ancillary molecules. Therefore,\nwe can significantly improve the signal-to-noise ratio inside the\ncompartments by washing out fluorescent background. Furthermore, these\ncore–shell compartments remain intact in aqueous solution and\nare able to exchange materials with the ambient environment. This\ncritical feature offers the capability to execute multistep assays\nin simple operational settings, enabling the demonstration of multitarget\nsingle-bacteria quantification in our platform. We further show that\nthe assay can be paused with samples preserved for >2 weeks between\ndifferent detection steps thanks to the excellent biochemical stability\noffered by the core–shell compartments. We envision PECS-dPCR\nbecoming a versatile platform supporting multiple-reaction-step digital\nassays, offering high-quality detection signals and long-term stability.