Permeability-Engineered Compartmentation System-Enabled Digital PCR (PECS-dPCR): A Digital Platform toward Multistep...

Droplet-based digital PCR has emerged as a powerful platform for nucleic acid-based detection. However, the formation of droplet compartments and the subsequent amplification process in oil present significant drawbacks: instability under harsh thermal conditions, high background fluorescent noise inside droplets, and major difficulty in supporting multistep assays. Alternatively, droplets made of a hydrogel, or other advanced materials, have been adopted and demonstrate promising improvement over conventional droplet-based platforms. In this context, we present permeability-engineered compartmentation system-enabled digital PCR (PECS-dPCR), a novel digital platform that facilitates multistep biomolecular assays with thermal stability, minimized background noise, and long-term preservation capability. We achieve compartmentalization by forming a core-shell structure using the aqueous two-phase system (ATPS). The hydrogel shell provides exceptional mechanical strength and thermal stability to these compartments. The permeability of the shell can be fine-tuned to retain larger DNA targets while sieving out smaller ancillary molecules. Therefore, we can significantly improve the signal-to-noise ratio inside the compartments by washing out fluorescent background. Furthermore, these core-shell compartments remain intact in aqueous solution and are able to exchange materials with the ambient environment. This critical feature offers the capability to execute multistep assays in simple operational settings, enabling the demonstration of multitarget single-bacteria quantification in our platform. We further show that the assay can be paused with samples preserved for >2 weeks between different detection steps thanks to the excellent biochemical stability offered by the core-shell compartments. We envision PECS-dPCR becoming a versatile platform supporting multiple-reaction-step digital assays, offering high-quality detection signals and long-term stability.