Accurate and Microfluidics-Free Digital Nucleic Acid Quantification with Crescent Microbeads-Templated Emulsions

Droplet digital polymerase chain reaction (ddPCR) enables the absolute quantification of nucleic acids by counting individual molecules, obviating the need for standard curves and endogenous controls. However, existing ddPCR methods heavily depend on complex microfluidic setups and skilled operators, while alternative non-microfluidic dependent methods face challenges with limited reaction volume and inconsistent droplet sizes. Here, we present an accurate droplet digital quantification strategy utilizing a crescent microbead-based microfluidics-free droplet generation setting. The crescent microbeads were produced by leveraging the aqueous two-phase system (ATPS) principle and demonstrated high size uniformity. Following the optimization of aqueous surfactant types, pipetting cycles, and washing procedures, we produced high-quality crescent microbead-laden droplets utilizing a pipetting-based droplet-making method, which were consistently 2.7 μm larger than the crescent microbeads alone. The crescent microbeads strategy facilitates precise quantification of DNA gradient dilution samples, achieving accuracy performance comparable to commercial ddPCR systems, and demonstrates an overall 57.3% positive ratio quantification performance improvement over the spherical microbeads quantification strategy. Additionally, we validated this platform by accurately quantifying synthetic KRAS gene mutations and clinical HPV samples. The crescent microbead-based digital quantification strategy significantly enhances the accessibility and applicability of ddPCR in resource-constrained environments, potentially transforming nucleic acid diagnostics by overcoming current technological and financial barriers.