Development of a non-transfer microdroplet generation chip and digital molecular detection system†

Abstract

Droplet digital PCR (ddPCR) is a transformative technology for nucleic acid detection, offering exceptional sensitivity and absolute quantification. In this study, we developed a non-transfer microdroplet generation chip based on a cross-junction flow-focusing mechanism, enabling precise production of uniformly sized water-in-oil droplets. This chip integrates droplet generation and PCR amplification in a closed workflow, eliminating manual transfer steps and reducing contamination risks. Combined with a custom fluorescence reader, the system achieves a dynamic range of 1–10⁴ copies per μL with high reproducibility (intra-/inter-batch CV <10%).Comparative analysis with the Bio-Rad QX200 system using ALB4 DNA standards revealed comparable accuracy (recovery rates: 95–129% vs. 95–131%) and linearity (R² = 0.998 vs. 0.999). Critically, our platform offers three key advantages: (1) cost-effectiveness (lower chip production costs), (2) elimination of transfer steps: (avoids a major source of contamination and droplet loss), and (3) simplified workflow (reduction in hands-on time). These innovations position the system as a practical solution for applications demanding reliable low-abundance nucleic acid detection, such as liquid biopsy and pathogen surveillance.