Highly sensitive and portable digital microfluidics for on-site quantitative analysis of soil microbial counts

Precise quantification of soil microorganisms is essential for understanding their ecological functions. However, conventional culture-based methods often produce biased results because most soil microbes are non-culturable. Although digital PCR (dPCR) enables absolute quantification, droplet-based dPCR (ddPCR) requires complex fluidic control systems, limiting its portability and applicability for on-site analysis. To address these limitations, we developed a simplified dPCR chip and detection system without the need for droplet generation devices. Autonomous sample partitioning was achieved via capillary action, eliminating the need for auxiliary components such as constant-pressure pumps and thereby reducing system complexity and cost. An aluminum alloy thermal cycling chamber with high thermal conductivity enabled efficient temperature control, allowing PCR amplification and fluorescence detection to be completed within approximately 1.5 h. Using Bacillus subtilis 168 as a model organism, the platform demonstrated good quantitative linearity over the range of 10²-10⁵ CFU/mL (R² = 0.996) and proved capable of reliably detecting target concentrations as low as 10² CFU/mL. The system was further validated using real soil samples, including black soil, fluvo-aquic soil, and paddy soil, yielding bacterial abundances of (1.54 ± 0.49) × 10³ CFU/g, (1.49 ± 0.49) × 10³ CFU/g, and (0.85 ± 0.25) × 10³ CFU/g, respectively. This platform provides a practical approach for rapid and sensitive on-site quantification of soil microorganisms and supports the transition from laboratory-based analyses to in situ field monitoring.