Self-Interference Digital Optofluidic Genotyping for Integrated and Automated Label-Free Pathogen Detection

Pathogen, prevalent in both natural and human environments, cause approximately 4.95 million deaths annually, ranking them among the top contributors to global mortality. Traditional pathogen detection methods, reliant on microscopy and cultivation, are slow and labor-intensive and often produce subjective results. While nucleic acid amplification techniques such as polymerase chain reaction offer genetic accuracy, they necessitate costly laboratory equipment and skilled personnel. Consequently, isothermal amplification methods like recombinase polymerase amplification (RPA) have attracted interest for their rapid and straightforward operations. However, these methods face challenges in specificity and automated sample processing. In this study, we introduce a self-interferometric digital optofluidic platform incorporating asymmetric direct solid-phase RPA for real-time, label-free, and automated pathogen genotyping. By integration of digital microfluidics with a DNA monolayer detection method using hyperspectral interferometry, this platform enables rapid, specific, and sensitive pathogen detection without the need for exogenous labeling or complex procedures. The system demonstrated high sensitivity (10 CFU·mL^-1), specificity (differentiating four <i>Candida</i> species), detection efficiency (fully automated within 50 min for Gram-negative bacteria), and throughput (simultaneous detection of four indices). This integrated approach to pathogen quantitation on a single microfluidic chip represents a significant advancement in rapid pathogen diagnostics, providing a practical solution for timely pathogen detection and analysis.