Label-free recombinase polymerase amplification with hyperspectral digital optofluidics

Nucleic acid detection techniques have played a crucial role in identifying specific genetic indicators or species, with Polymerase Chain Reaction (PCR) being the established gold standard in this field. However, PCR's dependence on specialized equipment and skilled personnel has limited its utility in resource-limited or field settings, and its multitemperature stage protocol hinders rapid nucleic acid detection. The emergence of isothermal amplification methods, particularly recombinase polymerase amplification (RPA), has addressed some of these limitations, offering high sensitivity and efficiency. Nevertheless, the challenge of RPA amplicon detection, typically reliant on labeling methods, has persisted, potentially introducing false positives and increased costs. This study introduces an innovative approach to nucleic acid detection, harnessing hyperspectral quantitative interference for label-free, isothermal nucleic acid detection within a remarkably short 25-minute timeframe. By employing a solid-phase RPA amplification process that transforms the product into a DNA molecule layer, and leveraging Fourier domain optical slice separation and spectral phase shift analysis, this method enables the semi-quantitative determination of amplification results. The integration of digital microfluidic technology further enhances the method's performance, enabling parallel, integrated, and clinical multiindicator pathogen detection. Overall, this research presents a practical and rapid solution for label-free nucleic acid detection, addressing the current limitations associated with nucleic acid amplification techniques. This advancement holds promise for a wide range of applications, from point-of-care diagnostics to field-based pathogen detection, ultimately contributing to more accessible and efficient nucleic acid testing methodologies.