Overview and Future Perspectives of Microfluidic Digital Recombinase Polymerase Amplification (dRPA)

Digital recombinase polymerase amplification (dRPA) aims to quantify the initial amount of nucleic acid by dividing nucleic acid and all reagents required for the RPA reaction evenly into numerous individual reaction units, such as chambers or droplets. dRPA turns out to be a prominent technique for quantifying the absolute quantity of target nucleic acid because of its advantages including low equipment requirements, short time consumption, as well as high sensitivity and specificity. dRPA combined with microfluidics are recognized as simple, various, and high-throughput nucleic acid quantization systems. This paper classifies the microfluidic dRPA systems over the last decade. We analyze and summarize the vital technologies of various microfluidic dRPA systems (e.g., chip preparation process, segmentation principle, microfluidic control, and statistical analysis methods), and major efforts to address limitations (e.g., prevention of evaporation and contamination, accurate initiation, and reduction of manual operation). In addition, this paper summarizes key factors and potential constraints to the success of the microfluidic dRPA to help more researchers, and possible strategies to overcome the mentioned challenges. Lastly, actual suggestions and strategies are proposed for the subsequent development of microfluidic dRPA.