Rotational Microfluidic Paper-Based Chip Platform Integrating Nucleic Acid Sequence-Based Amplification for Visual and Sensitive Detection of Viable Pathogens

Sensitive, simple, and rapid detection of pathogens is essential in preventing the transmission of related diseases and protecting human health. In this study, a rotational microfluidic paper-based analytical device integrating nucleic acid sequence-based amplification (NASBA) and colorimetric analysis was developed for the detection of viable pathogens. Biotin- and HRP (horseradish peroxidase)-labeled DNA single strands were used as the capture probe and detection probe, respectively. The capture probe was anchored on the surface of the paper chip based on the interaction between biotin and streptavidin. The target mRNA of the pathogen could be exponentially amplified through NASBA isothermal amplification, and then the product of amplification was combined with the capture probe and detection probe according to nucleic acid hybridization. With a simple and rapid RNA extraction method, isothermal amplification, and colorimetric analysis, this rotational microfluidic paper-based analytical platform could accomplish the whole process of pathogen detection within 110 min using simple equipment. The limit of detection for E. coli was as few as 10 CFU/mL (with a 95% positive detection rate in 20 replicate samples). The quantitative analysis of pathogens could be carried out by detecting the gray value on the paper chip with a smartphone. This portable platform has great potential for the on-site, rapid, and visual detection of viable pathogens such as bacteria and viruses.