Capillary Flow Velocity-based Length Identification of PCR and RPA Products on Paper Microfluidic Chips

This work demonstrates a novel, non-fluorescence approach to the length identification of polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) products, utilizing capillary flow velocities on paper microfluidic chips. It required only a blank paper chip, aminated microspheres, and a smartphone, with a rapid assay time and under ambient lighting. A smartphone evaluated the initial capillary flow velocities on the paper chips for the PCR and RPA products from various bacterial samples, where the pre-loaded aminated microspheres differentiated their flow velocities. Flow velocities were analyzed at different time frames and compared with the instantaneous flow velocities and interfacial tension (γ) data. Subsequent error analysis justified the use of the initial time frames. A robust linear relationship could be established between the initial flow velocities against the square root of the product lengths, with R values of 0.981 for PCR and 0.993 for RPA. The assay seemed not to have a significant dependency on the cycle numbers and initial target concentrations. This novel method can be potentially used with various paper microfluidic methods of nucleic acid amplification tests towards rapid and handheld assays.