Fig. 1. Schematic diagram of overall structure and reaction of paper-based recombinant polymerase amplification (RPA). A, Fabrication of the layers of paper chips. B, Operation of RPA paper chip. The black area represents hydrophobic zones and the white area represents hydrophilic zones.

Fig. 2. Diagram of experimental results. A, Comparison of fluorescence intensity of recombinant polymerase amplification (RPA) in different paper films. ∆S represents the change in fluorescence intensity of the reaction pad before and after the RPA reaction, and ∆N is the change in fluorescence intensity of the control pad before and after the RPA reaction without sample DNA. B, Effect of the ratio of iron powder to activated carbon on temperature and time. C, Effect of the ratio of iron powder to activated carbon on the exothermic rate during the first 5 min. D, Effect of the ratio of iron powder to vermiculite on temperature and time. E, Effect of vermiculite insulation on the rate of heat release between 10‒25 min. F, Variation in heating layer temperature profile at different ambient temperatures. G, Effect of complex solution volume on the activation of pre-deposited reactants and assay outcomes. H, Comparison of paper-based RPA and tube-based RPA outcomes. I, Comparison of RPA results from self-heating paper chips (Group B) with those from ordinary paper chips using external heating equipment (Group A). J, Specific detection of different pathogens (Xanthomonas oryzae pv. Oryzae, X. oryzae pv. oryzicol, and Acidovorax avenae subsp. Avenae) using a self-heating paper chip device. I0 represents fluorescence signal intensity in the paper-based detection region when there is no response, and ∆I represents the difference in fluorescence signal intensity between the reacted and non-reacted paper-based detection regions. K, Receiver operating characteristic (ROC) analysis of samples detected by paper-based self-heating RPA chip. L, Comparison of reliability of this chip method with RPA conventional tube-based solutions and standard paper-based chips using an externally heated platform, as evaluated by ROC curves. M, Physical appearance of paper-based and tube-based RPA results. ‘c’ refers to control group, ‘+’ for positive, and ‘-’ for negative. N, Physical view of paper chip heating layer and overall device. Fluorescence photometric values of the detection area on the paper and test tube substrate were measured under 495 nm wavelength irradiation and 520 ± 10 nm wavelength acquisition (at the end of the assay, images of all the detection areas were captured with a smartphone and converted from colour intensity to optical density using ImageJ software). Data were expressed as Mean ± SD of three replicates.