Microfluidic paper-based analytical device for point-of-care nucleic acid quantification of malaria

Malaria continues to pose significant global health challenges, necessitating the development of sensitive and rapid diagnostic tools to address the limitations of current diagnostic methods, particularly in low-resource settings. This study aims to develop a microfluidic paper-based analytical device (μPAD) that integrates recombinase polymerase amplification (RPA) directly on the paper substrate for the detection of Plasmodium species, enabling simultaneous amplification and detection on a single device. The μPADs were designed with specified primers and probes for Plasmodium 18S rRNA, modified as solid support on the device. Genomic DNA extracted from infected blood samples and RPA reagents were applied to the μPADs, where amplification occurs. A wax gate mechanism was incorporated to temporarily retain amplification reagents, allowing controlled incubation under constant temperature conditions. After amplification, a running buffer containing surfactants was introduced to open the wax gate, enabling the flow of amplicon products through the device. The resulting amplicons are bound to anti-DIG/gold nanoparticles, producing a visible red signal at designated test spots, indicating malaria presence. The μPADs demonstrated high sensitivity, achieving a limit of detection as low as 28 parasites mL􀀀 1 for P. falciparum. The overall amplification and detection time was approximately 35 min. The device was successfully validated using P. falciparum cultures and blood samples from patients infected with P. falciparum, P. vivax, P. knowlesi, and P. malariae. The developed μPADs represent a promising tool for rapid malaria diagnostics, particularly in low-resource settings, due to their high sensitivity, simplicity, and short detection time. This innovation has the potential to significantly improve malaria diagnosis and management in endemic regions.