Development of a Recombinase Polymerase Amplification Assay on Digital Microfluidics for Point-of-Care Detection of <i>Plasmodium falciparum</i>

Abstract Malaria is a dangerous mosquito-borne infectious disease that causes millions of death cases each year worldwide, especially in Southeast Asia. Current P. falciparum detection methods (e.g., blood smear, enzyme-linked Immunosorbent assay, polymerase chain reaction (PCR), etc.) have many disadvantages, such as being time-consuming, dependent on the operator's experience, and relying on sophisticated instruments that hinder the point-of-care (POC) application. To overcome those drawbacks, we developed a colorimetric recombinase polymerase amplification (RPA) reaction on a digital microfluidic (DMF) platform using SYBR Green I as the indicator for a simple result readout step, thus simplifying the diagnostic procedure. Using primers specific for the lactate dehydrogenase (LDH) gene, P. falciparum plasmids were successfully detected under optimal conditions for RPA reaction. For the implementation of RPA on the DMF platform, 0.1% Tween-20 was added to support droplet movement on the chip without interfering with the reaction. The droplet pathway was designed so the system can carry out up to five samples simultaneously on one single DMF device, thus providing a time-efficient point-of-care method for detecting P. falciparum and laying a foundation for developing a simple diagnostic procedure for other diseases.