Multiplexed detection of respiratory virus RNA using optical pH sensors and injection-molded centrifugal microfluidics

The application is demonstrated of injection-molded centrifugal microfluidic chips with integrated optical pH sensors for multiplexed detection of respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, and influenza B RNA. The optical pH sensors generated sensitive fluorescent readouts from diagnostic reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions; limits of detection for influenzas A and B, and SARS-CoV-2 of 89, 245, and 38 RNA copies per reaction, respectively, were attained. Results were obtainable within 44 min for SARS-CoV-2 and influenza A, and 48 min for influenza B. We implemented a data processing strategy based on numerical derivatives of the fluorescence curves that allowed for reliable, quantitative thresholds for deciding reaction outcomes and enabled 100% specificity. This work demonstrates the utility of optical pH sensors and injection-molded centrifugal microfluidics for multiplexed infectious disease diagnostics with point-of-care applications.