Multiplexed Detection of Respiratory Virus RNA Using Optical pH Sensors and Injection Molded Centrifugal Microfluidics

Abstract

Infectious pathogens, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), are a threat to global health and prosperity, with the coronavirus disease 2019 (COVID-19) pandemic causing deaths and negative economic impacts worldwide. Pathogens continuously mutate, evading vaccines and treatments; monitoring is therefore crucial to preventing future outbreaks. But there are still many shortcomings in available diagnostic technologies, and scalable and convenient point-of-care technologies are highly demanded. In this work, we demonstrate the application of injection molded centrifugal microfluidic chips with integrated optical pH sensors for multiplexed detection of 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 minutes for SARS-CoV-2 and influenza A, and 48 minutes for influenza B. We implemented a data processing strategy that allowed for reliable, quantitative thresholds for deciding reaction outcomes based on numerical derivatives of the fluorescence curves, enabling 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.