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.(click the image to learn more)
In this work, we demonstrate an inexpensive method of prototyping microfluidics using a desktop injection molding machine. A centrifugal microfluidic device with a novel central filling mechanism was developed to demonstrate the technique. We overcame the limitations of desktop machines in replicating microfluidic features by variotherm heating and cooling the mold between 50 °C and 110 °C within two minutes. Variotherm heating enabled good replication of microfeatures, with a coefficient of variation averaging only 3.6 % attained for the measured widths of 100 µm wide molded channels. Using this methodology, we produced functional polystyrene centrifugal microfluidic chips, capable of aliquoting fluids into 5.0 µL reaction chambers with 97.5% accuracy. We performed allele-specific loop-mediated isothermal amplification (AS-LAMP) reactions for genotyping CYP2C19 alleles on these chips. Readouts were generated using optical pH sensors integrated onto chips, by drop-casting sensor precursor solutions into reaction chambers before final chip assembly. Positive reactions could be discerned by decreases in pH sensor fluorescence, thresholded against negative control reactions lacking the primers for nucleic acid amplification and with time-to-results averaging 38 minutes. Variotherm desktop injection molding can enable researchers to prototype microfluidic devices more cost-effectively, in an iterative fashion, due to reduced costs of smaller, in-house molds. Designs prototyped this way can be directly translated to mass production, enhancing their commercialization potential and positive impacts. (click the image to learn more)
Improved diagnostics are needed to manage the ongoing COVID-19 pandemic. In this study, we enhanced the color changes and sensitivity of colorimetric SARS-CoV-2 RT-LAMP assays based on triarylmethane dyes. We determined a mechanism for the color changes and obtained sensitivities of 10 RNA copies per microliter. (click the image to learn more)
Embodiments of the invention relate to centrifugal fluidic devices, apparatus, and methods. Embodiments disclosed are fluidic devices, and associated apparatus and methods, which can partition a fluid sample from a single inlet or plurality of inlets into a plurality of chambers via their fluid inlets. Each chamber possesses a fluid outlet and a gas outlet. Partitioned fluid can be further distributed under centrifugal pressure to downstream fluidics modules, permitting various multiplexed assays to be performed including nucleic acid amplification tests.
