Sample-to-answer centrifugal microfluidic droplet PCR platform for quantitation of viral load

Abstract

Droplet digital polymerase chain reaction (ddPCR) stands out as a highly sensitive diagnostic technique that is gaining traction in infectious disease diagnostics due to its ability to quantitate very low numbers of viral gene copies. By partitioning the sample into thousands of droplets, ddPCR enables precise and absolute quantification without relying on a standard curve. Despite these advantages, current ddPCR systems often exhibit relatively low levels of integration, and the analytical process remains dependent on elaborate workflows for up-front sample preparation. Here, we introduce a fully-integrated system seamlessly combining viral lysis, nucleic acid extraction, emulsification, reverse transcription (RT) ddPCR, and fluorescence readout within a sample-to-answer format. The system comprises a disposable microfluidic cartridge housing buffers and reagents required for the assay, and a centrifugal platform that allows for pneumatic actuation of liquids during rotation, enabling automation of the workflow. Highly monodisperse droplets (~50 µm in diameter) are produced using centrifugal step emulsification and automatically transferred to an integrated heating module for amplification, thus limiting shear-induced merging of droplets during thermal cycling. The platform is equipped with a miniature fluorescence microscope enabling on-chip automated read-out of droplets after RT-ddPCR. As a use case, we demonstrate sample-to-answer detection of SARS-CoV-2 N and E genes, along with RNase P endogenous reference, using hydrolysis probes and multiplexed amplification within single droplets, achieving a low limit of detection of 0.1 copy/µL. We also tested 14 nasopharyngeal swab specimens from patients and were able to accurately distinguish positive and negative SARS-CoV-2 samples with 100% accuracy, surpassing results obtained by conventional real-time amplification. Being fully integrated, the assay facilitates potential deployment outside of specialized laboratories, opening new possibilities for quantitative, high-sensitivity detection of pathogens.