Microfluidics-Enabled Digital Isothermal Cas13a Assay

Abstract The isothermal molecular diagnosis with CRISPR has attracted particular interest for the sensitive, specific detection of nucleic acids. However, most of the assays with Cas enzymes were performed in bulk assays using multistep approaches and hard to realize quantitative detection. Herein, we report Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA), a digital format of SHERLOCK with enhanced robustness and sensitivity. We first address the macromolecular crowding problems when combining the recombinase polymerase amplification (RPA) and Cas13a detection into a one-pot SHERLOCK assay. After the assay optimization, the enhanced one-pot SHERLOCK (E-SHERLOCK) achieves high robustness and 200-fold increased sensitivity. Leveraging droplet microfluidics, we streamline the E-SHERLOCK to eliminate undesired input targets caused by pre-amplification before partition, enabling background-free absolute quantification. From the real-time monitoring, MEDICA enables qualitative detection within 10 min and absolute quantification within 25 min. For the proof of concept, we applied MEDICA to quantify HPV 16 and 18 viral loads in 44 clinical samples, indicating perfect accordance with qPCR results. MEDICA highlights the CRISPR-based isothermal assays are promising for the next generation of point-of-care diagnostics.