- hochgeladen 25. Oktober 2019
This high-resolution version of the "On-chip CRISPR/Cas13a cleavage" video visualizes the “one-for-all” sensor chip handling and cleavage process and is the Supplementary video of article "CRISPR/Cas13a-Powered Electrochemical Microfluidic Biosensor for Nucleic Acid Amplification-Free miRNA Diagnostics", published in Advanced Materials (DOI: 10.1002/adma.201905311).
In this work, we have programmed the enzyme Cas13a, a microbial clustered regularly interspaced short palindromic repeats (CRISPR) effector, with a target-specific CRISPR RNA (crRNA) to detect the miRNA miR-19b and miR-20a (tumor biomarker candidates) electrochemically without nucleic acid amplification. Our sensing system is specifically engineered for on-site testing and requires low volumes of measurement samples, less than 600 nanoliters, enabled by the microfluidic technology. The entire test takes less than 4 hours along with a 9-minute readout time. The method proposed is able to detect miR-19b within the physiologically relevant range of circulating miRNAs in serum and down to 2 pM. Moreover, one of the most important features of the system proposed is that it can be scaled to create a “one-for-all” biosensor that can quantify a multitude of miRNAs with a single chip. In order to measure different miRNAs, only the target-specific crRNA would need to be replaced during sample preparation.