This high-resolution version of the animation visualizes off-chip cleavage and subsequent electrochemical detection of miRNAs using CRISPR-Biosensor X and is the Supplementary video of article "CRISPR-powered electrochemical microfluidic multiplexed biosensor for target amplification-free miRNA diagnostics", published in Biosensors and Bioelectronics (https://doi.org/10.1016/j.bios.2020.112887).
In this study, we design and implement different multiplexed versions of our electrochemical microfluidic biosensor by dividing its channel into subsections, creating four novel chip designs for the amplification-free and simultaneous quantification of up to eight miRNAs on the CRISPR-Biosensor X ('X' highlighting the multiplexing aspect of the device). We then use a one-step model assay followed by amperometric readout in combination with a 2-minute-stop-flow-protocol to explore the fluidic and mechanical characteristics and limitations of the different versions of the device. The sensor showing the best performance, is subsequently used for the Cas13a-powered proof-of-concept measurement of two miRNAs (miRNA-19b and miRNA-20a) from the miRNA-17~92 cluster, which is dysregulated in the blood of pediatric medulloblastoma patients. Quantification of the latter, alongside simultaneous negative control measurements are accomplished on the same device. We thereby confirm the applicability of our platform to the challenge of amplification-free, parallel detection of multiple nucleic acids.
Credit: Ella Maru Studio