Magnetofluid-integrated biosensors based on DNase-dead Cas12a for visual point-of-care testing of HIV-1 by an up and down chip

The CRISPR Cas system, as a novel nucleic acid detection tool, is often hindered by cumbersome experimental procedures, complicated reagent transfer processes, and associated aerosol pollution risks. In this study, an integrated nucleic acid detection platform named "up and down chip" was developed, which combined RT-RAA technology for nucleic acid amplification, DNase-dead Cas12a-modified magnetic beads for specific recognition of target nucleic acid, and HRP-TMB chromogenic reaction for signal output in different chambers of a single microfluidic chip. The magnetic beads were migrated in an up-and-down manner between different chambers through magnetic driving, achieving a "sample-in, result-out" detection mode. By introducing a homemade heating box for temperature control during the reaction and using the naked eye or a smartphone APP for color-based signal reading, no professional or precise instruments were required in this platform. Using this platform, highly sensitive detection of the HIV-1 genome as low as 250 copies (CPs) per mL was achieved within 100 min while maintaining good detection performance against common variants as well as excellent specificity and anti-interference ability. In addition, compared with qRT-PCR, it also exhibited good accuracy for 56 spiked plasma samples, indicating its promising potential for clinical application. Magnetofluid-integrated biosensors based on DNase-dead Cas12a termed "up and down chip" was established for visual point-of-care testing of HIV-1.