CRISPR/Cas-Based Electrochemical Biosensor for Human Immunodeficiency Virus-1 Nucleic Acid Amplification-Free Detection to the Attomolar Level

Aiming at realizing field detection during HIV-1 patient screening in developing countries and point of care testing (POCT) of virus levels in HIV-1 patients receiving medication, an urgent demand for portable nucleic acid detection technology with low cost and sensitivity is raised. To solve this, a CRISPR/Cas13a-based electrochemical detection platform by a multiple combined crRNAs strategy is developed. This sensing platform is based on the Ion Current Rectification regulation through a Porous Anodic Alumina membrane decorated with ssRNA chains, which are trans-cleavage substrates for activated Cas13a and become shorter when the target gene exists, resulting in an altered motion of transmembrane ions. This platform was proven to be feasible and ultrasensitive for tracing the HIV-1 virus by multiple applicable crRNAs combination, and its clinical sample detection limit arrives at 60 copies/μL for HIV-1 patients' serum samples. Importantly, this method is amplification-free, and its sensitivity is beyond the nonamplification fluorescence-based nucleic acid strategy by one order of magnitude. This sensor is facile to use and cost-effective, and can also be extended to other RNA-based pathogenic microorganism detections. This POCT method facilitates self-monitoring of drug resistance in HIV-1 patients, regardless of medication status.