Exploring the trans-cleavage activity of CRISPR-Cas12a for the development of a Mxene based electrochemiluminescence biosensor for the detection of Siglec-5

Abstract

Sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs) is a type I transmembrane receptor on the cell surface. Siglec-5, as one of the Siglecs family, play an important role as an inhibitory receptor for leukocytes in the human body. The development of novel siglec-5 assays can help to study the pathogenesis of related diseases as well as to develop novel therapeutic drugs. We use catalytic hairpin assembly (CHA) amplification strategy combined with CRISPR-Cas12a′s side-cutting feature to build a 2D ultra-thin Ti3C2Tx (MXene) based electrochemiluminescence (ECL) biosensor for the detection of Siglec-5. By using this ECL biosensor, the cleavage of CRISPR-Cas12a is reasonably combined with CHA-mediated isothermal amplification, thereby realizing the sensitive amplification assay Siglec-5 with 20.22 fM sensitivity. By introducing pairs of sites that are not in the same double-stranded DNA into the DNA duplex, the hybridization sequence of CRISPR-Cas12a complements the targeting mechanism to enhance indirect Siglec-5 amplification assay. Also, the double-strand DNA (dsDNA) design based on CRISPR-Cas12a amplification allows the same CRISPR RNA (crRNA, also known as guide RNA (gRNA)) to detect the output of DNA duplexes from different intermediate DNAs, which provides a common way for biomarker detection based on the conversion of protein analytes to intermediate DNA strategy. This work extends the application scope of CRISPR-Cas12a to the construction of ECL biosensors, evaluates the role of lectins, which can be used for the biochemical research and clinical diagnosis of protein markers. This is the first investigative work exploring the Trans-Cleavage activity of CRISPR-Cas12a for Mxene-based ECL biosensor establishment to the best of our knowledge.