Highly sensitive electrochemiluminescence biosensor for miRNA-21 based on cascaded signal amplification and magnetic separation

MicroRNA-21 (miRNA-21) can serve as an early biomarker for acute kidney injury (AKI), it is important to develop sensitive and selective detection methods for miRNA-21 detection. In this study, an electrochemiluminescence (ECL) biosensor for miRNA-21which synergistically integrates T7 RNA polymerase-driven transcriptional amplification with CRISPR/Cas13a-mediated collateral cleavage and magnetic separation strategy had been designed. T7 RNA polymerase-driven transcriptional amplification has the characters of low requirements for conditions and high amplification efficiency. A cascaded signal amplification strategy which coupled enzymatic target recycling with CRISPR/Cas13a-mediated exponential signal amplification was proposed to achieve high detection sensitivity. And T7 promoter specificity and CRISPR/Cas13a sequence targeting had been adopted to ensure high specificity of the developed system. Furthermore, magnetic bead separation had been adopted to reduce the background signal. The parameters which may affected the performance of the biosensor had been optimized. Under optimized conditions, the ECL response of the system has a linear relationship with the logarithmic value of miRNA-21 concentration in the range of 1.0 fM to 1.0 nM, with a calculated limit of detection(LOD) of 0.33 fM (S/N = 3). The proposed methodology has been adopted to detect the target in clinical urine specimens with satisfied results.