Signal-on electrochemiluminescence resonance energy transfer biosensor for miRNA-543 based on CRISPR/Cas13a and magnetic separation
Zixin Li, Jing Wang, Ke Shen, Xiaoying Zhao, Zhenyu Lin, Yi Huan
Abstract
In this study, an electrochemiluminescence resonance energy transfer (ECL-RET) biosensor with high sensitivity and strong resistance to interference was constructed based on the CRISPR/Cas13a system and magnetic separation for ovarian cancer biomarker miR-543 detection. Mesoporous silica nanoparticles embedded with Ru(bpy) 3 2+ (Ru@SiO 2 ) have high electrochemiluminescence (ECL) response was chosen as energy donor. Single-stranded DNA S1 containing "rUrU" motif was immobilized on AuNRs (AuNRs-S1), which hybridized with single-stranded DNA S2 modified SAMBs (SAMBs-S2) to form AuNRs-S1/S2-SAMBs complex, this has been used as energy acceptor. In the absence of the target, Cas13a remained inactive, preventing the cleavage of S1, thereby maintaining the association of AuNRs with SAMBs . Then they were added in Ru@SiO 2 solution after magnetic separation. The electrostatic adsorption between the negatively charged AuNRs and the positively charged Ru@SiO 2 cause the occurrence of ECL-RET and low ECL signal had been detected. When the target was added, Cas13a was activated and resulted in the non-specifically cleaving of S1, so AuNRs detached from SAMBs. After magnetic separation, fewer AuNRs participated in ECL-RET, leading to an enhanced ECL signal detected. The change in ECL intensity ( Δ ECL) exhibited a linear correlation with the logarithm of miR-543 concentration within the range of 10 fM to 10 nM, with a detection limit of 6.91 fM. The biosensor had been applied to detect miR-543 in clinical samples with high accuracy.