Gold Nanoparticles/Mo₂C/MoO₂-Modified Electrodes for Nucleic Acid Detection through CRISPR/Cas12a Photoelectrochemical Assay

Abstract

Recently, RNA-guided CRISPR/Cas12a systems have been applied in sequence-specific DNA/RNA targeting and detection. However, the CRISPR/Cas12a system mainly relies on fluorescence readout, and the signal conversion depends on the nonspecific cleavage of single-stranded DNA. In this study, a CRISPR/Cas12a assay with site-specific double-stranded DNA (dsDNA) cleavage was developed. A self-powered photoelectrochemical (PEC) detection based on redox cycling with paminophenol and Ru(bpy) 3 Cl 2 on a gold nanoparticles/Mo 2 C/ MoO 2 -modified glassy carbon electrode was used for signal readout. The nucleic acid was detected by an enhancement of the PEC signal produced upon the activation of Cas12a dsDNase activity by dsDNA produced from the recycling of target DNA. dsDNA was cleaved to release alkaline phosphatase to catalyze the generation of p-aminophenol, which can be reused to achieve the PEC signal conversion. Under the optimal conditions, this PEC-CRISPR/Cas12a assay accurately identifies as low as 0.4 fM target HIV nucleic acid, and the dynamic range reaches from 1 to 700 fM. The relative standard deviation was 3.2% at 10 fM. As for the FMS-like tyrosine kinase 3 gene, the limit of detection was 58 aM with a linearity range from 0.2 to 100 fM. This assay enables stable and accurate determination to measure other nucleic acids, indicating important potential in future nucleic acid detection.

Gold Nanomaterials‐Implemented CRISPR‐Cas Systems for Biosensing

“…Reproduced with permission. [ 110 ] Copyright 2021, American Chemical Society Publishing Group. D) The principle of the SERS biosensor for the detection of nucleic acids of pathogenic bacteria.…”

“…AuNPs‐containing nanocomposites can serve as photoelectroactive nanomaterials to construct a CRISPR‐Cas12a‐based biosensor for the detection of nucleic acid (Figure 9C). [ 110 ] In the assay, AuNPs/MoO 2 /Mo 2 C glassy carbon electrode was prepared by the electrodeposition of AuNPs. The H1‐MBs probe was covalently assembled on the electrode, which could bind with the target DNA to form an H1‐target complex.…”

Research progress of CRISPR-based biosensors and bioassays for molecular diagnosis

“…They work by generating substances on the electrode surface that change from stable precursors to unstable intermediates and consequently form luminescent excited states via electron transfer involving the conversion of electrical and radiative energy (Richter, 2004). ECL improves on conventional electrochemical sensors with high sensitivity, low background interference, electrochemical controllability, and excellent selectivity (He et al, 2020), which can build a more stable bioassay platform and play an essential role in the field of medical examinations (Qin et al, 2021;Suea-Ngam et al, 2021;Zhang et al, 2021).…”

A Label-Free Photoelectrochemical Biosensor Based on CRISPR/Cas12a System Responsive Deoxyribonucleic Acid Hydrogel and “Click” Chemistry

“…Compared to conventional electrochemical and spectroscopic detection, PEC owns the merit of low background signal, high sensitivity, and easy miniaturization. − At present, there are relatively few reports on PEC sensors based on CRISPR technology. , Tang’s group developed a PEC sensor based on the destruction of (TiO 2 )-Au-CdS QDs by activated CRISPR-Cas12a . Hun’s group utilized the dsDNA cleavage character of Cas12a, preparing the PEC sensor amplified by redox cycling between Ru­(bpy) 3 Cl 2 and paminophenol . In Liu et al.’s work, dsDNA modified with HPR were fixed on the electrode .…”

“…35 Hun's group utilized the dsDNA cleavage character of Cas12a, preparing the PEC sensor amplified by redox cycling between Ru(bpy) 3 Cl 2 and paminophenol. 36 In Liu et al's work, dsDNA modified with HPR were fixed on the electrode. 28 After the cis-cleavage of Cas12a, a biocatalytic precipitation (BCP)-based PEC biosensor was obtained.…”