Photoswitchable CRISPR/Cas12a-Amplified and Co₃O₄@Au Nanoemitter Based Triple-Amplified Diagnostic Electrochemiluminescence Biosensor for Detection of miRNA-141

Abstract

In this work, a CRISPR/Cas12a initiated switchable ternary electrochemiluminescence (ECL) biosensor combined with a Co3O4@Au nanoemitter is presented for the in vitro monitoring of miRNA-141. Benefiting from the advantages of high-throughput cargo payload capability and superconductivity, three-dimensional reduced graphene oxide (3D-rGO) was designated as an introductory conducting stratum of a paper working electrode (PWE). With the collaborative participation of Co3O4@Au NPs, the transmutation of TPrA in the Ru­(bpy)3 2+/TPrA system can be riotously expedited into exorbitant free radical ions TPrA•, which provoked the exaggeration of the ECL signal. Moreover, the programmable enzyme-free hybrid chain reaction (HCR) amplifier on the PWE surface accurately anchored the assembly of nucleic acid tandem and accomplished the secondary recursion of the signal. Impressively, the multifunctional CRISPR/Cas12a with nonspecific cis/trans-splitting decomposition manipulated the photoswitch of the “on–off” signal state that avoided the false-positive diagnosis. The presented multistrategy cooperative biosensor demonstrated extraordinary sensitivity and specificity, with a low detection limit of 3.3 fM (S/N = 3) in the concentration scope from 10 fM to 100 nM, which fully corresponded to the expectation. Overall, this innovative methodology paved a generous avenue for evaluating multifarious biotransformations and provided a tremendous impetus to the development of real-time diagnosis and clinical detection of other biomarkers.

A one-tube dual-readout biosensor for detection of nucleic acids and non-nucleic acids using CRISPR-ALP tandem assay

“…[9][10][11] In this regard, various CRISPR-based diagnostic platforms, including colorimetry, electrochemistry, electrochemiluminescence, and chemiluminescence, have been proposed. [12][13][14][15][16][17][18][19][20][21] Although the aforementioned biosensors can provide highly sensitive, selective, fast, and convenient testing, they are only available in a single-readout mode that is not conducive to the accuracy of detection due to the influence of environmental and instrumental factors. There is an urgent demand for extending and building a novel CRISPR/Cas12abased multiple-readout biosensor.…”

Ultrasensitive miRNA Detection Based on Magnetic Upconversion Nanoparticle Enhancement and CRISPR/Cas13a-Driven Signal Amplification

“…In brief, the Cas13a protein assembles with crRNA and forms a Cas13a/crRNA complex to specifically recognize the target RNA, followed by activating its trans-cleavage activity to nonspecifically cleave the RNA molecules at a rate of ∼4854 turnovers per second. , Owing to its remarkable sensitivity and specificity, the CRISPR/Cas13a system shines in the field of molecular diagnostics. More and more researchers have developed various molecular detection methods combined with electrochemiluminescence, − fluorescence, − quantum dots, , and other nanomaterials , based on CRISPR/Cas13a. However, the applications of these techniques in the detection of complex biological samples are often limited on account of their characteristics such as photobleaching, instability, and high background signal of traditional fluorescent probes as well as high toxicity of quantum dots.…”

Metal–Organic Framework-DNA Bio-Barcodes Amplified CRISPR/Cas12a Assay for Ultrasensitive Detection of Protein Biomarkers

“…The clustered regularly interspaced short palindromic repeats (CRISPR/Cas) system has attracted extensive attention in genome editing and biosensing. − Among the CRISPR/Cas family, CRISPR/Cas12a has been widely deployed in molecular diagnosis due to its fascinating trans-cleavage activity, which can be activated to cleave single-stranded DNA (ssDNA) in a nonsequence-specific manner. , Recently, the CRISPR/Cas12 system exhibited great potential in detecting several targets, such as proteins, nucleic acids, − bacteria, metal ions, virus, and other small molecules, , with the advantages of being rapid, simple, and low-cost . As the programmability of the CRISPR/Cas system depends on the interaction between guiding the RNA and nucleic acid, some nucleic acid amplification technologies, including polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAPM), were employed to enhance the signal output of nucleic acid detection . However, these methods are not only time-consuming, laborious, and costly but also prone to cross-contamination between samples.…”

“…17 As the programmability of the CRISPR/Cas system depends on the interaction between guiding the RNA and nucleic acid, some nucleic acid amplification technologies, including polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAPM), 18 were employed to enhance the signal output of nucleic acid detection. 19 However, these methods are not only time-consuming, laborious, and costly but also prone to cross-contamination between samples. Thus, it urgently needs efficient strategies to achieve signal conversion and amplification to broaden the application of the CRISPR/Cas12a system in non-nucleic acid target detection.…”