CRISPR/Cas12a Powered DNA Framework‐Supported Electrochemical Biosensing Platform for Ultrasensitive Nucleic Acid Analysis

Abstract

Nucleic acid analysis using ultrasensitive and simple methods is critically important for the early‐stage diagnosis and treatment of diseases. The CRISPR/Cas proteins, guided by a single‐stranded RNA have shown incredible capability for sequence‐specific targeting and detection. Herein, in order to improve and expand the application of CRISPR/Cas technology to the electrochemical interface‐based nucleic acids analysis, the authors develop a CRISPR/Cas12a powered DNA framework‐supported electrochemical biosensing platform via the cis and trans cleavage of Cas12a on the heterogeneous carbon interface (the existing publications which commonly adopted trans‐cleavage). Their solid‐liquid interface is first immobilized by 3D tetrahedral framework nucleic acids (FNAs) with specific DNA recognition probe. Based on the recognition of the complementary target through protospacer adjacent motif (PAM) confirmation and CRISPR‐derived RNA (crRNA) matching, the easily formed Cas12a/crRNA duplex can get access to the interface, and the cis and trans cleavage of Cas12a can be easily activated. In combination with the enzyme catalyzed reaction, they achieved an ultralow limit of detection (LOD) of 100 fm in HPV‐16 detection without pre‐amplification. Furthermore, the platform is compatible with a spike‐in human serum sample and has superior stability. Thus, their reported platform offers a practical, versatile, and amplification‐free toolbox for ultrasensitive nucleic acid analysis.

Harnessing Multiplex crRNA in the CRISPR/Cas12a System Enables an Amplification-Free DNA Diagnostic Platform for ASFV Detection

“…CRISPR/Cas12a, an RNA-guided DNase, demonstrates ssDNase ability (also called collateral effect or collateral cleavage) when the Cas12a/crRNA complex is activated by the target dsDNA. − In the CRISPR/Cas12a detection system, ssDNA is usually dual-labeled with a fluorophore and quencher so that the dsDNA input can be transformed into the measurable fluorescent output. ,, CRISPR/Cas12a is advantageous for being both temperature-resilient and highly specific . Cas12a can be activated even at room temperature and performs low mismatch tolerance. , These features enable the CRISPR/Cas12a system to be an efficient and programable biosensing platform. − However, the CRISPR/Cas12a system alone is challenged for its limited sensitivity. , A typical CRISPR/Cas12a system can hardly provide detectable signals when the concentration of the dsDNA substrate is lower than 100 pM . To compensate for such limitations, the standard strategy is priorly using nucleic-acid amplification to increase the quantity of the target dsDNA substrate in the CRISPR/Cas12a system.…”

A smartphone-based three-in-one biosensor for co-detection of SARS-CoV-2 viral RNA, antigen and antibody

“…Stability is important to evaluate the biosensor performance. 18,20 The results showed that our biosensor has acceptable stability. The recovery tests showed that three targets of SARS-CoV-2 could be detected by the three-in-one biosensor (Table S2, ESI†).…”

Interfacial DNA Framework-Enhanced Background-to-Signal Transition for Ultrasensitive and Specific Micro-RNA Detection

“…Signal Enhancement Using the etFNA Sensor. To verify the signal enhancement of our etFNA sensor, we designed three different capture probes, including 1D thiolated ssDNA, 2D polyA (30), and 3D tFNA probe, with the identical recognition domain (10 nt) complementary to target miR21 in part (Figure 3a). The prehybridization of miR21 with biotin-DNA probes allows the surface-confined 1D−3D capture probes to complement with the heteroduplex in one step via base-stacking.…”

“…There are two gold-standard methods, including sequencing and quantitative reverse transcription PCR (qRT-PCR), suitable for miRNAs analysis, but they suffer from large volume samples, cDNA synthesis, and false positive . Recently, some PCR-free and sequencing-free schemes have been deployed in miRNA biosensors, whereas they pay more attention on the capture probes design, for example, implementing electrically neutral peptide nucleic acid (PNA) and high-affinity locked nucleic acid (LNA) to enhance the capture ability, and amplifying the detection signal with enzyme-based or enzyme-free DNA extension reactions, as well as the ultrasensitive detectors. ,− Despite the progress, most of these strategies ignore the potential contribution of capture probe-based background-to-signal transformation to signal-amplified detection of miRNAs.…”