Cas12a-assisted RTF-EXPAR for accurate, rapid and simple detection of SARS-CoV-2 RNA.

Developing highly accurate and simple approaches to rapidly identify and isolate SARS-CoV-2 infected patients is important for the control of the COVID-19 pandemic. We, herein, reported the performance of a Cas12a-assisted RTF-EXPAR strategy for the identification of SARS-CoV-2 RNA. This assay combined the advantages of RTF-EXPAR with CRISPR-Cas12a can detect SARS-CoV-2 within 40 min, requiring only isothermal control. Particularly, the simultaneous use of EXPAR amplification and CRISPR improved the detection sensitivity, thereby realizing ultrasensitive SARS-CoV-2 RNA detection with a detection limit of 3.77 aM (∼2 copies/µL) in an end-point fluorescence read-out fashion, and at 4.81 aM (∼3 copies/µL) level via a smartphone-assisted analysis system (RGB analysis). Moreover, Cas12a increases the specificity by intrinsic sequence-specific template recognition. Overall, this method is fast, sensitive, and accurate, needing minimal equipment, which holds great promise to meet the requirements of point-of-care molecular detection of SARS-CoV-2.

Rapid and sensitive detection of Ebola RNA in an unamplified sample based on CRISPR-Cas13a and DNA roller machine.

A fast and simple Cas13a-based assay approach for direct detecting Ebola RNA in unamplified samples is reported. The procedure (named Cas-Roller) is comprised of a 10-min Cas13a-mediated cleavage protocol, followed by a DNA roller running for 30 min. This involves Cas13a collateral cleaving a suitably designed substrate in the presence of Ebola virus RNA sequence, and the cleavage product is used for DNA roller to amplify and generate fluorescent signals. After optimization of the conditions, the assay is able to achieve a limit of detection as low as 291 aM (∼175 copies RNA/µL) along with excellent anti-interfering performance in human serum and blood detection, which is ∼310-fold improved compared with the direct CRISPR assay. The entire workflow can be completed in ∼40 min at 37 °C without any pre-amplification, transcription, or centrifugation steps, thus avoiding the generation of false-negative or positive results. In addition, the downstream roller reaction is independent of the target sequence, this method can be applied to detect any other RNA by merely redesigning the hybridization regions of the crRNA. Overall, this strategy gives a new idea for the construction of simple and accurate Cas13a-based assays for the direct detection of RNA.

Rapid, ultrasensitive and non-enzyme electrochemiluminescence detection of hydrogen peroxide in food based on the ssDNA/g-C3N4 nanosheets hybrid.

Hydrogen peroxide (H2O2) is usually used as a fungicide in food, it is carcinogenic, accelerates aging or inducing toxic effects such as cardiovascular disease. Herein, to meet the demand for effective and fast detection of H2O2 in food, a novel non-enzymatic electrochemiluminescence (ECL) sensor based on single-stranded DNA (ssDNA)/g-C3N4 nanosheets (NS) was established. The ssDNA/g-C3N4 NS hybrid was prepared by simple mixing g-C3N4 NS and ssDNA solution together. The prepared ssDNA/g-C3N4 NS exhibited improved peroxidase-like activity and was modified on a glassy carbon electrode to catalyze the ECL reaction of luminol-H2O2 to amplify the luminescence signal. Under the optimized conditions, the proposed sensor exhibits high sensitivity with a limit of detection (LOD) as low as 33 aM H2O2, which is much lower than the vast majority of reported methods. This method enables the reliable responding to H2O2 from the milk samples within 1 min.

Cas12a-based electrochemiluminescence biosensor for target amplification-free DNA detection.

CRISPR/Cas system have drawn increasing attention in accurate and sensitive nucleic acids detection. Herein, we reported a novel Cas12a-based electrochemiluminescence biosensor for target amplification-free human papilloma virus subtype (HPV-16) DNA detection. During this detection process, Cas12a employed its two-part recognition mechanism to improve the specificity and trans-cleavage capability to achieve signal amplification, while L-Methionine stabilized gold nanoclusters (Met-AuNCs) were served as high-efficiency ECL emitters to achieve ECL signal transition. Given the unique combination of Cas12a with ECL technique, the detection limit was determined as 0.48 pM and the whole detection could be completed within 70 min. We also validated the practical application of the proposed biosensor by using undiluted human blood samples, which gives impetus to the design of new generations of CRISPR/Cas detection system beyond the traditional ones with ultimate applications in sensing analysis and diagnostic technologies.

Simple Tripedal DNA Walker Prepared by Target-Triggered Catalytic Hairpin Assembly for Ultrasensitive Electrochemiluminescence Detection of MicroRNA.

In contrast to common DNA walkers, multipedal DNA walkers exhibit larger walking area and faster walking kinetics and provide increased amplification efficiency. Consequently, they have received a considerable amount of attention in biosensing. However, most of them are synthesized by immobilizing multiple DNA walking strands on the surface of Au nanoparticles, which is tedious and time-consuming. Simple preparation of multipedal DNA walkers remains a challenge. Herein, we adopted a simple enzyme-free target-triggered catalytic hairpin assembly (CHA) circuit to synthesize a tripedal DNA walker. By walking on a DNA track-functionalized electrode, a sensitive electrochemiluminescence DNA nanomachine biosensor was constructed for sensing miRNA-21. The DNA walker was powered by toehold-mediated strand displacement; the whole process did not need the assistance of enzymes, thus avoiding tedious procedures and enzyme degradation under unfavorable environmental conditions. Specifically, a superior detection limit of 4 aM and a broad linear range of 10 aM to 1 pM were achieved. This CHA-tripedal DNA walker biosensor was then applied for the detection of miRNA-21 in human serum and showed high selectivity and excellent reproducibility, demonstrating its practical application in bioanalysis. In particular, the Y-shaped tripedal DNA walker comes from the DNA circuit, which makes the approach ideally suited for biosensing of small nucleic acid targets.