Herein, a chemiluminescence (CL) biosensor based on CRISPR-Cas12a and cation exchange reaction was constructed to detect the biomarker microRNA-21 (miRNA-21). The rolling circle amplification (RCA) reaction was introduced to convert each target RNA strand into a long single-stranded DNA with repeated sequences, which acted as triggers to initiate the transcleavage activity of CRISPR-Cas12a. The activated Cas12a could cleave the biotinylated linker DNA of CuS nanoparticles (NPs) to inhibit the binding of CuS NPs to streptavidin immobilized on the surface of the microplate, which strongly reduced the generation of Cu2+ from a cation exchange between CuS NPs and AgNO3, and thus efficiently suppressed the CL of Cu2+-luminol-H2O2 system, giving a "signal off" biosensor. With the multiple amplification, the detection limit of the developed sensor for miRNA-21 reached 16 aM. In addition, this biosensor is not only suitable for a professional chemiluminescence instrument but also for a smartphone used as a detection tool for the purpose of portable and low-cost assay. This method could be used to specifically detect quite a low level of miRNA-21 in human serum samples and various cancer cells, indicating its potential in ultrasensitive molecular diagnostics.

Chemiluminescence Sensor for miRNA-21 Detection Based on CRISPR-Cas12a and Cation Exchange Reaction.

Herein, by directly limiting the reaction space, an ingenious three-dimensional (3D) DNA walker (IDW) with high walking efficiency is developed for rapid and sensitive detection of miRNA. Compared with the traditional DNA walker, the IDW immobilized by the DNA tetrahedral nanostructure (DTN) brings stronger kinetic and thermodynamic favorability resulting from its improved local concentration and space confinement effect, accompanied by a quite faster reaction speed and much better walking efficiency. Once traces of target miRNA-21 react with the prelocked IDW, the IDW could be largely activated and walk on the interface of the electrode to trigger the cleavage of H2 with the assistance of Mg2+, resulting in the release of amounts of methylene blue (MB) labeled on H2 from the electrode surface and the obvious decrease of the electrode signal. Impressively, the IDW reveals a conversion efficiency as high as 9.33 × 108 in 30 min with a much fast reaction speed, which is at least five times beyond that of typical DNA walkers. Therefore, the IDW could address the inherent challenges of the traditional DNA walker easily: slow walking speed and low efficiency. Notably, the IDW as a DNA nanomachine was utilized to construct a sensitive sensing platform for rapid miRNA-21 detection with a limit of detection (LOD) of 19.8 aM and realize the highly sensitive assay of biomarker miRNA-21 in the total RNA lysates of cancer cell. The strategy thus helps in the design of a versatile nucleic acid conversion and signal amplification approach for practical applications in the areas of biosensing assay, DNA nanotechnology, and clinical diagnosis.

High-Efficiency 3D DNA Walker Immobilized by a DNA Tetrahedral Nanostructure for Fast and Ultrasensitive Electrochemical Detection of MiRNA.

Electrochemiluminescence detection of miRNA-21 based on dual signal amplification strategies: Duplex-specific nuclease -mediated target recycle and nicking endonuclease-driven 3D DNA nanomachine.

Herein, an electrochemiluminescence (ECL) and electrochemical (EC) dual-mode biosensor platform with a self-powered DNAzyme walking machine was established for accurate and sensitive detection of miRNA-21. By employing a magnesium ion (Mn2+)-dependent DNAzyme cleavage cycling reaction, the walking machine was built by assembling DNAzyme walking strands and ferrocene (Fc)-labeled substrate strands on the Au nanoparticles and graphitic carbon nitride nanosheet (g-C3N4 NS)-covered electrode. The DNAzyme walking strand was first prohibited by a blocker strand. After the addition of target miRNA-21 and Mn2+, the DNAzyme walker could be activated and produce autonomous movements along the electrode track fueled by Mn2+-dependent DNAzyme-catalyzed substrate cleavage without additional energy supply. Notably, each walking step resulted in the cleavage of a substrate strand and the release of a Fc-labeled DNA strand fragment, allowing us to acquire an extreme ECL signal recovery of g-C3N4 inhibited by Fc. Meanwhile, numerous Fc-labeled DNA fragments escaped from the surface of the electrode, directly producing an obvious decrease in the square wave voltammetry (SWV) signal from Fc on the same sensing platform. This work not only avoided difficultly assembling various signal indicators but also significantly improved the sensitivity through using self-powered DNAzyme-walker amplification. Moreover, the proposed design employed the same reaction to produce two signal output modes, which could eliminate the interference from diverse reactive pathways on the outcome to mutually improve the accuracy. Therefore, the dual-mode miRNA-21 biosensor exhibited wide detection ranges of 100 aM to 100 nM with low detection limits of 54.3 and 78.6 aM by ECL and SWV modes, respectively, which provided an efficient and universal biosensing approach with extensive applications in early disease diagnosis and bioanalysis.

Self-Powered DNAzyme Walker Enables Dual-Mode Biosensor Construction for Electrochemiluminescence and Electrochemical Detection of MicroRNA.

Electrochemical analysis of microRNAs with hybridization chain reaction-based triple signal amplification

In this work, an intelligent multiregion linear DNA walker (MLDW) with a high walking rate and a high amplification efficiency was explored for ultrasensitive detection of miRNA. Significantly, amounts of functional domain could be concentrated in a long linear DNA obtained by the target miRNA-mediated rolling-circle amplification to simultaneously increase the local concentration and collision probability, resulting in an obviously improved reaction rate. Impressively, the MLDW can accomplish the reaction within 30 min, which is at least 4 times beyond that of traditional single-leg and multiple-leg DNA walkers. As a proof of concept, the high-efficiency MLDW was used to develop an electrochemical biosensing platform for ultrasensitive detection of target miRNA-21 with a low detection limit down to 36 aM. Therefore, the MLDW we designed puts forward an innovative insight to construct a functional DNA nanodevice and promote the investigation of the inherent performance of nucleic acid signal amplification for ultimate application in the detection of biomolecules and clinical disease diagnosis.

Tong-Lin Hou

Papers

Multiregion Linear DNA Walker-Mediated Ultrasensitive Electrochemical Biosensor for miRNA Detection.