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

Abstract

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.

Photoactivated Controlled Dnazyme Platform for on-Demand Activation Sensitive Electrochemiluminescence mRNA Analysis

“…Then, the strongly reduced intermediate was transformed into an excited state K-doped g-C 3 N 4 * (eq ), which could return to the ground state accompanied by strong ECL emission via intramolecular electron and energy (eq ). The ECL emission was recorded during cathodic polarization based on previous literature and our discussion, as shown in the following ECL reactions: normalS 2 normalO 8 2 − + normale − → SO 4 • − + SO 4 2 − normalK ‐doped g normal‐C 3 normalN 4 + normale − → normalK‐ doped g normal‐C 3 normalN 4 • − SO 4 • − + normalK‐ doped g‐ normalC 3 normalN 4 • − → SO 4 2 − + normalK‐ doped g‐ normalC 3 normalN 4 * normalK‐ doped g‐ normalC 3 normalN 4 * …”

“…Then, the strongly reduced intermediate was transformed into an excited state K-doped g-C 3 N 4 * (eq ), which could return to the ground state accompanied by strong ECL emission via intramolecular electron and energy (eq ). The ECL emission was recorded during cathodic polarization based on previous literature and our discussion, as shown in the following ECL reactions: …”

“…First, bulk g-C 3 N 4 was prepared according to refs and , and the specific steps were as follows: 24 mmol of melamine was placed in a porcelain cup and heated to 550 °C for 4 h in a muffle furnace with the heating rate and cooling rate of 3 and 10 °C/min, respectively. Then, the obtained product was dissolved in water (0.5 mg/mL) and treated with ultrasound for more than 12 h. Second, the preparation process of K-doped g-C 3 N 4 was the same as that of bulk g-C 3 N 4 , but 0.24 mmol of KCl and melamine needed to be ground and mixed evenly before calcination.…”

Thermodynamic Allosteric Switch-Actuated 3D DNA Nanomachine for Ultrasensitive Electrochemical/Fluorescent Dual-Mode Biosensing of a Transcription Factor

“…In order to improve the accuracy of the biosensing, dual-mode biosensors have spurred tremendous enthusiasm on account of their self-calibration function to exclude the influence of systematic error; the interverifiable dual-response generated by independent signal transduction endows the biosensor with self-calibration function, demonstrating higher reliability than single-mode system. − Notably, electrochemical (EC)/fluorescent (FL) dual-mode system has the advantages of simplicity and rapidity in EC measurement, while FL sensing adopts light as the output signal to increase sensitivity. − So far, most EC/FL dual-mode biosensors are manufactured by integrating two signal probes and relatively independent signal transduction patterns . Notwithstanding no interference between the two separate signaling routes, the adoption of diverse signal probes will bring inevitable blemishes during the construction of biosensors, such as cumbersome signal probe assembly and uncontrollable fluctuation in different batches .…”

DNA Polymerase-Steered Self-Propelled and Self-Enhanced DNA Walker for Rapid and Distinctly Amplified Electrochemical Sensing

“…With the advancement of DNA nanotechnology, DNA-based molecular machines have attracted extensive attention and they have been developed for the widespread applications in biosensing, biocomputing, bioimaging, and nanomedicine. − DNA walkers, as one of the most attractive DNA machines, could be autonomously and progressively operated on a solid interface upon the stimulus of external input without human intervention. − Owing to DNA’s highly predictable and programmable assembly or hybridization process, the DNA walkers could be flexibly harnessed to control its moving behaviors. Now, DNA walkers have been developed rapidly for the fabrication of DNA-based biosensors toward the analysis of various biomolecules such as nucleic acids, proteins, and small molecules. − It provides a powerful means for the fast, reliable, and amplified dynamic response related with target recognition events. The driving forces for the operation of DNA walkers are mostly based on the use of various enzymes (endonuclease, exonuclease, and DNAzyme). − The stepwise and specific enzyme cleavage toward the predesigned DNA track pushes forward the DNA movement.…”