An electrochemical biosensor equipped with a logic circuit as a smart automaton for two-miRNA pattern detection.

Detecting multiple targets in complex cellular and biological environments yields more reliable results than single-label assays. Here, we introduced an electrochemical biosensor equipped with computing functions, acting as a smart automaton to enable computing-based detection. By defining the logic combinations of miR-21 and miR-122 as detection patterns, we proposed the corresponding AND and OR detection automata. In both logic gate modes, miR-21 and miR-122 could be replaced with single-stranded FO or FA, modified with Fc, binding to the S chain on the electrode surface. This process led to a significant decrease in the square wave voltammetry (SWV) of Fc on the same sensing platform, as numerous ferrocene (Fc)-tagged DNA fragments escaped from the electrode surface. Experimental results indicated that both automata efficiently and sensitively detected the presence of the two targets. This strategy highlighted how a small amount of target could generate a large current signal decrease in the logic automata, significantly reducing the detection limit for monitoring low-abundance targets. Moreover, the short-stranded DNA components of the detection automata exhibited a simple composition and easy programmability of probe sequences, offering an innovative detection mode. This simplified the complex process of detection, data collection, computation, and evaluation. The direct detection result ("0" or "1") was exported according to the embedded computation code. This approach could be expanded into a detection system for identifying other sets of biomarkers, enhancing its potential for clinical applications.

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

Programming ultrasensitive and stimuli-responsive DNAzyme-based probes holds great potential for on-demand biomarker detection. Here, an optically triggered DNAzyme platform was reported for on-demand activation-sensitive electrochemiluminescence (ECL) c-myc mRNA analysis. In this design, the sensing and recognition function of the split DNAzyme (SDz) probe was silent by engineering a blocking sequence containing a photocleavable linker (PC-linker) group at a defined site that could be indirectly cleaved by 302 nm ultraviolet (UV) light. When the SDz probes were assembled on the Au nanoparticles and potassium (K) element doped graphitic carbon nitride nanosheet (K-doped g-C3N4) covered electrode, UV light activation induces the configurational switching and consequently the formation of an active DNAzyme probe with the help of target c-myc mRNA, allowing the cleavage of the substrate strand by magnesium ions (Mg2+). Thus, the release of a ferrocene (Fc)-labeled DNAzyme 2 strand contributed to an extreme ECL signal recovery. In the meantime, the released target c-myc mRNA combined another inactive SDz motif to form active DNAzyme and repeat the cyclic cleavage reaction, resulting in the signal amplification. Furthermore, according to the responses toward two other designed nPC-SDz and m-SDz probes, we demonstrated that controlled UV light mediated photoactivation of the DNAzyme biosensor "on demand" effectively constrained the ECL signal to the mRNA of interest. Moreover, false positive signals could also be avoided due to such a photoactivation design with UV light. Therefore, this study provided a simple methodology that may be broadly applicable for investigating the mRNA-associated physiological events that were difficult to access using traditional DNAzyme probes.

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

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.