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Herein, a novel ratiometric electrochemical biosensor with methylene blue (MB) as the only one signal tag was proposed for highly reliable and ultrasensitive detection of microRNA-21 (miRNA-21) under the assistance of an intelligent targetinduced dual signal amplification (T-DSA). First, a small amount of target miRNA-21 could produce abundant mimic targets DNA S1 and Zn 2+ through target-induced recycle and acid dissolution, respectively. Then, S1 triggered rolling circle amplification (RCA) to generate functional DNA nanospheres (DSP) encoded by DNAzyme and substrate sequence for loading numerous signal tag MB with a remarkable electrochemical signal (signal on), and the Zn 2+ cofactor mediated the nonviolent DNAzyme-catalyzed cleavage of DSP to sharply release MB with obviously reduced electrochemical responses (signal off). Impressively, our strategy could controllably load and release the only signal tag MB through the well-designed DSP to effectively avoid the false positive responses caused by the non-ideal upright state of DNA probes connected to electrodes in traditional distance-dependent signal adjustment ratiometric strategies with two different signal tags. Meanwhile, with the aid of innovative T-DSA recycle and RCA-produced functional DSP, the detection sensitivity of this sensing platform was significantly improved. As a result, the proposed biosensor successfully realized highly reliable and ultrasensitive detection of miRNA-21 with a detection limit down to 26.7 aM, which shows exceptional promise in biological analysis and medical diagnosis.

Herein, we designed a dual 3D DNA nanomachine (DDNM)-mediated catalytic hairpin assembly (DDNM-CHA) to construct an electrochemical biosensor for ultrasensitive detection of miRNA, which possesses quite a faster reaction rate and much higher amplification efficiency than those of traditional catalytic hairpin assembly (CHA). Impressively, since the DDNM skillfully increases the local concentration of reactants and decreases the steric hindrance of substrates simultaneously, the DDNM-CHA could be endowed with higher collision efficiency and more effective reaction compared with traditional CHA, resulting in a hyper conversion efficiency up to 2.78 × 10 7 only in 25 min. This way, the developed DDNM-CHA could easily conquer the main predicaments: long reaction time and low efficiency. As a proof of the concept, we adopt the gold nanoparticles (AuNPs) and the magnetic nanoparticle (Fe 3 O 4 ) as the kernel of DNM-A and DNM-B, respectively, and harness the magnetic electrode to directly adsorb the products H1−H2/Fe 3 O 4 for constructing an immobilization-free biosensor for high-speed and ultrasensitive detection of miRNA with a detection limit of 0.14 fM. As a result, the DDNM-CHA we developed carves out a new insight to design a functional DNA nanomachine and evolve the analysis method for practical amplification in the sensing area and promotes the deeper exploration of the nucleic acid signal amplification strategy and DNA nanobiotechnology.

Herein, a programmable dual-catalyst hairpin assembly (DCHA) for realizing the synchronous recycle of two catalysts is developed, displaying high reaction rate and outstanding conversion efficiency beyond traditional nucleic acid signal amplifications (NASA). Once catalyst I interacts with the catalyst II, the DCHA can be triggered to realize the simultaneous recycle of catalysts I and II to keep the highly concentrated intermediate product duplex I-II instead of the steadily decreased one in typical NASA, which can accomplish in about only 16 min and achieves the outstanding conversion efficiency up to 4.54 × 10 8 , easily conquering the main predicaments of NASA: time-consuming and low-efficiency. As a proof of the concept, the proposed DCHA as a high-speed and hyper-efficiency DNA signal magnifier is successfully applied in the rapid and ultrasensitive detection of miRNA-21 in cancer cell lysates, which exploits the new generation of universal strategy for the applications in biosensing assay, clinic diagnose, and DNA nanobiotechnology.