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Background: Amplified imaging of microRNA (miRNA) in cancer cells is essential for understanding of the underlying pathological process. Synthetic catalytic DNA circuits represent pivotal tools for miRNA imaging. However, most existing catalytic DNA circuits can not achieve the reactant recycling operation in cells and in vivo. Additionally, specificity miRNA imaging in tumor site also is a challenge. Herein, inspired by "turning waste into wealth", we report a DNA sensor for imaging of miRNA in target cells based on apurinic/apyrimidinic endonuclease 1 (APE1)-activated reactant recycling catalytic DNA circuit., Results: The sensing function of the DNA sensor is suppressed firstly through engineering an apurinic/apyrimidinic (AP) site. In the presence of APE1, the AP site undergoes hydrolysis, thereby activating sensing activity and triggering the strand displacement reaction (SDR) under miRNA assistance. In this catalytic DNA circuit, the next reaction cycle can be initiated when the duplex strand waste products are reverted into active components, which allows it to be performed continuously just consuming fuel DNA without depleting the reactant. Noteworthy, the liposome plays important role in overcome biological barriers for nucleic acid delivery. The amplified miRNA imaging strategy is carried out in vivo by this DNA sensor with reducing off-tumor signal under assistance with APE1, and enhances tumor-to-normal tissue contrast compared with traditional catalytic DNA circuit., Significance and Novelty: Firstly, APE1-activated reactant recycling catalytic DNA circuit is developed. Secondly, miRNA image in cell and in animal is achieved with high spatial selectivity. Thirdly, the signal-to-background ratio for imaging is improved in vitro and in vivo. Lastly, our strategy provides an automatic yet versatile approach for the development of more efficient and selective DNA circuits capable of differentiating miRNA in cancer cells from those in normal cells, promising to be valuable in cancer diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

DNAzyme-based cascade networks are effective tools to achieve ultrasensitive detection of low-abundance miRNAs. However, their designs are complicated and costly, and the operation is time-consuming. Herein, a novel simple noncascade DNAzyme network is designed and its amplification effect is comparable to or even better than many cascading ones. It is a nonenzymatic, isothermal, bicirculating amplification network consisting of two toehold-mediated strand-displacement reactions and a localized DNAzyme amplification strategy. Taking microRNA-122 as a target model, this ultrasensitive fluorescence biosensor has a detection limit of 84 zmol L -1 , which is 8-orders of magnitude lower than that of the nonamplification one. The ultrasensitivity mainly benefits from the exclusive design and positive self-feedback mechanism of the ingenious bicirculating DNAzyme amplification network. In addition, the utilization of superparamagnetic Fe 3 O 4 @SiO 2 particles not only helps for the localization of DNAzymes but also facilitates the rapid separation of signal probes (output DNA-CdTe QDs). This fluorescent biosensor also has the advantages of specificity, speed, thermal stability, and low cost. This novel design paves a new way to simple and effective bioamplification strategy, which may be very attractive for biosensors, DNA logic gates, and DNA computers.

A biosensor based on solid-state nanochannels of anodic aluminum oxide (AAO) membrane for both electrochemical and naked-eye detection of microRNA-31 (MiR-31) is proposed. For this purpose, MoS 2 nanosheets, which possess different adsorption capabilities to single-stranded and double-stranded nucleic acids, are deposited onto the top surface of the AAO membrane. Moreover, multi-functional DNA nanostructure have been designed by linking a G-rich sequence for folding to a G-quadruplex at three vertices and a complementary sequence of MiR-31 at the other one vertex of a DNA tetrahedron. In the absence of MiR-31, the tetrahedron DNAzyme probe formed after the addition of hemin can mediate the deposition of insoluble on MoS 2 /AAO, which not only enables the color change of the membrane but also gates the transport of K 3 [Fe(CN) 6 ] across the nanochannels. Therefore, the detection of MiR-31 is realized by both visual observation of the brown color and measuring the electrochemical redox current of [Fe(CN) 6 ] 3- . Using this biosensor, a detection limit as low as 0.06 fM is achieved. The dual-mode detection method also exhibits good specificity, reproducibility, and stability, demonstrating potential application in the diagnosis of oral squamous cell carcinoma and other related biological purposes., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The concentration elevation of myocardial microRNA (miRNA) biomarker is associated with the pathogenic process of acute myocardial infarction (AMI), and sensitive quantification of myocardial miRNA biomarker plays an important role for early AMI diagnosis and its treatment. In response, this work describes an ultrasensitive and non-label electrochemical biosensor for the assay of myocardial miRNA based on cascade signal amplifications integrated by DNAzyme walker and hemin/G-quadruplex nanowires. The DNAzyme walker is activated by presence of target miRNAs to move along the electrode surface to cyclically cleave the substrate hairpins to release G-quadruplex segments, which further trigger the in situ formation of many hemin/G-quadruplex nanowires. The large amounts of hemin intercalated into the DNA nanowires subsequently generate drastically magnified electrochemical current signals for highly sensitive label-free assay of myocardial miRNAs down to 15.7 fM within dynamic range of 100 fM to 10 nM. Such a biosensor also has high selectivity and can monitor myocardial miRNAs in diluted serums at low levels, providing a sensitive and reliable platform for diagnosing infarct-associated cardiovascular diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In situ monitoring microRNA (miRNA) expression in vivo holds immense potential for directly visualizing the occurrence and progression of tumors. However, the significant barrier to developing a probe that can overcome the low abundance of miRNAs while providing an output signal with unlimited tissue penetration depth remains formidable. In this study, we developed a DNA machine-based magnetic resonance imaging nanoprobe (MRINP) for amplified detection of miR-21 in vivo. The MRINP was constructed with superparamagnetic Fe 3 O 4 nanoparticles (NPs), paramagnetic Gd-DOTA complexes, and miR-21-activated DNA machines; the DNA machine was composed of hairpin DNAzyme (HD) strands serving as the DNAzyme walker and hairpin substrate (HS) strands serving as the track. Once uptake into tumor cells, the intracellular miR-21 specifically recognized and hybridized with the HD strand, restoring the activity of DNAzyme. Subsequently, the DNAzyme walker autonomously traveled on the surface of MRINP, and each step movement of the DNAzyme walker resulted in the cleavage of its substrate strands and the ensued release of the Gd-DOTA complex-labeled oligonucleotides, turning on the T 1 signal of Gd-DOTA complexes for in situ imaging of miR-21 in tumor-bearing mice. This strategy would offer a promising approach for mapping tumor-specific biomarkers in vivo with unlimited penetration depth., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Precise and spatiotemporally controllable analysis of microRNA-21 in living cells is crucial for accurate diagnosis and effective treatment of related diseases. Herein, a near-infrared (NIR)-photoactivatable DNA nanomachine (PUCNPs-NH 2 /PEG-ZL-DNA) was constructed for the precise analysis and diagnosis of microRNA-21 in tumor cells. Peanut-shaped upconversion nanoparticles (PUCNPs) were employed as the carriers and activators for the intelligent DNA probe, specifically enabling the cleavage of the photocleavable linker (PC-linker) from the hairpin DNA probe (Hp-Dzy) upon exposure to 808 nm irradiation. In the presence of the target microRNA-21, the locker DNA hybridized with microRNA-21 and the DNAzymes was freed to hybridize with the looped portion of the hairpin DNA (Hp-1). Mg 2+ was employed as the cofactor, facilitating the precise cleavage of Hp-1, which triggered the restoration of fluorescence signals. Subsequently, DNAzymes exhibited the competency to selectively recognize and engage with additional Hp-1, and the fluorescence signals were effectively amplified by the recycling process. Consequently, the DNA nanomachine exhibited a linear response to microRNA-21 concentrations ranging from 0.5 nM to 1.0 μM, achieving a remarkable detection limit (LOD) of 1.19 nM under the optimal conditions. This strategy is realized through the integration of photocontrollable upconversion nanotechnology with the signal amplification approach, showing feasible prospects for spatiotemporally precise and highly sensitive monitoring of microRNA in tumor cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Insight into the epigenetic modulation-correlated molecule interactions has significant implications for the in-depth understanding of intracellular intricate biological networks. However, there is currently a lack of reliable biological tools for elucidating the potential correlation between epigenetic regulators and relevant genes, e.g., microRNAs (miRNAs). Herein, an alkB homologue 5 (ALKBH5, a key epigenetic regulator)-modulated catalytic DNA circuit (ACD) was constructed by grafting a N6-methyladenosine (m 6 A)-caged I-R3 DNAzyme into the circuitry components for achieving the on-site miRNA imaging in living cells. Specifically, the catalytic activity of I-R3 DNAzyme could be effectively suppressed by the m 6 A modification situated at its highly sequence-conserved core region and then be selectively restored through the ALKBH5-mediated demethylation pathway. And the ALKBH5-activated I-R3 DNAzyme allowed the highly efficient DNA cleaving reaction in the presence of DNAzyme cofactors, resulting in the liberation of catalytic hairpin assembly (CHA) reactants. Subsequently, target miRNA triggered the CHA circuit to produce a duplex DNA product while releasing the miRNA analyte. The liberated miRNA could autonomously trigger the next round of the CHA assembly cycle for generating the amplified fluorescence readout. By virtue of the stimuli-responsive activation and the CHA amplification circuit, the ACD system achieved highly specific and sensitive imaging of miRNA in tumor cells. Moreover, this efficiently and reliably ALKBH5-activated DNA circuit is demonstrated to reveal the underlying relationship between activator ALKBH5 and miRNA. Overall, the developed ACD system provides a promising tool for the robust on-site profiling of epigenetic-involved signal pathways, thus displaying great potential in bioanalytical applications.

We present a novel DNA molecular machine (RCA-D-Walker) that integrates a DNAzyme-based molecular beacon with RCA-based vectors for miRNA imaging in tumor cells. It can accurately target tumor cells through the sgc8 aptamer. The target miRNA can restore the DNAzyme's ability to cleave the substrate, which in turn produces an amplified fluorescent signal. The RCA-D-Walker exhibits enhanced tumor cell targeting, improved cell permeability, and greater resistance to nuclease degradation. Utilizing this strategy, we achieved accurate and efficient imaging of miRNA-21 in tumor cells.

Aging is a critical global issue that contributes to the high incidence of Alzheimer's disease (AD). Blood screening emerges as the most promising measure for early diagnosis and intervention of AD due to its noninvasive and low cost. However, the practical application of AD blood screening confronts two significant challenges. First, due to the blood-brain barrier, the concentration of AD biomarkers in blood is much lower than that in cerebrospinal fluid. Second, simultaneous quantitative analysis of multiple biomarkers is necessary due to the low specificity of individual biomarkers. Herein, we propose DNAzyme-based 3D DNA walkers for the sensitive and multiplex detection of five AD-associated miRNA biomarkers: hsa-miR-125b, hsa-miR-342-3p, hsa-miR-29b, hsa-miR-191-5p, and hsa-miR-7d-5. The DNAzyme-based 3D DNA walkers provide highly efficient and autonomous amplification of the minimal biomarkers' quantities. The walking-released metal isotopes 89 Y, 165 Ho, 139 La, 140 Ce, and 159 Tb can be sensitively detected by elemental mass spectrometry without any spectral overlap. The detection limit was achieved to be as low as 1.0 fmol. The proposed method was successfully applied to human serum samples with satisfactory spiked recoveries. With its high sensitivity and multiplexity capabilities, this metal isotope strategy may contribute to the early diagnosis and intervention of AD.

While the intracellular imaging of miRNA biomarkers is of significant importance for the diagnosis and treatment of human cancers, DNA assembled nanoprobe has recently attracted considerable attention for imaging intracellular biomolecules. However, the complex construction process, intrinsic vulnerability to nuclease degradation and the limited signal transduction efficiency hamper its widespread application. In this contribution, based on persistent autonomous molecular motion of DNAzyme walker along a nano-substrate track, a DNA nanosphere probe (PNLD) is developed for the sensitive intracellular miR-21 imaging. Specifically, DNA nanosphere (called PN, single-molecule nano-track) is assembled from only one palindromic substrate, into which the locking strand-silenced DNAzymes (LD) are installed in a controlled manner. PNLD (made of PN and LD) can protect all DNA components against nuclease attack and maintain its structural integrity in serum solution over 24 h. Upon the activation by target miRNA, DNAzyme walker can move on the substrate scattered within PNLD (or on the surface) and between different PNLD objects and cleave many DNA substrates, generating an amplified signal. As a result, miR-21 can be detected down to 6.83 pM without the detectable interference from co-existing nontarget miRNAs. Moreover, PNLD system can accurately screen the different expression levels of miR-21 within the same type of cells and different types of cells, which is consistent with gold standard polymerase chain reaction (PCR) assay. Via changing the target recognition sequence, the PNLD system can be suitable for the intracellular imaging of miR-155, exhibiting the desirable universality. In addition, the DNAzyme walker-based PNLD system can be used to distinguish cancer cells from healthy cells, implying the potential application in cancer diagnosis and prognosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cascaded amplification showed promising potential for detection of trace target miRNAs in molecular diagnosis and prevention of many diseases. In this study, miRNA21 was chosen as the target, and rolling circle amplification (RCA)-based DNA nanoscaffold was integrated with target triggered RNA-cleaving DNAzyme for sensitive detection of miRNA21. That is, the H1 probe was bound with the long-chain product of RCA to self-assemble into DNA nanoscaffold. Target miRNA21 triggered the hybridization chain reaction (HCR) located on the nanoscaffold, and led to rapid proximity of DNAzyme fragments modified at both ends of the H2 probe, which realized the cyclic cleavage of self-quenching substrate probe efficiently, and the fluorescence signal was restored. The results demonstrated that the proposed assay was sensitive, 0.76 pM of miRNA21 can be detected. The proposed assay was specific; only one-base mismatched miRNA21 can be effectively recognized, other nucleic acid sequence and the serum matrix did not cause any interference. The proposed assay was accurate; recoveries from 82.1 to 115.0% can be obtained in the spiked fetal bovine serum (FBS). The flexible and programmable characteristics of DNA nanoscaffold and DNAzyme provide a confident and robust strategy for more sensitive nucleic acid detection, and can be developed to be a universal sensing platform for detecting other miRNAs just needing modification on the corresponding sequence of H1 probe in HCR., Competing Interests: Declarations. Ethics Approval: Not applicable. Consent to Participate: Not applicable. Consent for Publication: Not applicable. Conflict of Interest: The authors declare no conflict of interest., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Simple yet specific miRNA detection remains an enormous challenge due to its low abundance in samples and the high similarity among homologous miRNAs. Here, we propose a novel fluorescent approach for miRNA detection with greatly elevated accuracy by utilizing exonuclease-iii (Exo-iii) assisted twice target recognition. The proposed method involves a "Sensing probe" engineered with two loop sections to facilitate dual target miRNA recognition. The collaboration between Exo-iii and miRNA initiates target recycling for signal amplification, resulting in the formation of complete DNAzyme. The intact DNAzyme connects with the "Signal probe" and creates a nicking site within its loop region. The fluorescence signal of the "Signal probe" reemerges, correlating with the quantity of miRNA in the sensing system. The suggested technique demonstrates great selectivity for target miRNA and can readily differentiate sequences with a one-base mismatch, based on dual-target identification. Furthermore, the Exo-iii-assisted signal recycling imparts the approach with great sensitivity and a low detection limit of 548 aM. This method has the potential to be a robust alternative for the detection of miRNAs in real samples due to its high accuracy, simplicity, and resistance to potential fluorescence interferences., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A product-induced catalytic amplification (PICA) strategy had been developed for miRNA-21 detection based on DNA tetrahedron module (DTM). The produced DNA fragment could open hairpin structure and increase the concentration of catalyst, accelerating the circular cleavage reaction on DTM by DNAzyme cleavage. The continuously cleavage of DNAzyme on DTM resulted the greatly enhancement of signal. A favorable linear range was achieved from 20 pM to 5 nM with a limit of detection of 7 pM. Furthermore, through the implementation of the PICA strategy, the overall reaction time experienced a noticeable decrease to 30 min. The assessments of the amplification rate and kinetic constant of the PICA strategy were also conducted. These results highlighted the promising potential of the PICA strategy for practical utilization in serum samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Biomolecules play essential roles in regulating the orderly progression of biochemical reaction networks. DNA-based biocircuits supplement an attractive and ideal approach for the visual imaging of endogenous biomolecules, yet their sensing performance is commonly encumbered by the undesired signal leakage. To solve this issue, here we proposed a glutathione (GSH)-activated DNA circuit for achieving the spatio-selective microRNA imaging through the successive response of a GSH-specific activation procedure and a non-enzymatic catalytic signal amplification procedure. In this design, by incorporating a disulfide bond into the pre-sealed nucleic acid probe, the uncontrolled circuitry leakage could be effectively ameliorated. In target cancer cells with high-abundant GSH and miR-21, endogenous GSH recognized and cleaved the pre-installed disulfide bond within DNA probes, thereby restoring the activity of circuitry components. The miR-21 then catalyzed the specific operation of circuitry for generating an amplified readout signal. We demonstrate that this system not only enables the effective discriminations of various cell types, but also contributes to the exploration of the correlationship between GSH and miR-21. This on-site activated DNA circuit can be extended to the robust analysis and exploration of different biomolecular interactions, offering a reliable reference for the in-depth understanding of biochemical interaction networks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As a post transcriptional regulator of gene expression, microRNAs (miRNA) is closely related to many major human diseases, especially cancer. Therefore, its precise detection is very important for disease diagnosis and treatment. With the advancement of fluorescent dye and imaging technology, the focus has shifted from in vitro miRNA detection to in vivo miRNA imaging. This concept review summarizes signal amplification strategies including DNAzyme catalytic reaction, hybrid chain reaction (HCR), catalytic hairpin assembly (CHA) to enhance detection signal of lowly expressed miRNAs; external stimuli of ultraviolet (UV) light or near-infrared region (NIR) light, and internal stimuli such as adenosine triphosphate (ATP), glutathione (GSH), protease and cell membrane protein to prevent nonspecific activation for the avoidance of false positive signal; and the development of fluorescent probes with emission in NIR for in vivo miRNA imaging; as well as rare earth nanoparticle based the second near-infrared window (NIR-II) nanoprobes with excellent tissue penetration and depth for in vivo miRNA imaging. The concept review also indicated current challenges for in vivo miRNA imaging including the dynamic monitoring of miRNA expression change and simultaneous in vivo imaging of multiple miRNAs., (© 2024 Wiley-VCH GmbH.)

Acrylamide (AA) in heat-processed foods has emerged as a global health problem, mainly carcinogenic, neurotoxic, and reproductive toxicity, and an increasing number of researchers have delved into elucidating its toxicological mechanisms. Studies have demonstrated that exposure of HepG2 by AA in a range of concentrations can induce the upregulation of miR-21 and miR-221. Monitoring the response of intracellular miRNAs can play an important role in unraveling the mechanisms of AA toxicity. Here, multicolor aggregation induced emission nano particle (AIENP) probes were constructed from three AIE dyes for simultaneous imaging of intracellular AA and AA-induced miR-21/miR-221 by combining the recognition function of AA aptamers and the signal amplification of a DNAzyme walker. The surface of these nanoparticles contains carboxyl groups, facilitating their linkage to a substrate chain modified with a fluorescent quencher group via an amide reaction. Optimization experiments were conducted to determine the optimal substrate-to-DNAzyme ratio, confirming its efficacy as a walker for signal amplification. Sensitive detection of AA, miR-21 and miR-221 was achieved in extracellular medium, with detection limits of 0.112 nM for AA, 0.007 pM and 0.003 pM for miR-21 and miR-221, respectively, demonstrating excellent selectivity. Intracellularly, ZIF-8 structure collapsed, releasing Zn 2+ , activating DNAzyme cleavage activity, and the fluorescence of multicolor AIENPs within HepG2 cells gradually recovered with increasing stimulation time (0-12 h) and concentrations of AA (0-500 μM). This dynamic response unveiled the relationship between AA exposure and miR-21/miR-221 expression, further validating the carcinogenicity of AA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sensitive imaging of microRNAs (miRNAs) in tumor cells holds great significance in the domains of pathology, drug development, and personalized diagnosis and treatment. DNA nanostructures possess excellent biostability and programmability and are suitable as carriers for intracellular imaging probes. With its highly controllable motion mechanism and remarkable target recognition specificity, the DNA walker is an ideal tool for living cell imaging. Here, we report a DNA nanowire based-DNAzyme Walker (D-Walker), which loads the DNAzyme based-molecular beacon (D-MB) onto DNA nanowires (NWs) functionalized with aptamers. The experimental results demonstrated that the intracellular target miRNA can specifically activate the pre-locked DNAzyme through a strand displacement reaction, thereby triggering the cleavage of its substrate molecular beacon (MB) and subsequent fluorescence emission. NWs decorated with aptamers can effectively prevent the degradation of the D-Walker by nuclease, and can enter target cells without any transfection reagents, which enhances the stability and reliability of cell imaging. Furthermore, the D-Walker exhibited a remarkable sensitivity with a limit of detection (LOD) of 61 pM and was capable of distinguishing miRNA-21 from other closely related family members. This study provides a novel strategy for intracellular miRNA imaging, offering a promising tool for cancer diagnosis and treatment.

MicroRNAs (miRNAs) play an important role in the process of heart failure (HF) and are emerging biomarkers that can be used for the auxiliary diagnosis of HF. However, it is very challenging to accurately analyze the expression levels of trace miRNAs in complex clinical samples. Here, we developed an enzyme-free colorimetric sensor for the ultrasensitive detection of miRNA-423-5p (HF-associated miRNA) based on three-dimensional DNA walkers constructed from functional nucleic acids and gold nanoparticles (AuNPs). DNAzyme with cleavage activity was specifically activated by miRNA-423-5p to sustainably cleave the substrate, thereby releasing the trigger sequence to initiate the subsequent mismatched catalytic hairpin assembly (MCHA) cycle. Then, as the MCHA cycle proceeded to continuously expose the G-quadruplex (GQ) sequence, the sequence bound with hemin to form a large amount of GQ/hemin DNAzyme on the surface of the AuNPs, which rapidly catalyzed the chromogenic oxidation of 3,3',5,5'-tetramethylbenzidine to yield an amplified colorimetric signal readout. The colorimetric sensor exhibited an ultralow detection limit (32 fM), showed excellent specificity and performed well in serum samples. The sensor was applied to detect miRNA-423-5p in clinical plasma samples from healthy individuals and HF patients, and the results revealed its good clinical application in HF diagnosis. Thus, the developed colorimetric sensor provides a convenient detection tool for early screening and diagnosis of HF, as well as for pathophysiological studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this work, we reported an electrochemical biosensor with target-assisted self-cleavage DNAzyme function for signal amplified detection of miRNA. The target-recycling amplification led to significant signal enhancement and thus offers high detection sensitivity.

A target-induced multiregion MNAzyme nanowire system is designed for the ultrasensitive and homogeneous detection of microRNAs (miRNAs). miRNA-21 and miRNA-375 are chosen as analytes, and a miRNA-induced primer exchange reaction (PER) is utilized to construct a long DNA strand with repetitive sequences. This innovative design enables the efficient anchoring of numerous MNAzymes. This unique architecture significantly boosts the effective local concentration of MNAzymes, thereby enhancing the sensitivity and efficiency of miRNA detection. Notably, the limit of detection (LOD) achieved with our target-induced multiregion MNAzyme nanowire approach is over an order of magnitude lower than most other MNAzyme-based methods, while the MNAzyme reaction time is reduced from several hours to 50 min. The method has demonstrated successful applications in quantitatively determining the expression levels of two miRNAs in cell lysates of MCF-7, HeLa and MCF-10 A cells, highlighting its potential for assaying miRNA biomarkers in clinical samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this work, by ingeniously integrating catalytic hairpin assembly (CHA), double-end Mg 2+ -dependent DNAzyme, and hybridization chain reaction (HCR) as a triple cascade signal amplifier, an efficient concatenated CHA-DNAzyme-HCR (CDH) system was constructed to develop an ultrasensitive electrochemical biosensor with a low-background signal for the detection of microRNA-221 (miRNA-221). In the presence of the target miRNA-221, the CHA cycle was initiated by reacting with hairpins H1 and H2 to form DNAzyme structure H1-H2, which catalyzed the cleavage of the substrate hairpin H0 to release two output DNAs (output 1 and output 2). Subsequently, the double-loop hairpin H fixed on the electrode plate was opened by the output DNAs, to trigger the HCR with the assistance of hairpins Ha and Hb. Finally, methylene blue was intercalated into the long dsDNA polymer of the HCR product, resulting in a significant electrochemical signal. Surprisingly, the double-loop structure of the hairpin H could prominently reduce the background signal for enhancing the signal-to-noise ratio (S/N). As a proof of concept, an ultrasensitive electrochemical biosensor was developed using the CDH system with a detection limit as low as 9.25 aM, achieving favorable application for the detection of miRNA-221 in various cancer cell lysates. Benefiting from its enzyme-free, label-free, low-background, and highly sensitive characteristics, the CDH system showed widespread application potential for analyzing trace amounts of biomarkers in various clinical research studies.

Electrochemical detection of miRNA biomarkers in complex physiological samples holds great promise for accurate evaluation of tumor burden in the perioperative period, yet limited by reproducibility and bias issues. Here, nanosensors installed with hybrid probes that responsively release catalytic DNAzymes (G-quadruplexes/hemin) were developed to solve the fidelity challenge in an immobilization-free detection. miRNA targets triggered toehold-mediated strand displacement reactions on the sensor surface and resulted in amplified shedding of DNAzymes. Subsequently, the interference background was removed by Fe 3 O 4 core-facilitated magnetic separation. Binding aptamers of the electrochemical reporter (dopamine) were tethered closely to the catalytic units for boosting H 2 O 2 -mediated oxidation through proximity catalysis. The one-to-many conversion by dual amplification from biological-chemical catalysis facilitated sufficient homogeneous sensing signals on electrodes. Thereby, the nanosensor exhibited a low detection limit (2.08 fM), and high reproducibility (relative standard deviation of 1.99%). Most importantly, smaller variations (RSD of 0.51-1.04%) of quantified miRNAs were observed for detection from cell lysates, multiplexed detection from unprocessed serum, and successful discrimination of small upregulations in lysates of tumor tissue samples. The nanosensor showed superior diagnostic performance with an area under curve (AUC) of 0.97 and 94% accuracy in classifying breast cancer patients and healthy donors. These findings demonstrated the synergy of signal amplification and interference removal in achieving high-fidelity miRNA detection for practical clinical applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Homogeneous electrochemiluminescence (ECL) has gained attention for its simplicity and stability. However, false positives due to solution background interference pose a challenge. To address this, magnetic ECL nanoparticles (Fe 3 O 4 @Ru@SiO 2 NPs) were synthesized, offering easy modification, magnetic separation, and stable luminescence. These were utilized in an ECL sensor for miRNA-155 (miR-155) detection, with locked DNAzyme and substrate chain (mDNA) modified on their surface. The poor conductivity of long-chain DNA significantly impacts the conductivity and electron transfer capability of Fe 3 O 4 @Ru@SiO 2 NPs, resulting in weaker ECL signals. Upon target presence, unlocked DNAzyme catalyzes mDNA cleavage, leading to shortened DNA chains and reduced density. In contrast, the presence of short-chain DNA has minimal impact on the conductivity and electron transfer capability of Fe 3 O 4 @Ru@SiO 2 NPs. Simultaneously, the material surface's electronegativity decreases, weakening the electrostatic repulsion with the negatively charged electrode, resulting in the system detecting stronger ECL signals. This sensor enables homogeneous ECL detection while mitigating solution background interference through magnetic separation. Within a range of 100 fM to 10 nM, the sensor exhibits a linear relationship between ECL intensity and target concentration, with a 26.91 fM detection limit. It demonstrates high accuracy in clinical sample detection, holding significant potential for clinical diagnostics. Future integration with innovative detection strategies may further enhance sensitivity and specificity in biosensing applications.

Intracellular detection and imaging of microRNAs (miRNAs) with low expression usually face the problem of unsatisfactory sensitivity. Herein, a novel dual-function DNA nanowire (DDN) with self-feedback amplification and efficient signal transduction was developed for the sensitive detection and intracellular imaging of microRNA-155 (miRNA-155). Target miRNA-155 triggered catalytic hairpin assembly (CHA) to generate plenty of double-stranded DNA (dsDNA), and a trigger primer exposed in dsDNA initiated a hybridization chain reaction (HCR) between four well-designed hairpins to produce DDN, which was encoded with massive target sequences and DNAzyme. On the one hand, target sequences in DDN acted as self-feedback amplifiers to reactivate cascaded CHA and HCR, achieving exponential signal amplification. On the other hand, DNAzyme encoded in DDN acted as signal transducers, successively cleaving Cy5 and BHQ-2 labeled substrate S to obtain a significantly enhanced fluorescence signal. This efficient signal transduction coupling self-feedback amplification greatly improved the detection sensitivity with a limit of detection of 160 aM for miRNA-155, enabling ultrasensitive imaging of low-abundance miRNA-155 in living cells. The constructed DDN creates a promising fluorescence detection and intracellular imaging platform for low-expressed biomarkers, exhibiting tremendous potential in biomedical studies and clinical diagnosis of diseases.

Accurate microRNA (miRNA) detection is pivotal in the diagnosis and monitoring of cancer. Entropy-driven catalysis (EDC) has attracted widespread attention as an enzyme-free, isothermal technique for miRNA detection owing to its inherent simplicity and reliability. However, conventional EDC is a single-output mode, limiting the efficiency of signal amplification. In this study, a novel EDC dual-output mode was employed in conjunction with DNAzyme, resulting in the development of an EDC dual-end DNAzyme (EDC-DED) approach for highly sensitive miRNA detection. In this system, miRNA-21 initiated the EDC reaction, producing a large amount of catalytically active dual-end Mg 2+ -dependent DNAzyme. The DNAzyme further cleaved the reporter cyclically, generating a notably amplified fluorescence signal. The proposed method achieved a low detection limit of 2 pM. Compared with the traditional EDC single-end DNAzyme (EDC-SED) strategy, the present method exhibited superior amplification efficiency, enhancing detection sensitivity by approximately 46.5-fold. Furthermore, this platform demonstrated ideal specificity, satisfactory reproducibility and acceptable detection capabilities in clinical serum samples. Therefore, the straightforward and convenient strategy is a potential tool for miRNA analysis, which may provide a new perspective for biological analysis and clinical application., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) can serve as biomarkers for early-diagnosis, therapy, and postoperative care of cervical cancer. Sensitive and reliable quantification of miRNA remains a huge challenge due to its low expressing levels and background interference. Herein, we propose a novel exonuclease-III (Exo-III)-propelled DNAzyme cascade for sensitive and high-efficient miRNA analysis. This method involves the engineering of compact DNAzyme hairpin probes, including the H1 probe and H2 probe. The H1 probe is designed with exposed analyte recognition subunits that can specifically recognize target miRNA. This recognition triggers two processes: Exo-iii-assisted target regeneration and successive substrate cleavage catalyzed by DNAzyme. The unique character of Exo-III that catalyzes removal of mononucleotides from the blunt or recessed 3'-OH termini of dsDNA confers the approach with a minimal background signal. The multiple signal cycles provided an abundant signal amplification and consequently, the method exhibited a low limit of detection of 3.12 fM, and a better specificity over several homologous miRNAs. In summary, this powerful Exo-III driven DNAzyme cascaded system offers broader and more adaptable methods for comprehending the activities of miRNA in various biological occurrences., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The primer-guided entropy-driven high-throughput evolution of the DNA-based constitutional dynamic network, CDN, is introduced. The entropy gain associated with the process provides a catalytic principle for the amplified emergence of the CDN. The concept is applied to develop a programmable, spatially localized DNA circuit for effective in vitro and in vivo theranostic, gene-regulated treatment of cancer cells. The localized circuit consists of a DNA tetrahedron core modified at its corners with four tethers that include encoded base sequences exhibiting the capacity to emerge and assemble into a [2 × 2] CDN. Two of the tethers are caged by a pair of siRNA subunits, blocking the circuit into a mute, dynamically inactive configuration. In the presence of miRNA-21 as primer, the siRNA subunits are displaced, resulting in amplified release of the siRNAs silencing the HIF-1α mRNA and fast dynamic reconfiguration of the tethers into a CDN. The resulting CDN is, however, engineered to be dynamically reconfigured by miRNA-155 into an equilibrated mixture enriched with a DNAzyme component, catalyzing the cleavage of EGR-1 mRNA. The DNA tetrahedron nanostructure stimulates enhanced permeation into cancer cells. The miRNA-triggered entropy-driven reconfiguration of the spatially localized circuit leads to the programmable, cooperative bis-gene-silencing of HIF-1α and EGR-1 mRNAs, resulting in the effective and selective apoptosis of breast cancer cells and effective inhibition of tumors in tumor bearing mice.

MicroRNA is regarded as a significant biomarker for cancer diagnosis, disease process evaluation and therapeutic guidance, and dual-parameter measurement may contribute to a more accurate and realistic assessment. To meet the urgent need for simultaneous detection of multiple biomarkers, we combined three-dimensional DNAzyme motors with single molecule imaging technique to construct a convenient, intuitive, and sensitive approach for the simultaneous detection of dual miRNAs in the free state or in extracellular vesicles. Quantification of target miRNAs can be realized through the detection of amplified fluorescence signals generated by the target miRNA-initiated cleavage of fluorescent substrate strands by the DNAzyme motors. The practicability was systematically validated with microRNA-21-5p and microRNA-10b-5p as targets, acquiring a satisfactory sensitivity sufficient to detect low abundance targets at 0.5 or 1 pM to 100 pM. Besides, the extracellular vesicular miRNAs can be conveniently detected without extraction. The clinical applicability was verified with a series of extracellular vesicles from clinical samples, which exhibited good distinguishability between colorectal cancer patients and healthy donors. In addition to the advantages of good specificity and high sensitivity, the system has potential to be easily adapted by minor alteration of the DNA sequences and fluorophore sets for detection of multiple miRNAs and even other types of biomarkers such as proteins. Therefore, it shows promise to be widely applied in various fields such as early diagnosis of cancer and its prognostic assessment.

MicroRNA (miRNA) is demonstrated to be associated with the occurrence and development of various diseases including cancer. Currently, most miRNA detection methods are confined to in vitro detection and cannot obtain information on the temporal and spatial expression of miRNA in relevant tissues and cells. In this work, we established a novel enzyme-free method that can be applied to both in vitro detection and in situ imaging of miRNA by integrating DNAzyme and catalytic hairpin assembly (CHA) circuits. This developed CHA-Amplified DNAzyme miRNA (CHAzymi) detection system can realize the quantitively in vitro detection of miR-146b (the biomarker of papillary thyroid carcinoma, PTC) ranging from 25 fmol to 625 fmol. This strategy has also been successfully applied to in situ imaging of miR-146b both in human PTC cell TPC-1 and clinical samples, showing its capacity as an alternative diagnostic method for PTC. Furthermore, this CHAzymi system can be employed as a versatile sensing platform for various miRNAs by revising the relevant sequences. The results imply that this system may expand the modality of miRNA detection and show promise as a novel diagnostic tool in clinical settings, providing valuable insights for effective treatment and management of the disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: MicroRNA (miRNA) emerges as important cancer biomarker, accurate detection of miRNA plays an essential role in clinical sample analysis and disease diagnosis. However, it remains challenging to realize highly sensitive detection of low-abundance miRNA. Traditional detection methods including northern blot and real-time PCR have realized quantitative miRNA detection. However, these detection methods are involved in sophisticated operation and expensive instruments. Therefore, the development of novel sensing platform with high sensitivity and specificity for miRNA detection is urgently demanded for disease diagnosis., Results: In this work, a novel electrochemical biosensor was constructed for miRNA detection based on target-driven cascade amplified assembly of electroactive covalent organic frameworks (COFs) on tetrahedral DNA nanostructure with multiplex recognition domains (m-TDN). COFs were employed as nanocarriers of electroactive prussian blue (PB) molecules by the "freeze-drying-reduction" method without the use of DNA as gatekeeper, which was simple, mild and efficient. The target-triggered catalytic hairpin assembly (CHA) and glutathione reduction could convert low-abundance miRNA into a large amount of Mn 2+ . Without the addition of exogenous Mn 2+ , the dynamically-generated Mn 2+ -powered DNAzyme cleavage process induced abundant PB-COFs probe assembled on the four recognition domains of m-TDN, resulting in significantly signal output. Using miRNA-182-5p as the model target, the proposed electrochemical biosensor achieved ultrasensitive detection of miRNA-182-5p in the range of 10 fM-100 nM with a detection limit of 2.5 fM., Significance and Novelty: Taking advantages of PB-COFs probe as the enhanced signal labels, the integration of CHA, Mn 2+ -powered DNAzyme and m-TDN amplification strategy significantly improved the sensitivity and specificity of the biosensor. The designed sensing platform was capable of miRNA detection in complex samples, which provided a new idea for biomarker detection, holding promising potential in clinical diagnosis and disease screening., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) have been involved in many biological processes and are regarded as promising biomarkers. The short sequence, low abundance and highly homologous interference sequences greatly hinder the accurate detection of miRNAs. Here, a cascade branch migration-triggered strand displacement amplification (CBM-TSDA) strategy was developed for the first time for specific and sensitive detection of miRNA-155 (miR-155). In the presence of target miR-155, the CBM was initiated and two Y-shaped probes were eventually produced. Next, the Y-shaped probes were transformed into three-way junction (3WJ) structures and triggered the SDA to produce a large number of G-quadruplex (G4) structures. Finally, the increased fluorescence signal of G4/Thioflavin T (ThT) was used to quantify miR-155. Meanwhile, the colorimetric responses of the G4-hemin DNAzyme could be used as supplementary detection to obtain a dual-mode signal readout. This detection strategy showed high detection sensitivity, and the limit of detection was 0.28 pM in the fluorescence detection mode and 0.34 pM in the colorimetric detection mode. Notably, it showed high detection specificity, being able to discriminate the single-base mutations of the target with a high discrimination factor. The strategy also possessed excellent capacity for miR-155 detection in cell lysates and real human blood samples. The developed strategy provides a promising detection platform for miRNA, which may be applied to early clinical diagnosis.

Herein, a dual self-protected DNAzyme-based 3D DNA walker (dSPD walker), composed of activated dual self-protected walking particles (ac-dSPWPs) and track particles (TPs), was constructed for ultrasensitive and ultrahigh-speed fluorescence detection and imaging of microRNAs (miRNAs) in living cells. Impressively, compared with the defect that "one" target miRNA only initiates "one" walking arm of the conventional single self-protected DNAzyme walker, the dSPD walker benefits from the secondary amplification and spatial confinement effect and could guide "one" target miRNA to generate " n " secondary targets, thereby initiating " n " nearby walking strands immediately, realizing the initial rate over one-magnitude-order faster than that of the conventional one. Moreover, in the process of relative motion between ac-dSPWPs and TPs, the ac-dSPWPs could cleave multiple substrate strands simultaneously to speed up movement and reduce the derailment rate, as well as combine with successive TPs to facilitate a large amount of continuous signal accumulation, achieving an ultrafast detection of miRNA-221 within 10 min in vitro and high sensitivity with a low detection limit of 0.84 pM. In addition, the DNA nanospheres obtained by the rolling circle amplification reaction can capture the Cy5 fluorescence dispersed in liquids, which achieves the high-contrast imaging of miRNA-221, resulting in further ultrasensitive imaging of miRNA-221 in cancer cells. The proposed strategy has made a bold innovation in the rapid and sensitive detection as well as intracellular imaging of low-abundance biomarkers, offering promising application in early diagnosis and relevant research of cancer and tumors.

Epigenetic modification plays an indispensable role in regulating routine molecular signaling pathways, yet it is rarely used to modulate molecular self-assembly networks. Herein, we constructed a bioorthogonal demethylase-stimulated DNA circuitry (DSC) system for high-fidelity imaging of microRNA (miRNA) in live cells and mice by eliminating undesired off-site signal leakage. The simple and robust DSC system is composed of a primary cell-specific circuitry regulation (CR) module and an ultimate signal-transducing amplifier (SA) module. After the modularly designed DSC system was delivered into target live cells, the DNAzyme of the CR module was site-specifically activated by endogenous demethylase to produce fuel strands for the subsequent miRNA-targeting SA module. Through the on-site and multiply guaranteed molecular recognitions, the lucid yet efficient DSC system realized the reliably amplified in vivo miRNA sensing and enabled the in-depth exploration of the demethylase-involved signal pathway with miRNA in live cells. Our bioorthogonally on-site-activated DSC system represents a universal and versatile biomolecular sensing platform via various demethylase regulations and shows more prospects for more different personalized theragnostics.

DNA nanostructures with diverse biological functions have made significant advancements in biomedical applications. However, a universal strategy for the efficient production of DNA nanostructures is still lacking. In this work, a facile and mild method is presented for self-assembling polyethylenimine-modified carbon dots (PEI-CDs) and DNA into nanospheres called CANs at room temperature. This makes CANs universally applicable to multiple biological applications involving various types of DNA. Due to the ultra-small size and strong cationic charge of PEI-CDs, CANs exhibit a dense structure with high loading capacity for encapsulated DNA while providing excellent stability by protecting DNA from enzymatic hydrolysis. Additionally, Mg 2+ is incorporated into CANs to form Mg@CANs which enriches the performance of CANs and enables subsequent biological imaging applications by providing exogenous Mg 2+ . Especially, a DNAzyme logic gate system that contains AND and OR Mg@CANs is constructed and successfully delivered to tumor cells in vitro and in vivo. They can be specifically activated by endogenic human apurinic/apyrimidinic endonuclease 1 and recognize the expression levels of miRNA-21 and miRNA-155 at tumor sites by logic biocomputing. A versatile pattern for delivery of diverse DNA and flexible logic circuits for multiple miRNAs imaging are developed., (© 2024 Wiley‐VCH GmbH.)

Manipulating the expression of cellular genes through efficient CRISPR/Cas9 delivery is rapidly evolving into a desirable tumor therapeutics. The exposure of CRISPR/Cas9 to a complex external environment poses challenges for conventional delivery carriers in achieving responsive and accurate release. Here, we report a Trojan horse-like nanocapsule for the on-demand delivery of CRISPR/Cas9 in a microRNA-responsive manner, enabling precise tumor therapy. The nanocapsule comprises a nanoassembled, engineered DNAzyme shell encasing a Cas9/sgRNA complex core. The DNAzyme, functioning as a catalytic unit, undergoes a conformational change in the presence of tumor-associated microRNA, followed by activating a positive feedback-driven autonomous catabolic cycle of the nanocapsule shell. This catabolic cycle is accomplished through chain reactions of DNAzyme "cleavage-hybridization-cleavage", which ensures sensitivity in microRNA recognition and effective release of Cas9/sgRNA. Utilizing this Trojan horse-like nanocapsule, as low as 1.7 pM microRNA-21 can trigger the on-demand release of Cas9/sgRNA, enabling the specific editing of the protumorigenic microRNA coding gene. The resulting upregulation of tumor suppressor genes induces apoptosis in tumor cells, leading to significant inhibition of tumor growth by up to 75.94%. The Trojan horse-like nanocapsule, with superior programmability and biocompatibility, is anticipated to serve as a promising carrier for tailoring responsive gene editing systems, achieving enhanced antitumor specificity and efficacy.

Small extracellular vesicle-derived microRNAs (sEV-miRNAs) have emerged as promising noninvasive biomarkers for early cancer diagnosis. Herein, we developed a molecular probe based on three-dimensional (3D) multiarmed DNA tetrahedral jumpers (mDNA-Js)-assisted DNAzyme activated by Na + , combined with a disposable paper-based electrode modified with a Zr-MOF-rGO-Au NP nanocomplex (ZrGA) to fabricate a novel biosensor for sEV-miRNAs Assay. Zr-MOF tightly wrapped by rGO was prepared via a one-step method, and it effectively aids electron transfer and maximizes the effective reaction area. In addition, the mechanically rigid, and nanoscale-addressable mDNA-Js assembled from the bottom up ensure the distance and orientation between fixed biological probes as well as avoid probe entanglement, considerably improving the efficiency of molecular hybridization. The fabricated bioplatform achieved the sensitive detection of sEV-miR-21 with a detection limit of 34.6 aM and a dynamic range from100 aM to 0.2 µM. In clinical blood sample tests, the proposed bioplatform showed results highly consistent with those of qRT-PCRs and the signal increased proportionally with the NSCLC staging. The proposed biosensor with a portable wireless USB-type analyzer is promising for the fast, easy, low-cost, and highly sensitive detection of various nucleic acids and their mutation derivatives, making it ideal for POC biosensing., (© 2024. The Author(s).)

Catalytic DNA circuits are desirable for sensitive bioimaging in living cells; yet, it remains a challenge to monitor these intricate signal communications because of the uncontrolled circuitry leakage and insufficient cell selectivity. Herein, a simple yet powerful DNA-repairing enzyme (APE1) activation strategy is introduced to achieve the site-specific exposure of a catalytic DNA circuit for realizing the selectively amplified imaging of intracellular microRNA and robust evaluation of the APE1-involved drug resistance. Specifically, the circuitry reactants are firmly blocked by the enzyme recognition/cleavage site to prevent undesirable off-site circuitry leakage. The caged DNA circuit has no target-sensing activity until its circuitry components are activated via the enzyme-mediated structural reconstitution and finally transduces the amplified fluorescence signal within the miRNA stimulation. The designed DNA circuit demonstrates an enhanced signal-to-background ratio of miRNA assay as compared with the conventional DNA circuit and enables the cancer-cell-selective imaging of miRNA. In addition, it shows robust sensing performance in visualizing the APE1-mediated chemoresistance in living cells, which is anticipated to achieve in-depth clinical diagnosis and chemotherapy research.

Advancements in DNA computation have unlocked molecular-scale information processing possibilities, utilizing the intrinsic properties of DNA for complex logical operations with transformative applications in biomedicine. DNA computation shows promise in molecular diagnostics, enabling precise and sensitive detection of genetic mutations and disease biomarkers. Moreover, it holds potential for targeted gene regulation, facilitating personalized therapeutic interventions with enhanced efficacy and reduced side effects. Herein, we have developed six DNAzyme-based logic gates able to process YES, AND, and NOT Boolean logic. The novelty of this work lies in their additional functionalization with a common DNA scaffold for increased cooperativity in input recognition. Moreover, we explored hierarchical input binding to multi-input logic gates, which helped gate optimization. Additionally, we developed a new design of an allosteric hairpin switch used to implement NOT logic. All DNA logic gates achieved the desired true-to-false output signal when detecting a panel of miRNAs, known for their important role in malignancy regulation. This is the first example of DNAzyme-based logic gates having all input-recognizing elements integrated in a single DNA nanostructure, which provides new opportunities for building DNA automatons for diagnosis and therapy of human diseases.

The identification of Chinese medicinal herbs occupies a crucial part in the development of the food and drug market. Although molecular identification based on real-time PCR offers good versatility and uniform digital standards compared with traditional methods, such as morphology, the dependence on large-scale equipment hinders spot detection and marketable applications. In this study, we developed a DNA nanoclaw for colorimetric detection and visible on-site identification of Chinese medicines. When specific miRNA is present, the DNAzyme is activated and cleaves the substrate strand, triggering the catalytic hairpin assembly (CHA) reaction and forming branched DNA junctions on AuNP-I. This can then capture AuNP-II through hybridization and facilitate their aggregation, resulting in a noticeable color change that is observable to the naked eye. By harnessing the dual amplification of DNAzyme and CHA, this highly sensitive nanoprobe successfully achieved specific identification of Chinese medicines. This offers a new perspective for on-site testing in the herbal market.

While three-dimensional (3D) DNA walking amplifiers hold considerable promise in the construction of advanced DNA-based fluorescent biosensors for bioimaging, they encounter certain difficulties such as inadequate sensitivity, premature activation, the need for exogenous propelling forces, and low reaction rates. In this contribution, a variety of profitable solutions have been explored. First, a catalytic hairpin assembly (CHA)-achieved nonenzymatic isothermal nucleic acid amplification is integrated to enhance sensitivity. Subsequently, one DNA component is simply functionalized with a photocleavage-bond to conduct a photoresponsive manner, whereby the target recognition occurs only when the biosensor is exposed to an external ultraviolet light source, overcoming premature activation during biodelivery. Furthermore, a special self-propelling walking mechanism is implemented by reducing biothiols to MnO 2 nanosheets, thereby propelling forces that are self-supplied to a Mn 2+ -reliant DNAzyme. By carrying the biosensing system with a DNA molecular framework to induce a unique concentration localization effect, the nucleic acid contact reaction rate is notably elevated by 6 times. Following these, an ultrasensitive in vitro detection performance with a limit of detection down to 2.89 fM is verified for a cancer-correlated microRNA biomarker (miRNA-21). Of particular importance, our multiple concepts combined 3D DNA walking amplifier that enables highly efficient fluorescence bioimaging in live cells and even bodies, exhibiting a favorable application prospect in disease analysis.

Simultaneous quantifications of multiple miRNAs in the single-sampling system would be conducive to the accurate diagnosis of diseases in contrast with single miRNA analysis. In this work, a stochastic DNAzyme motor on spherical nucleic acids (SNAs) for simultaneous fluorescence assay of double miRNAs was established. Hairpin 1 (H1)-FAM-7a and H1-TAMRA-133a-functionalized magnetic beads (MBs) as SNAs were mixed. Targets (let-7a and miRNA-133a) reacted with two different S1 and S2, triggering the formation of two types of metal DNAzymes. The DNAzymes can further react with H1 stem-loop DNA on SNAs to release the two fluorescent DNA-FAM and DNA-TAMRA fragments in the presence of Mg 2+ . Meanwhile, the DNAzyme as DNA motors were separated from the previous H1 probe to participate the next cycling operations, resulting in the signal amplification toward the simultaneous and sensitive detection of let-7a and miRNA-133a. SNAs with three dimensional nanostructures provided enough space for the operation of DNAzyme walker, promoting the sensitivity of this proposed analytical system. The two mixed SNAs enable one-step and specific quantification of miRNA let-7a and miRNA-133a with lower detection limits of 90.5 fM and 74.9 fM, respectively. Finally, this proposed strategy was employed to simultaneously detect double miRNAs in practical applicability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The engineering of catalytic hybridization DNA circuits represents versatile ways to orchestrate a complex flux of molecular information at the nanoscale, with potential applications in DNA-encoded biosensing, drug discovery, and therapeutics. However, the diffusive escape of intermediates and unintentional binding interactions remain an unsolved challenge. Herein, we developed a compact, yet efficient, self-regulatory assembly circuit (SAC) for achieving robust microRNA (miRNA) imaging in live cells through DNA-templated guaranteed catalytic hybridization. By integrating the toehold strand with a preblocked palindromic fragment in the stem domain, the proposed miniature SAC system allows the reactant-to-template-controlled proximal hybridization, thus facilitating the bidirectional-sustained assembly and the localization-intensified signal amplification without undesired crosstalk. With condensed components and low reactant complexity, the SAC amplifier realized high-contrast intracellular miRNA imaging. We anticipate that this simple and template-controlled design can enrich the clinical diagnosis and prognosis toolbox.

Improving the accuracy, sensitivity and speed of intracellular miRNA imaging is essential for early diagnosis of cancer. To achieve this goal, we herein present a strategy for imaging two distinct miRNAs by DNA tetrahedron-based catalytic hairpin assembly (DCHA). Two nanoprobes, DTH-13 and DTH-24, were prepared by one-pot synthesis. The resultant structures were DNA tetrahedrons functionalized with two sets of CHA hairpins, which respectively responded to miR-21 and miR-155. Using these structured DNA nanoparticles as the carriers, the probes could easily enter living cells. The presence of miR-21 or miR-155 could trigger CHA between DTH-13 and DTH-24, leading to independent fluorescence signals of FAM and Cy3. In this system, the sensitivity and kinetics were significantly enhanced owing to the strategy of DCHA. The sensing performance of our method was thoroughly investigated in buffers, fetal bovine serum (FBS) solutions, living cells, and clinical tissue samples. The results validated the potential of DTH nanoprobes as a diagnostic tool for early stages of cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Autocatalytic biocircuit are powerful tools for analysing intracellular biomarkers, but these tools are constrained by limitations in amplification capacity and intracellular delivery efficiency. In this work, we developed a DNAzyme-based dual-feedback autocatalytic exponential amplification biocircuit sustained by a honeycomb MnO 2 nanosponge (EDA 2 @hMNS) for live-cell imaging of intracellular low-abundance microRNAs (miRNA). The EDA 2 biocircuit comprises a blocked DNAzyme (b-DNAzyme), a Fuel strand and a Substrate strand. In the EDA 2 biocircuit, target miRNAs are recycled and feedback for rounds of DNAzymatic amplification, and the DNAzymatic reactions continuously generate target miRNA analogues for dual-feedback to achieve multiple parallel cascade DNAzymatic reactions that improve amplification capacity substantially. In addition, the hMNS ensures high loading and delivery efficiency of biocircuit probes into living cells and also provides sufficient Mn 2+ DNAzyme cofactor from in situ decomposition by intracellular glutathione (GSH). The EDA 2 @hMNS realized a detection limit of 17 pM, which is 288-fold lower than the b-DNAzyme lacking the DNAzymatic amplification. These results demonstrate the great promise for this critical tool in analysing low-abundance biomarkers and cancer diagnostics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The working efficiency of traditional 3D DNA nanomachines is extremely restricted due to the complex DNA components modified on nanoparticles in the same spatial height. Herein, an ultrafast dual-layer 3D DNA nanomachine (UDDNM) based on catalytic hairpin assembly (CHA) was developed by assembling two different lengths of hairpin DNA on the surface of gold nanoparticles, the long hairpin 1 (H1), to capture the trigger, and the short hairpin 2 (H2), as the signal probe, to recycle the trigger. Compared to the traditional single-layer 3D DNA nanomachine, the dual-layer 3D DNA nanostructure greatly enhances the effective collision between trigger and targeted DNA probe, H1, since the H1 located in outer layer would react with the trigger, inhibiting the invalid collision between the trigger and residual DNA component, H2, and remarkably decreasing the steric hindrance associated with the nucleic acids layer around the nanoparticles. Especially, when the distance of two layers was fixed at 3 nm, the corresponding UDDNM could accomplish the overall reaction only in 3 min with a dramatically high initial rate of up to 5.93 × 10 -7  M s -1 , which was at least 5-fold beyond that of the typical single-layer 3D DNA nanomachines. As a proof of concept, the described UDDNM was successfully applied in ultrasensitive fluorescence detection and sensitive intracellular imaging of miRNA-21. Consequently, our strategy, based on the creation of dual-layer 3D DNA nanostructure, may create a new approach to designing the next generation of DNA nanomachine and has enormous potential for applications in bio-analysis, logic gate operations, and clinical diagnoses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Chemiluminescence resonance energy transfer (CRET)-based assays have shown great potential in biosensing due to their negligible background autofluorescence, yet are still limited by their low sensitivity and short half-life luminescence. Herein, a multistage CRET-based DNA circuit was constructed with amplified luminescence signals for accurate miRNA detection and fixed reactive oxygen species (ROS) signals for cell imaging. The DNA circuit is designed through an ingenious programmable catalytic hairpin assembly (CHA), hybridization chain reaction (HCR), and the use of DNAzyme to realize target-triggered precise regulation of distance between the donor and acceptor for CRET-mediated excitation of photosensitizers. In detail, the analyte catalyzes the hybridization of CHA reactants, which leads to the assembly of multiple HCR-mediated DNAzyme nanowires. Subsequently, DNAzymes catalyze the oxidation of luminol by H 2 O 2 , and the adjacent photosensitizer chlorin e6 (Ce6) anchored on the DNA nanostructure is stimulated by the CRET process, resulting in the amplified long-wavelength luminescence and the generation of single oxygen signals through further energy transfer to oxygen. The biomarker miRNA can be detected with great sensitivity by integrating the recognition module into a universal platform. Furthermore, the DNA circuit enables CRET-mediated intracellular miRNA imaging, by detecting singlet oxygen signals through a ROS probe. The significant amplification effect is attributed to the robust multiple recognition of the target and the guaranteed transduction of the CRET signal through programmable engineering of DNA nanostructures. The CRET-based DNA circuit achieves amplified long-wavelength luminescence for accurate miRNA detection with low background and ROS-mediated signal fixation for cell imaging, making it a promising candidate for early diagnosis and theranostics.

As a typical biomarker, the expression of microRNA is closely related to the occurrence of cancer. However, in recent years, the detection methods have had some limitations in the research and application of microRNAs. In this paper, an autocatalytic platform was constructed through the combination of a nonlinear hybridization chain reaction and DNAzyme to achieve efficient detection of microRNA-21. Fluorescently labeled fuel probes can form branched nanostructures and new DNAzyme under the action of the target, and the newly formed DNAzyme can trigger a new round of reactions, resulting in enhanced fluorescence signals. This platform is a simple, efficient, fast, low-cost, and selective method for the detection of microRNA-21, which can detect microRNA-21 at concentrations as low as 0.004 nM and can distinguish sequence differences by single-base differences. In tissue samples from patients with liver cancer, the platform shows the same detection accuracy as real-time PCR but with better reproducibility. In addition, through the flexible design of the trigger chain, our method could be adapted to detect other nucleic acid biomarkers., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

DNAzyme motors are widely used for the sensitive detection of intracellular miRNAs due to their excellent signal response. Generally, the addition of exogenous mental ions to DNAzyme motors is crucial for the efficient operation of the system. Moreover, the position of the DNAzyme relative to the substrate has a significant impact on the cleavage rate during the reaction. Herein, we proposed a highly loaded Na + -fueled linear programmable DNAzyme nanostructure (LPDN) composed of long, single-strand DNA produced by rolling circle amplification reactions that served as binding partners for Na + -specific DNAyme and substrate. In the meantime, the long, programmable scaffolds can precisely control the position of the DNAzyme and substrate for the optimal effect. During the assay, miR-21 and endogenous Na + can specifically trigger multiple adjacent substrate-cleaving reactions, resulting in a significant recovery of the Cy3 fluorescence signal in living cells. This method could enable in situ real-time imaging and biocompatibility-enhancing evaluation of intracellular miR-21-level changes. Furthermore, LPDN's ability to distinguish normal cells from cancer cells makes it a promising candidate for early cancer diagnosis and imaging analysis of cancer.

Circulating microRNAs (miRNAs) can be regarded as reliable noninvasive biomarkers in body fluids for the early diagnosis, prognosis, and monitoring of cancers. By combining target triggered bipedal DNAzyme walker cleavage cycling amplification and planar intercalated methylene blue (MB) molecules amplification, a versatile ratiometric electrochemical biosensing system is constructed for miRNAs detection. Using the microRNA-21 (miRNA-21) as a triggered model target from breast cancer cells (MCF-7) and cervical cancer cells (HeLa), the sensitivity and feasibility of the ratiometric biosensing strategy were verified on the basis of decreased streptavidin-conjugated cupric sulfide@platinum (CuS@Pt-SA) nanozyme signal with cleaved Zn 2+ -dependent DNAzyme walkers as well as enhanced duplex section of MB signal, which were assisted by the modification of high electronic conductivity and specific surface area of metallic WSe 2 nanoflowers on the electrode. Hence, the introduced sensing strategy of higher cleavage activity of the bipedal DNAzyme walker cyclic amplification resulted into the remarkable sensitive measurement which had a detection limit of 0.16 fM from 1 fM to 1 nM for miRNA-21. Benefiting from the precise design of the capture Hairpin DNA, this proposed method showed excellent specificity to distinguish miRNA-21 from other miRNAs sequences, in addition to possessing good stability and reproducibility. Thus, this versatility platform can be utilized to sense various miRNAs biomarkers by simple of the redesigning the capture Hairpin DNA, hence presents a great promise in clinical application towards early cancer diagnosis, biological analysis and prognosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs), useful biomarkers for cancer diagnosis, play an important role in tumorigenesis and progression, but many of the current analysis methods can suffer from excessive protease dependence, being time-consuming and unsatisfactory performance. Therefore, a reliable sensing strategy for the protein-free, ultrasensitive analysis of tumor-associated miRNAs is desired. The proposed dual-walker biosensing strategy based on an entropy-driven catalytic (EDC) walker coupled with a smart-responsive DNAzyme walker was demonstrated for the dual-amplification detection of miRNA-21. Namely, the target miRNA-21 initiates the three-stranded substrate complex of the traditional EDC circuit to release the input trigger of the Dz walker, which recognizes the circular binding domain to restore the cleavage activity of the DzS-AuNP walker. The fluorescence signal continuously released from the AuNPs was recorded by a fluorescence reader for miRNA-21 sensing. The optimized dual-walker exhibited appreciable sensitivity with a detection limit of 70 fM, satisfactory flexibility, fine specificity and ideal stability for clinical serum sample assays. The proposed strategy may open a new avenue for the development of powerful DNA molecular tools for cancer diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)