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Background: β-lactoglobulin (β-Lg), a major allergen in dairy products, can trigger severe allergic reactions and even fatal outcomes in infants. In this work, we develop a new low background current redox recycling strategy by conjugating the electrochemical mediator to trimetallic hybrid nanoparticles (NPs)-dispersed graphene as the signal tag, which is coupled with DNAzyme amplifications to construct highly catalytic and ultrasensitive β-Lg aptasensor., Results: Target β-Lg molecules bind aptamers in DNAzyme/aptamer duplexes to release active DNAzymes to initiate cyclic cleavage of hairpin substrates. This subsequently leads to confinement of many probes for signal generation on electrode. Assisted by K 3 [Fe(CN) 6 ] in detection buffer, catalytic redox recycling of mediators induced by trimetallic hybrid NPs thus yield considerably magnified currents for detecting β-Lg with detection limit of 5.4 pg/mL. Our results show that attachment of redox mediator to NP-dispersed graphene can effectively and significantly lower the background current compared with its presence in buffer. At the meantime, the coupling of the DNAzyme amplification with catalytic hybrid NPs can enhance the current signal, leading to high signal-to-noise ratio and sensitivity. Such aptasensor also exhibits high selectivity and can achieve detection of low levels of β-Lg in infant rice cereal., Significance: The simultaneous background current reduction and dual catalytic signal amplification of our biosensor strategy leads to highly improved signal-to-noise ratio and sensitivity, which suggesting its promising potential for the monitoring of different trace molecular biomarkers for diverse 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 © 2025 Elsevier B.V. All rights reserved.)

As a common pollutant, cadmium (Cd) poses a serious threat to the growth and development of plants. Currently, there is no effective method to elucidate the protective mechanism of Cd 2+ in plant cells. For the first time, we designed a Cd 2+ fluorescent probe to observe the adsorption and sequestration of Cd 2+ in rice cell walls and vacuoles. Specifically, Cd 2+ is blocked by the Casparian strip and electrostatically attracted to hemicellulose, which is abundantly adsorbed and fixed to the cell walls of the endodermis. For Cd 2+ that successfully entered the endodermis, one part entered the cells and was compartmentalised and fixed in the vacuoles, while the other part entered the vascular bundles and precipitated in the cell walls of the sclerenchyma through the ion exchange effect. Furthermore, with prolonged exposure to Cd 2+ , compartmentalised bodies that were strongly labelled by fluorescence gradually appeared in the vacuoles, which were assumed to be a new heavy metal protective mechanism activated by plants in response to continuous Cd 2+ exposure. In conclusion, this study provides an innovative and effective method for the detection of adsorption, transportation, and accumulation of Cd 2+ in plant tissues, which can be employed for the rapid identification of crops with low Cd accumulation., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

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.)

Herein, a dual-mode fluorometric and colorimetric biosensor for Pax-5a gene was developed based on zinc-doped molybdenum disulfide quantum dots (Zn-MoS 2 QDs) by coupling exonuclease-assisted recycling amplification and peroxidase-mimic DNAzyme. In the presence of Pax-5a gene, the exonuclease III can cleave the duplexes formed by Pax-5a gene and the hairpin DNA (HP), releasing the output DNA (oDNA). G-rich DNA and magnetic beads (MBs) labeled with capture DNA (cDNA) can hybridize with oDNA to form the MBs-cDNA/oDNA/G-rich DNA sandwich complex. The remaining G-rich DNA in the supernatant through magnetic separation could bind hemin to produce G-quadruplex/hemin peroxidase-mimicking DNAzyme, which catalyzed the oxidization of 3,3'-diaminobenzidine (DAB) by H 2 O 2 . The generated brown oxidation product (oxDAB) had a distinct absorption peak at 464 nm and could quench the fluorescence of Zn-MoS 2 QDs at 406 nm. The high peroxidase activity of DNAzyme, recycling amplification strategy and magnetic separation technique led to excellent sensitivity and specificity of this detection platform. The detection limits of Pax-5a gene by fluorometric and colorimetric methods were 0.52 pM and 1.12 pM, respectively. Furthermore, this sensing system was successfully applied to Pax-5a gene determination in human serum samples, which had promising potential in biochemical analysis and clinical 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.)

Pb 2+ has numerous sources in cosmetics, industrial pollution and other environments. Therefore, sensitive and accurate detection of Pb 2+ content is extremely important in food safety. In this work, bifunctional nanomaterials Ti 3 C 2 @PtPd NPs with fluorescence quenching effect and peroxidase activity were prepared by in situ growth of platinum‑palladium nanoparticles (PtPd NPs) on the surface of 2D material Ti 3 C 2 . Combining the DNA enzyme recognition element with magnetic separation technology, we constructed a fluorescence/colorimetric dual-channel for the sensitive detection of Pb 2+ . Under the optimal conditions, the detection ranges of this fluorescence/colorimetric bimodal sensing strategy were 0.1-1000 nmol/L and 0.5-1000 nmol/L, respectively. The LOD of the fluorescence method was 23 pmol/L, and that of the colorimetric method was 74 pmol/L, and the results of the detection were visible to the naked eye. This dual-mode sensing method provides a new platform for accurate, reliable and visualized detection of Pb 2+ ., 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 Ltd. All rights reserved.)

There is an urgent demand for a simple yet extremely accurate biosensor to analyze tumorigenesis. Herein, we present a novel fluorescent and enzyme-free approach for detecting p53 gene cascading proximity ligation-mediated catalytic hairpin assembly and DNAzyme-assisted signal reaction. When the target p53 gene is present, the interaction between p53 and L1 and L2 chains initiates catalytic hairpin assembly and subsequently exposes DNAzyme in the P3 probe. The exposed DNAzyme binds with the loop region of the P4 probe and generates a nicking site, resulting in the release of a significant amount of ATMND that is conjugated in the stem section of P4. This leads to an amplified fluorescence response, which serves as a fluorescence signal for the detection of the p53 gene. This method allows for the accurate and sensitive identification of the p53 gene, exhibiting a linear reaction range of 1 fM to 1 nM, with a limit of detection as low as 0.23 fM. Furthermore, this fluorescent method has been utilized for the examination of clinical samples with a favorable recovery rate. Crucially, this versatile platform may be expanded to analyze different targets by changing the corresponding recognition unit, showing great potential for point-of-care testing in tumorigenesis analysis., 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 development of intelligent nanotheranostic technology that integrates diagnostic and therapeutic functions holds great promise for personalized nanomedicine. However, most of the nanotheranostic agents exhibit "always-on" properties and do not involve an amplification step, which may largely limit imaging contrast and restrict therapeutic efficacy. Herein, we construct a novel nanotheranostic platform (Hemin/DHPs/PDA@CuS nanocomposite) by assembling DNA hairpin probes (DHPs) and hemin on the surface of PDA@CuS nanosheets that enables amplified fluorescence imaging and activatable chemodynamic therapy (CDT) of tumors. The cancer-relevant APE1 triggers nucleic acid amplification with DHPs to generate activatable and amplified fluorescence signals for discriminating cancer cells from normal cells. Meanwhile, excessive G-quadruplex/hemin-based DNAzyme are also activated, and they function as Fenton-like catalysts to catalyze the production of highly toxic hydroxyl radicals (•OH) for CDT. Moreover, owing to the excellent photothermal conversion efficiency in the near-infrared-II (NIR-II) window, the PDA@CuS not only improves the catalytic performance of CDT but also furnishes PTT. A remarkable antitumor therapeutic effect is demonstrated both in vitro and in vivo . Therefore, the Hemin/DHPs/PDA@CuS nanocomposite is expected to provide a promising avenue for precise imaging-guided antitumor therapy.

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.

DNAzyme represents a promising gene silencing toolbox yet is obstructed by the poor substrate accessibility in specific cells. Herein, a compact DNA nanoassembly, incorporating multimeric therapeutic DNAzyme, was prepared for selective delivery of gene-silencing DNAzyme with requisite cofactors and auxiliary chemo-drugs. By virtue of the sequence-conservative duplex-specific nuclease, the endogenous miRNA catalyzes the successive and site-specific cleavage of DNA nanoassembly substrate (nominated as the localized RNA walking machine) and thus ensures the liberation/activation of therapeutic agents with high accuracy and efficacy. The miR-10b-stimulated DNAzyme was designed to downregulate the TWIST transcription factor, an upstream promotor of miR-10b, thus acquiring the self-sufficient downregulation of TWIST/miR-10b signaling nodes (self-adaptive negative feedback loop) for abrogating tumor metastasis and chemo-resistance issues., (© 2024 Wiley-VCH GmbH.)

Enzymes encapsulated in extracellular vesicles (EVs) hold promise as biomarkers for early cancer diagnosis. However, precise measurement of their catalytic activities within EVs remains a notable challenge. Here, we report an enzymatically triggered spherical DNA nanomotor (EDM) that enables one-pot, cascaded, and highly sensitive analysis of the activity of EV-associated or free apurinic/apyrimidinic endonuclease 1 (APE1, a key enzyme in base excision repair) across various biological samples. The EDM capitalizes on APE1-triggered activation of DNAzyme (Dz) and its autonomous cleavage of substrates to achieve nonlinear signal amplification. Using EDM, we demonstrate a strong correlation between APE1 activity in EVs and that of their parental cancer cells. Additionally, EV APE1 mirrors the fluctuation of cellular APE1 activity in response to chemotherapy-induced DNA damage. In a pilot clinical study (n=63), the EDM-based assay reveals that more than 80 % of active APE1 in serum samples is EV-encapsulated. Notably, EV APE1 can differentiate early prostate cancer (PCa) patients from healthy donors (HDs) with an overall accuracy of 92 %, outperforming free APE1 in sera. We anticipate that EDM will become a versatile tool for quantifying EV-associated enzymes., (© 2024 Wiley-VCH GmbH.)

The host-guest recognition system is incorporated into the core region of the 10-23 DNAzyme for precise regulation of its functionality. Biochemical experiments demonstrate reversible and orthogonal control of the DNAzyme function using cucurbit(7)uril and its competitive guests. Furthermore, cellular experiments indicate the gene expression can be effectively manipulated through this ligand-controllable DNAzyme.

Flap endonuclease 1 (FEN1) plays a vital role in cancer by modulating DNA repair mechanisms, inducing genomic instability, and serving as a promising biomarker for cancer diagnosis and prognosis. In this work, we present the development of a novel DNAzyme signal amplification-directed point-of-care sensing system (Dz-PGM) for the sensitive and specific detection of FEN1. The Dz-PGM system utilizes DNAzyme signal amplification in conjunction with a personal glucose meter (PGM) for reporting, capitalizing on a biochemical cascade initiated by FEN1 recognition. Benefiting from enzymatic recognition, magnetic separation, and DNAzyme based signal amplification, this Dz-PGM system demonstrates exceptional specificity and achieves a low detection limit of 3.56× 10 -2 U/mL for FEN1. The versatility of the system is further demonstrated through its ability to screen FEN1 inhibitor drugs and to distinguish FEN1 levels in real cell samples with high accuracy. The article underscores the potential of the Dz-PGM system as a promising tool for early cancer diagnosis, and in constructing sensitive detection platforms for DNA repair enzymes., 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.)

Although DNAzyme sensors have been widely developed for imaging metal ions, their application in specific subcellular compartments remains challenging due to low spatial controllability. Here we present a locally activatable, DNAzyme-based sensing technology that enables subcellular compartment-specific imaging of metal ions through ribosomal RNA (rRNA) regulated signal amplification. The system leverages a subcellularly encoded rRNA to locally activate DNAzyme-based sensors, and further drives signal amplification via multiple turnover cleavage of molecular beacons, to significantly enhance sensitivity and spatial precision for metal-ion imaging in specific organelles (e.g. mitochondria) or membraneless compartments (e.g. cytosol). Furthermore, we demonstrate that the system allows in situ monitoring of subcellular dynamics of mitochondrial Zn 2+ during ischemia and the drug intervention. This study expands the DNAzyme toolbox for investigating the role of subcellular metal-ion dynamics in disease processes., (© 2024 Wiley-VCH GmbH.)

The detection of pathogens is critical for preventing and controlling health hazards across clinical, environmental, and food safety sectors. Functional nucleic acids (FNAs), such as aptamers and DNAzymes, have emerged as versatile molecular tools for pathogen detection due to their high specificity and affinity. This review focuses on the in vitro selection of FNAs for pathogens, with emphasis on the selection of aptamers for specific biomarkers and intact pathogens, including bacteria and viruses. Additionally, the selection of DNAzymes for bacterial detection is discussed. The integration of these FNAs into colorimetric biosensors has enabled the development of simple, cost-effective diagnostic platforms. Both non-catalytic and catalytic colorimetric biosensors are explored, including those based on gold nanoparticles, polydiacetylenes, protein enzymes, G-quadruplexes, and nanozymes. These biosensors offer visible detection through color changes, making them ideal for point-of-care diagnostics. The review concludes by highlighting current challenges and future perspectives for advancing FNA-based colorimetric biosensing technologies for pathogen detection., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

In this study, we present an intelligent electromagnetic-actuated microfluidic chip integrated with a G-quadruplex DNAzyme-based biocatalysis platform for rapid and sensitive tetracycline (TC) detection. In this sensing system, TC significantly quenches fluorescent magnetic carbon dots (M-CDs) via the internal filtration effect and dynamic quenching (the excitation and emission wavelength at 350 and 440 nm, respectively). Then, the G-quadruplex on the M-CDs-Aptamer is exposed and bound with hemin to form hemin-G-quadruplex DNAzyme, catalyzing the conversion of 3,3',5,5'-tetramethylbenzidine to produce blue color. This enables the fluorescence/colorimetric detection of TC. Importantly, an automatic electromagnet-integrated microfluidic chip was designed to control the shuttling of magnetic materials in each function slot according to a programmed sequence. Under the optimal conditions, the detection limits of TC for fluorescence and colorimetric methods were 11 and 43 μmol/L, respectively. The detection results for tilapia ( Oreochromis nilotica ) were comparable to those of traditional high-performance liquid chromatography. This platform offers excellent performance for TC determination and potential for portable, intelligent detection of trace pollutants in food and the environment.

Herein, an electrochemical aptasensor for highly sensitive detection of acetamiprid (ACE) was constructed based on a one-step cascade amplification strategy. This innovative strategy integrated DNA walker containing DNAzyme sequence into entropy-driven catalysis (EDC) system. The trigger strand was released by aptamer-specific binding to ACE, initiating the EDC amplification circuit and delivering DNA walker strands. The dangling DNA walker continuously bound and cleaved hairpin substrate to form G-quadruplex fragments with the assistance of Mg 2+ . The G-quadruplex fragments folded and captured hemin to form multitudinous G-quadruplex/hemin complexes in the presence of K + , generating significantly enhanced current, enabling enzyme-free, label-free and highly sensitive detection of ACE, with a linear detection range of 100 fM to 50 nM and a detection limit of 68.36 fM (S/N = 3). The constructed aptasensor achieved the reliable detection of ACE in vegetable soil and cucumber samples, demonstrating its potential application prospects in environmental protection and food supervision., 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 Ltd. All rights reserved.)

Background: Deoxyribozymes or DNAzymes represent artificial short DNA sequences bearing many catalytic properties. In particular, DNAzymes able to cleave RNA sequences have a huge potential in gene therapy and sequence-specific analytic detection of disease markers. This activity is provided by catalytic cores able to perform site-specific hydrolysis of the phosphodiester bond of an RNA substrate. However, the vast majority of existing DNAzyme catalytic cores have low efficacy in in vivo experiments, whereas SELEX based on in vitro screening offers long and expensive selection cycle with the average success rate of ~ 30%, moreover not allowing the direct selection of chemically modified DNAzymes, which were previously shown to demonstrate higher activity in vivo. Therefore, there is a huge need in in silico approach for exploratory analysis of RNA-cleaving DNAzyme cores to drastically ease the discovery of novel catalytic cores with superior activities., Results: In this work, we develop a machine learning based open-source platform SequenceCraft allowing experimental scientists to perform DNAzyme exploratory analysis via quantitative observed rate constant (k obs ) estimation as well as statistical and clustering data analysis. This became possible with the development of a unique curated database of > 350 RNA-cleaving catalytic cores, property-based sequence representations allowing to work with both conventional and chemically modified nucleotides, and optimized k obs predicting algorithm achieving Q 2  > 0.9 on experimental data published to date., Conclusions: This work represents a significant advancement in DNAzyme research, providing a tool for more efficient discovery of RNA-cleaving DNAzymes. The SequenceCraft platform offers an in silico alternative to traditional experimental approaches, potentially accelerating the development of DNAzymes., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

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.)

Hematoma and secondary neuroinflammation continue to pose a significant challenge in the clinical treatment of intracerebral hemorrhage (ICH). This study describes a nanoregulator formed through the self-assembly of Mg 2+ and signal regulatory protein α (SIRPα) DNAzyme (SDz), aimed at enhancing hematoma resolution and inhibiting neuroinflammation in the treatment of ICH. The structure of SDz collapses in response to the acidic endo/lysosomal microenvironment of microglia, releasing Mg 2+ and the SIRPα DNAzyme. The Mg 2+ then acts as a cofactor to activate the SIRPα DNAzyme. By blocking the CD47-SIRPα signaling pathway, microglia can rapidly and effectively phagocytose red blood cells (RBCs), thereby promoting the clearance of the hematoma. Simultaneously, Mg 2+ reset the microglia to the M2 phenotype by inhibiting the MYD88/MAPK/NF-κB signaling pathway, thereby modulating the inflammatory microenvironment of ICH. This co-delivery and synergistic strategy resulted in a significant reduction in hematoma size, decreasing from 11.90 to 5.84 mm 3 , and promoted recovery from ICH with minimal systemic side effects. This simple yet highly effective nanoplatform, which involves complex synergistic mechanisms, proves to be effective for ICH therapy and holds great promise for introducing novel perspectives into clinical and translational approaches for ICH., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

As the most abundant modification in eukaryotic messenger RNA (mRNA) and long noncoding RNA (lncRA), N 6 -methyladenosine (m 6 A) has been shown to play essential roles in various significant biological processes and attracted growing attention in recent years. To investigate its functions and dynamics, there is a critical need to quantitatively determine the m 6 A modification fractions at a precise location. Here, we report a deoxyribozyme mediated CRISPR-Cas12a platform (termed "DCAS") that can directly quantify m 6 A fractions at single-base resolution. DCAS employs a deoxyribozyme (VMC10) to selectively cleave the unmodified adenine (A) in the RNA, allowing only m 6 A-modified RNA amplified by RT-PCR. Leveraging the CRISPR-Cas12a quantify the PCR amplification products, DCAS can directly determine the presence of m 6 A at target sites and its fractions. The combination of CRISPR-Cas12a with RT-PCR has greatly improved the sensitivity and accuracy, enabling the detection of m 6 A-modified RNA as low as 100 aM in 2 fM total target RNA. This robustly represents an improvement of 2-3 orders of magnitude of sensitivity and selectivity compared to traditional standard methods, such as SCARLET and primer extension methods. Therefore, this method can be successfully employed to accurately determine m 6 A fractions in real biological samples, even in low abundance RNA biomarkers., 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.)

Non-invasive monitoring of glucose levels in tears and saliva is crucial for diagnosing and predicting various illnesses, such as diabetic nephropathy. However, the capability of the current glucose detection methods to identify small amounts of glucose with a high sensitivity remains a significant obstacle. This study proposes a simple, visual technique for sensitively detecting glucose levels from tears and saliva using glucose oxidase (GOx) to catalyze glucose and pistol-like DNAzyme (PLDz) to enhance the signal. In particular, the β-D-glucose present in the samples serves as the initial molecule that GOx identifies and catalyzes to generate gluconic acid and hydrogen peroxide (H 2 O 2 ). The H 2 O 2 induces the self-cleavage of PLDz, activating the "part b" sequence. This activation initiates catalytic hairpin assembly (CHA) and releases the DNAzyme section in the H1 probe. The DNAzyme acts as a peroxidase analog, facilitating the catalysis of the 3,3',5,5'-tetramethylbenzidine (TMB)-hydrogen peroxide (H 2 O 2 ) system and resulting in color changes. The proposed method exhibits a broad detection range of six orders of magnitude and a low limit of 0.32 μM for glucose detection. Furthermore, the proposed method was highly effective in detecting glucose in saliva and tears, suggesting that it could potentially diagnose hyperglycemia-related disorders in clinical environments., Competing Interests: Declarations. Ethics Approval: Permission from the Institutional Animal Ethical Committee was received before making these experiments (No. SYXK-2023–0014). Consent to Participate: Not applicable. Consent for Publication: All authors agree to publish. Competing Interests: The authors declare that they have no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.)

Three-dimensional (3D) DNA walking nanomachines have been widely applied in molecular diagnostics and cancer therapy. Specially, DNA dendrimer with 3D nanostructure has gained increasing attention in biomedical applications because of its high biostability, excellent programmability, and outstanding biocompatibility. In this chapter, we developed a 3D DNA walking nanomachine by assembling all nanomachine components (DNAzyme and substrate) onto a DNA dendrimer for miRNA imaging in living cells. In this design, the number of DNAzyme and substrate on the surface of the DNA dendrimer could be precisely regulated to fully implement the function of the DNA nanomachine., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

There is an urgent need for novel strategies to accurately and reliably detect pathogenic bacteria to address the global epidemic of antibiotic resistance. This study proposes an innovative approach combining dual aptamer-based target recognition and proximity ligation assay (PLA) triggered DNAzyme recycling cleavage. This method allows for the precise identification and reliable detection of methicillin-resistant Staphylococcus aureus (MRSA). The fluorescence probe labeled with a fluorophore is modified on gold nanoparticles (AuNPs), resulting in the quenching of the fluorescent signal by the AuNPs. The interaction between MRSA and two aptamers leads to forming a Mg 2+ -dependent DNAzyme. The DNAzyme cleaves the fluorescence probe, causing the fluorescent fragment to detach from the surface of the AuNPs, in which the quenched fluorescence signal in the fluorescence probe reappears. The DNAzyme-assisted cleavage and rebinding process generates a processive strolling along the surface track of AuNPs. Consequently, the fluorescence intensity experiences a substantial recovery. A strong linear correlation is observed between the fluorescence intensity and MRSA concentration within 50 cfu/mL to 10 6  cfu/mL. We believe that implementing the novel integrated strategy will broaden the range of bacterial detection methods in the battlefield environment and stimulate the creation of potential new drugs in the future., 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.)

Sugarcane smut is a major disease affecting the yield and quality of sugarcane, and its early detection is crucial for the healthy development of sugarcane industry. In this work, a dual-modal biosensing platform is designed based on Au-V-MOF and 3D DNA walker for highly sensitive and precise detection of the sugarcane smut pathogen. This detection system utilizes the catalytic properties of biocatalysts and the precise cleavage of DNAzymes, along with 3D DNA walker nanotechnology and a designed "walking track", to achieve amplified detection signals and accurate target recognition. The detection platform utilizes catalytic hairpin assembly for target recycling. Output strand T1 interacts with DNAzyme via a 3D transporter, cleaving S1 in Mg 2+ presence. This triggers cascading hybridization chain reaction to form long double-stranded DNA structures that absorb substantial methylene blue (MB). This amplifies the electrical signal and causes a proportional color change due to the redox reaction of MB, which enables electrochemical and colorimetric detection of the target. The method shows a linear response range from 0.0001 to 10,000 pM with a detection limit of 56.76 aM (S/N = 3). It features high sensitivity, specificity, and accuracy, which offers significant potential for early warning and precise detection of sugarcane smut., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024. Published by Elsevier B.V.)

Developing cost-effective, consumer-accessible platforms for point-of-use environmental and clinical pathogen testing is a priority, to reduce reliance on laborious, time-consuming culturing approaches. Unfortunately, a system offering ultrasensitive detection capabilities in a form that requires little auxiliary equipment or training has remained elusive. Here, a colorimetric DNAzyme-crosslinked hydrogel sensor is presented. In the presence of a target pathogen, DNAzyme cleavage results in hydrogel dissolution, yielding the release of entrapped gold nanoparticles in a manner visible to the naked eye. Recognizing that Escherichia coli holds high relevance within both environmental and clinical environments, an E. coli-responsive DNAzyme is incorporated into this platform. Through the optimization of the hydrogel polymerization process and the discovery of bacteriophage-induced DNAzyme signal amplification, 10 1 CFU mL -1 E. coli is detected within real-world lake water samples. Subsequent pairing with an artificial intelligence model removed ambiguity in sensor readout, offering 96% true positive and 100% true negative accuracy. Finally, high sensor specificity and stability results supported clinical use, where 100% of urine samples collected from patients with E. coli urinary tract infections are accurately identified. No false positives are observed when testing healthy samples. Ultimately, this platform stands to significantly improve population health by substantially increasing pathogen testing accessibility., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)

Staphylococcus aureus ( S. aureus , SA) is one of the most common bacteria in nosocomial infections. Sensitive and efficient analysis of methicillin-resistance of SA is crucial for improving the nursing performance of pneumonia. However, methicillin-resistance analysis with favorable sensitivity and specificity in an enzyme-free manner remains a huge challenge. This paper presents the development of a new fluorescent biosensor for detecting mecA gene using a triple-branch catalytic hairpin assembly (CHA) triggered DNAzyme switch-based DNA tweezer. The SA from the samples are immobilized on the plate's surface using the protein A antibody. The biosensor possesses several key features. Firstly, it utilizes dual signal amplification processes, specifically the triple-branch CHA and DNAzyme controlled DNA tweezer-based signal recycling, to enable mecA detection on the plate. This design enhances the method's sensitivity, resulting in a low limit of detection of 1.5 fM. Secondly, the biosensor does not rely on enzymes for mecA analysis, ensuring a high level of stability during target analysis. Lastly, the method demonstrates a remarkable selectivity by accurately distinguishing target sequences from non-target sequences. The proposed biosensor, which does not require enzymes and has a high level of sensitivity, offers a viable platform for the rapid and simple quantification of mecA in SA.

A novel detection technique is introduced that offers sensitive and reliable ochratoxin A (OTA) detection. The method leverages the etching of gold nanorods (AuNRs) stabilized by hexadecyl trimethyl ammonium bromide (CTAB) using the oxidized form of 3,3',5,5'-tetramethyl benzidine sulfate (TMB 2+ ), creating a susceptible multicolor visual detection system for OTA. The visual detection is enabled by Mg 2+ -assisted DNAzyme catalysis combined with the catalytic hairpin assembly (CHA) signal amplification strategy. The presence of OTA triggers CHA and signaling responses along with the formation of G-quadruplex-hemin DNAzyme, which promotes the oxidation of TMB with H 2 O 2 , leading to the etching of AuNRs and a reduction in their aspect ratio. AuNRs experienced a blue shift in the longitudinal localized surface plasmon resonance peak, resulting in a color change. The technique has been shown to detect OTA with a low detection limit of 0.309 pg/mL, demonstrating high sensitivity and specificity. The detection technique offers versatility by enabling the detection of other pollutants through a simple replacement of the aptamer, expanding the range of detection platforms available for pollutant determinations., Competing Interests: Declarations. Ethical approval: Not applicable. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Aggregation-induced emission luminogen (AIEgens)-based photothermal therapy (PTT) has grown into a sparkling frontier for tumor ablation. However, challenges remain due to the uncoordinated photoluminescence (PL) and photothermal properties of classical AIEgens, along with hyperthermia-induced antiapoptotic responses in tumor cells, hindering satisfactory therapeutic outcomes. Herein, a near-infrared (NIR) spiro-AIEgen TTQ-SA was designed for boosted PTT by auxiliary DNAzyme-regulated tumor cell sensitization. TTQ-SA with a unique molecular structure and packing mode was initially fabricated, endowing it with a strong AIE effect, favorable PL quantum yield, and good photothermal performance. DNAzyme, as a gene silencing tool, could alleviate antiapoptosis response during PTT. By integrating TTQ-SA and DNAzyme into folate-modified poly(lactic- co -glycolic acid) (PLGA) polymer, the as-fabricated nanosystem could promote cell apoptosis and sensitize tumor cells to PTT, thereby maximizing the therapeutic outcomes. With the combination of spiro-AIEgen-based PTT and DNAzyme-based gene silencing, the as-designed nanosystem showed promising NIR and photothermal imaging abilities for tumor targeting and demonstrated significant cell apoptotic, antitumor, and antimetastasis effects against orthotopic breast cancer. Furthermore, a synergistic antitumor effect was realized in spontaneous MMTV-PyMT transgenic mice. These findings offer new insights into AIEgen-based photothermal theranostics and DNAzyme-regulated tumor cell sensitization, paving the way for synergistic gene silencing-PTT nanoplatforms in clinical research.

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.

The expression levels of small extracellular vesicles (sEVs) are closely associated with several significant biological processes, which can be used as a crucial biomarker for cancer diagnosis, such as colorectal cancer. More efforts are still necessary to amplify sEV detection sensitivity, as their expression is minimal during the early stages of colorectal cancer. Through the integration of a catalytic assembly-triggered DNAzyme motor and gold nanoparticle (AuNP) aggregation, we have developed a triple signal amplified biosensor for the detection of sEVs. In this method, the catalytic assembly triggered DNAzyme motor continuously cleaved on the hairpin probe which is fixed on the surface of AuNPs, leaving a single-stranded sequence on the surface of AuNPs to induce the aggregation. This approach employs a triple signal amplification process to enhance the efficiency of the reaction and circumvent the issue of expensive and readily degradable proteases. The signal output system is based on dynamic light scattering technology, which enables ultra-sensitive detection of sEVs with a detection limit of 3.08 particles per μL. The present strategy exhibits significant potential for the analysis of a variety of additional analytes in clinical research disciplines due to its appealing analytical capabilities.

As a common cancer biomarker, human epidermal growth factor receptor-2 (HER2) is highly expressed in breast cancer. Consequently, developing a simple and accurate HER2 sensing platform is of great significance for early diagnosis and treatment of breast cancer. Herein, we developed a rapid enzyme-free fluorescent assay biosensor based on MNAzyme signal amplification for breast cancer biomarker, HER2. The MNAzyme consists of multiple parts, including complementary DNA (cDNA) and two parts of DNAzyme (partzyme A/B). Initially, cDNA is blocked by combining with the HER2 aptamer to form a double-stranded DNA. When HER2 is present, cDNA is released as a result of the binding between HER2 and its aptamer. Due to the complementary sequences among cDNA and partzyme A/B, the MNAzyme is successfully assembled to cleave the substrate, recovering the fluorescence output. The MNAzyme biosensor exhibited a low detection limit of 0.02 ng mL -1 and excellent selectivity. Furthermore, the proposed biosensor can also change the recognition element by changing the aptamer sequence to detect various biomarkers, holding great potential for cancer diagnosis and other related biomedical applications.

Oligonucleotide gene therapy (OGT) can be used to suppress specific RNA in cells and thus has been explored for gene therapy. Despite extensive effort, there is no clinically significant OGT for treating cancer. Low efficiency of OGT is one of the problems. Earlier, we proposed to address this problem by suppressing the most vital genes in cancer cells e. g. housekeeping genes. To achieve specific activation of the OGT agents in cancer but not in normal cells, we designed a binary (split) DNAzyme (BiDz) activated by cancer-related nucleic acid sequences. This work is devoted to BiDz optimization for efficient cleavage of structured RNA targets upon activation with a cancer marker-related sequence. To achieve efficient binding of folded RNA, the BiDz was equipped with RNA binding/unwinding arms to produce 'Binary DNA-nanomachines' (BiDM). It was demonstrated that BiDM can improve both the rate and selectivity of RNA cleavage in comparison with BiDz. For the best selectivity, single-nucleotide variations should be recognized by the strand detached from the common DNA scaffold of BiDM. Further development of DNA nanotechnology-inspired agents can advance OGT in treating cancer, viral infections, and genetic disorders., (© 2024 Wiley-VCH GmbH.)

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.

Although DNAzyme is a promising gene therapy agent, low cellular uptake efficiency, poor biological stability, and the unsatisfactory effect of monotherapy limit its development. Herein, a multifunctional DNA nanoassembly (RCA product-aptamer-DNAzyme, RAD) was constructed for cancer cell detection and targeted delivery of doxorubicin (DOX) and DNAzyme. Briefly, the rolling circle amplification (RCA) product was employed as a scaffold, and each repeated sequence was designed to combine with three single-stranded DNA (ssDNA), which carried the aptamer AS1411 sequence, fluorescent group, and DNAzyme sequence, respectively. Up to 40 groups of ssDNA can be assembled into an RCA product, resulting in a high affinity for cancer cells and stronger fluorescent signals. Due to the high binding affinity, RAD displayed high sensitivity for the detection of HepG-2 cells (the limit of detection was 200 cells/mL). In addition, with the formation of the double helix structure, each RAD could load up to 200 DOX molecules. Subsequently, RAD could efficiently and selectively deliver DOX and DNAzyme into cancer cells through the multivalent interaction between the aptamers and membrane nucleolin. Then, the released DNAzyme could recognize and cleave survivin mRNA under the action of Mg 2+ , leading to the apoptosis of HepG-2 cells for gene therapy, while DOX inserted into intracellular DNA to inhibit cell proliferation, realizing chemotherapy. According to the results, RAD-DOX displayed enhanced therapeutic effects compared with individual gene therapy or chemotherapy, and RAD could protect membrane nucleolin-negative cells from the effects of DOX. Overall, given the enhanced serum stability, high drug-loading capacity, and excellent selective cellular uptake ability of RAD, this strategy shows great potential in the field of cancer therapy.

Leptospirosis is a neglected zoonosis caused by a pathogenic spirochete Leptospira. Diagnosis of leptospirosis in the early stage is difficult and can be easily confused with other infections. The existing detection methods are considered chronophagous and labor-intensive. Leptospira survives in the kidney tubules of reservoir animals such as rodents and shed into the environment through their urine. In this study, we developed an Aptamer-DNAzyme-based biosensor for detecting pathogenic Leptospira in environmental water samples. The cell-specific aptamer with an extensive affinity binds to the cell surface proteins to detect the Leptospira interrogans. The DNAzyme that mimics as a peroxidase enzyme, acts as a transducing agent in the colorimetric reaction positively conditioned by the presence of L. interrogans. The Leptospira-specific aptamer coupled with DNAzyme is coated onto carbon nanotubes, to provide a cost-effective nanomaterial-based detection platform. L. interrogans contamination in the samples is detected with a color change of a peroxidase substrate, ABTS. The dissociation constant of the aptazyme was found to be 356.6 nM. The aptazyme system was able to detect up to 119 CFU/mL of L. interrogans exhibiting a high range of selectivity towards the pathogenic spirochete. This simple detection methodology makes the system promising for the environmental monitoring of L. interrogans., Competing Interests: Declarations. Conflict of interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The development of synthetic molecular tools responsive to biological cues is crucial for advancing targeted cellular regulation. A significant challenge is the regulation of cellular processes in response to gaseous signaling molecules such as hydrogen sulfide (H 2 S). To address this, we present the design of Gas signaling molecule-Responsive Artificial DNAzyme-based Switches (GRAS) to manipulate cellular functions via H 2 S-sensitive synthetic DNAzymes. By incorporating stimuli-responsive moieties to the phosphorothioate backbone, DNAzymes are strategically designed with H 2 S-responsive azide groups at cofactor binding locations within the catalytic core region. These modifications enable their activation through H 2 S-reducing decaging, thereby initiating substrate cleavage activity. Our approach allows for the flexible customization of various DNAzymes to regulate distinct cellular processes in diverse scenarios. Intracellularly, the enzymatic activity of GRAS promotes H 2 S-induced cleavage of specific mRNA sequences, enabling targeted gene silencing and inducing apoptosis in cancer cells. Moreover, integrating GRAS with dynamic DNA assembly allows for grafting these functional switches onto cell surface receptors, facilitating H 2 S-triggered receptor dimerization. This extracellular activation transmits signals intracellularly to regulate cellular behaviors such as migration and proliferation. Collectively, synthetic switches are capable of rewiring cellular functions in response to gaseous cues, offering a promising avenue for advanced targeted cellular engineering., (© 2024 Wiley-VCH GmbH.)

We introduce a multicore DNA nanomachine (MDNM), utilizing four binary DNAzymes for nucleic acid detection without the need for a preamplification step. This innovation remarkably yields a reduction in limit of detection (LOD), over 5-fold, as compared to single-core systems. This reduces the required test time thus highlighting the potential of MDNM in advancing nucleic acid detection., (© 2024 Wiley-VCH GmbH.)

Biosensors, devices capable of detecting biomolecules or bioactive substances, have recently become one of the important tools in the fields of bioanalysis and medical diagnostics. A biosensor is an analytical system composed of biosensitive elements and signal-processing elements used to detect various biological and chemical substances. Biomimetic elements are key to biosensor technology and are the components in a sensor that are responsible for identifying the target analyte. The construction methods and working principles of biosensors based on synthetic biomimetic elements, such as DNAzyme, molecular imprinted polymers and aptamers, and their updated applications in biomedical analysis are summarised. Finally, the technical bottlenecks and future development prospects for biomedical analysis are summarised and discussed., 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 monitoring of human 8-oxyguanine DNA glycosylase (hOGG1) activity in living cells is helpful to understand its function in damage repair and evaluate its role in disease diagnosis. Herein, a functional DNA-Zn 2+ coordination nanospheres was proposed for sensitive imaging of hOGG1 in living cells. The nanospheres were constructed through the coordination-driven self-assembly of the entropy driven reaction (EDR) -deoxyribozyme (DNAzyme) system with Zn 2+ , where DNAzyme was designed to split structure and assembled into the EDR system. When the nanospheres entered the cell, the competitive coordination between phosphate in the cell and Zn 2+ leaded to the disintegration of the nanospheres, releasing DNA and some Zn 2+ . The released Zn 2+ acted as a cofactor of DNAzyme. In the presence of hOGG1, the EDR was completed, accompanied by fluorescence recovery and the generation of a complete DNAzyme. With the assistance of Zn 2+ , DNAzyme continuously cleaved substrates to produce plenty of fluorescence signals, thus achieving sensitive imaging of hOGG1 activity. The nanospheres successfully achieved sensitive imaging of hOGG1 in human cervical cancer cells (HeLa), human non-small cell lung cancer cells and human normal colonic epithelial cells, and assayed changes in hOGG1 activity in HeLa cells. This nanospheres may provide a new tool for intracellular hOGG1 imaging and related biomedical 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.)

The residue of tobramycin, a broad spectrum antibiotic commonly used in animal husbandry, has evitable impact on human health, which may cause kidney damage, respiratory paralysis, neuromuscular blockade and cross-allergy in humans. Sensitive monitoring of tobramycin in animal-derived food products is therefore of great importance. Herein, a new aptamer electrochemical biosensor for sensing tobramycin with high sensitivity is demonstrated via exonuclease III (Exo III) and metal ion-dependent DNAzyme recycling and hybridization chain reaction (HCR) signal amplification cascades. Tobramycin analyte binds aptamer-containing hairpin probe to switch its conformation to expose the toehold sequence, which triggers Exo III-based catalytic digestion of the secondary hairpin to release many DNAzyme strands. The substrate hairpins immobilized on the Au electrode (AuE) are then cyclically cleaved by the DNAzymes to form ssDNAs, which further initiate HCR formation of lots of long methylene blue (MB)-tagged dsDNA polymers on the AuE. Subsequently electro-oxidation of these MB labels thus exhibit highly enhanced currents for sensing tobramycin within the 5-1000 nM concentration range with an impressive detection limit of 3.51 nM. Furthermore, this strategy has high selectivity for detecting tobramycin in milk and shows promising potential for detect other antibiotics for food safety monitoring., 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.)

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.)

The development of non-biological applications of DNA has not only resulted in delicately shaped DNA-based nano-objects with complex functions but also spawned their use for novel catalytic applications. From the multitude of applications of DNAzymes that operate on a relatively simple substrate, we have witnessed the emergence of multifunctional catalytically active DNA-based nanostructures for one of the most challenging tasks known to a chemist: the controlled and precise modification of a wild-type protein in its natural environment. By incorporating various elements associated with post-translational modification (PTM) writer enzymes into complex nanostructures, it is now possible to chemically modify a specific protein in cell lysates under the influence of an externally added trigger, clearly illustrating the promising future for this approach., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Despite the significant potential of RNA-cleaving DNAzymes for gene regulation, their application is limited by low therapeutic efficacy and lack of cell-specific control. Here, a DNAzyme-based nanohybrid designed for ultrasound (US)-controlled, enzyme-activatable mRNA regulation is presented, enabling tumor cell-selective combination therapy. The nanohybrid is constructed by coordination-directed self-assembly of an enzymatically-triggerable therapeutic DNAzyme (En-Dz) and natural sonosensitizer hemoglobin (Hb). Controlled US exposure induces reactive oxygen species generation from Hb units, which not only facilitates efficient endosomal escape of En-Dz, but also promotes the translocation of specific enzyme from the nucleus to the cytoplasm, thereby enhancing gene regulation efficacy. Notably, the enzyme-triggered, spatiotemporally-controlled activation of En-Dz's catalytic activity results in enhanced cancer-cell selectivity in the therapeutic treatment. Furthermore, the combination of enzyme-activated mRNA regulation and sonodynamic therapy significantly enhances anti-tumor efficacy both in vitro and in vivo. This work highlights the potential of integrating a sonodynamic strategy to overcome the current limitations of DNAzyme-based gene regulators, providing a spatiotemporally-controlled approach for precise tumor treatment., (© 2024 Wiley‐VCH GmbH.)

Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated Proteins (CRISPR/Cas) system can accurately identify and cleave target DNA sequences, while the effective combination of DNA nanomatrix and entropy-driven self-assembled enzymes can significantly enhance the sensitivity, stability, and diversified functionality of sensors through highly ordered molecular arrangement and spontaneous efficient assembly processes. Herein, a carbon-encapsulated MoS 2 hollow nanorod (C-MoS 2 ) with excellent conductivity and multiple active sites is used to construct bioanode of biofuel cell by integrating it with an entropy-driven self-assembled enzyme-DNA nanomatrix cascade DNAzyme-CRISPR/Cas system. When thrombin binds aptamer, it exposes the trigger strand on the anode, initiating chain displacement. This activates DNAzyme, triggering a cascade reaction that cleaves and releases the probe strand. The probe then binds CrRNA, forming a multimeric complex with Cas protein.When non-specific cleavage of the single strand occurs, it leads to the release of glucose oxidase (GOD) from the DNA matrix and causes a significant decrease in the value of the system's open-circuit voltage (E OCV ). The E OCV values show a good negative correlation with the concentration of thrombin (TB) in the range of 0.00001-100 nM, achieving a limit of detection of 3.55 fM (S/N = 3)., 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.)

A two-color fluorescent sensing strategy based on a functionalized tetrahedral DNAzyme nanotweezer (FTDN) was developed to detect ochratoxin A (OTA) utilizing the multifunctional properties of DNA nanotechnology. The FTDN enables rapid OTA detection directly through a Cy5 fluorescent group, modified to respond to the target signal. Additionally, FTDN exhibits DNAzyme cutting activity in the presence of Mg 2 ⁺ ions, enabling it to traverse DNA nanoflower-functionalized magnetic beads. This process results in the continuous cleavage of DNA nanoflowers labeled with numerous FAM fluorescent groups, thereby amplifying the detection signal and enhancing OTA sensitivity. The linear ranges for the Cy5 and FAM signals in response to OTA were 5-1000 ng/mL and 0.05-100 ng/mL, respectively, with corresponding limits of detection (LOD) of 1.59 ng/mL and 0.03 ng/mL. This study demonstrates that dual-color fluorescence via Cy5 and FAM can effectively verify OTA detection in food, significantly reducing false-positive and false-negative rates. The proposed platform offers sensitive and accurate detection of mycotoxins in food and can be adapted for monitoring other trace contaminants by simply altering the aptamer., 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.)

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.)