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As a common heavy metal contaminant, Cd 2+ has adverse effects on food safety and consumer health. It is very important for human health to realize highly sensitive Cd 2+ detection methods. The self-powered sensing system based on enzyme biofuel cells (EBFCs) does not need an external power supply, which can simplify the experimental equipment and has great application value in portable detection. Thus, the biosensor is innovatively integrated into the screen-printed electrode to construct a new type of portable sensor suitable for on-site and real-time Cd 2+ detection. Hybridization chain reaction (HCR) combined with the Cd 2+ -dependent deoxyribose (DNAzyme) signal amplification strategy is used to enhance the detection sensitivity while specifically recognizing the Cd 2+ . Moreover, the self-powered sensor combines with smartphones to realize quantitative Cd 2+ detection without other instruments and has the characteristic of Effectively improving the hazard detection technology is essential to ensure food safety. Portability, simplicity, and speed are suitable for real-time Cd 2+ detection in the field. The dual mechanism and three quantitative modes combining colorimetric and two electrical signals output modes are adopted to realize the visualization and accurate detection. A series of research results confirm that this strategy is of great significance to strengthen the development of intelligent Cd 2+ technology, expand the application of self-powered sensing technology, and improve the safety detection system., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The Hybridization Chain Reaction (HCR) is an isothermal amplification technique widely used for sensing nucleic acids and small molecules. Despite its effectiveness, conventional linear HCR exhibits relatively slow kinetics and insufficient sensitivity. To address this challenge, we have innovatively combined HCR with DNAzyme technology to enhance nucleic acid detection. In this novel approach, the presence of a target molecule triggers the formation of DNAzyme, leading to the cleavage of substrate S, the initiation of HCR, and the production of DNA nanowires and labeled DNAzyme. The newly generated DNAzyme continuously cleaves substrate S, promoting sequential HCR amplification and significantly enhancing the fluorescence signal. This system offers a simple, sensitive, selective, and versatile method for nucleic acid detection, with a detection limit as low as 5 pM. When tested on classical swine fever virus (CSFV) samples, the system demonstrated detection accuracy comparable to RT-qPCR and exhibited superior repeatability.

DNA-templated silver nanoclusters (DNA-AgNCs) are novel nanomaterials with unique fluorescence characteristics. DNAzyme is a functional oligonucleotide that can catalyze the disruption of nucleic acid substrates. In this research, the effect of DNAzyme digestion on the fluorescence property of DNA-AgNCs was explored for the first time. A significant reduction in the fluorescence intensity of DNA-AgNCs after cleavage by DNAzyme was discovered. Further research found that the DNAzyme-catalyzed cleavage reduced the stability of DNA-AgNCs and led to their aggregation, accounting for a decline in fluorescence intensity up to 84%. Inspired by the above finding, a fluorescent aptasensor that integrates the benefits of DNA-AgNCs, exonuclease III (Exo III)-assisted signal amplification and DNAzyme was developed for sensitive detection of adenosine triphosphate (ATP). Under optimal conditions, the linear range was from 25 μM to 1000 μM and the detection limit was estimated to be 4.46 μM. Furthermore, this fluorescent aptasensor was effectively employed to quantify ATP levels in human serum samples, demonstrating its practicality in detecting ATP in biological matrices. The elucidation of DNAzyme-based fluorescence characteristic variation of DNA-AgNCs may provide insights into the interactions between DNAzyme and nanomaterials and has great prospects in the construction of fluorescent biosensors.

Cells use transient membraneless organelles to regulate biological reaction networks. For example, stress granules selectively store mRNA to downregulate protein expression in response to heat or oxidative stress. Models mimicking this active behavior should be established to better understand in vivo regulation involving compartmentalization. Here we use active, complex coacervate droplets as a model for membraneless organelles to spatiotemporally control the activity of a catalytic DNA (DNAzyme). Upon partitioning into these peptide-RNA droplets, the DNAzyme unfolds and loses its ability to catalyze the cleavage of a nucleic acid strand. We can transiently pause the DNAzyme activity upon inducing droplet formation with fuel. After fuel consumption, the DNAzyme activity autonomously restarts. We envision this system could be used to up and downregulate multiple reactions in a network, helping understand the complexity of a cell's pathways. By creating a network where the DNAzyme could reciprocally regulate the droplet properties, we would have a powerful tool for engineering synthetic cells., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Background: Enzymes with critical effects on life systems are regulated by expression and activation to modulate life processes. However, further insights into enzyme functions and mechanisms in various physiological processes are limited to concentration or activation analysis only. Currently, enzyme analysis has received notable attention, particularly simultaneous analysis of their concentration and activation in one system. Herein, N-methyl mesoporphyrin IX (NMM), a specific dye with notable structural selectivity for parallel G-quadruplex nucleic acid enzyme (G4h DNAzyme), is employed for the analysis of its concentration. In addition, the peroxidase activity of G4h DNAzyme is characterized based on G4h DNAzyme-catalyzed decomposition of H 2 O 2 to continuously consume luminol. Accordingly, an increased fluorescence (FL) response of NMM and a decreased FL response of luminol could be simultaneously employed to analyze the concentration and activation of G4h DNAzyme., Result: Herein, a novel concentration and activation biresponsive strategy is proposed using a G4h DNAzyme-based model that simultaneously employs a G4h structure-specific signal probe for enzyme concentration analysis and G4h DNAzyme-catalyzed reactions for enzyme activation analysis. Under optimal conditions, the biresponsive strategy can be effectively used for the simultaneous analysis of G4h DNAzyme concentration and activation, with detection limits of 718.7 pM and 233.4 nM respectively, delivering acceptable performances both in cell and in vitro., Significance: This strategy can not only be applied to concentration and activation analyses of G4h DNAzyme but can also be easily extended to other enzymes by simultaneously combining concentration analysis via target-induced direct reaction and activation analysis via target-induced catalytic reaction, offering deeper insights into various enzymes and enabling their effective implementation in bioanalysis and biochemistry., 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.)

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.)

Detection methods based on CRISPR/Cas12a have been widely developed in the application of pathogenic microorganisms to guarantee food safety and public health. For sensitive detection, the CRISPR-based strategies are often in tandem with amplification methods. However, that may increase the detection time and the process may introduce nucleic acid contamination resulting in non-specific amplification. Herein, we established a sensitive S. aureus detection strategy based on the CRISPR/Cas12a system combined with DNAzyme. The activity of Cas12a is blocked by extending the spacer of crRNA (bcrRNA) and can be reactivated by Mn 2+ . NH 2 -modified S. aureus-specific aptamer was loaded on the surface of Fe 3 O 4 MNPs (apt-Fe 3 O 4 MNPs) and MnO 2 NPs (apt-MnO 2 NPs) by EDC/NHS chemistry. The S. aureus was captured to form apt-Fe 3 O 4 MNPs/S. aureus/apt-MnO 2 NPs complex and then MnO 2 NPs were etched to release Mn 2+ to activate DNAzyme. The active DNAzyme can cleave the hairpin structure in bcrRNA to recover the activity of the CRISPR/Cas system. By initiating the whole detection process by generating Mn 2+ through nanoparticle etching, we established a rapid detection assay without nucleic acid extraction and amplification process. The proposed strategy has been applied in the ultrasensitive quantitative detection of S. aureus and has shown good performance with an LOD of 5 CFU/mL in 29 min. Besides, the proposed method can potentially be applied to other targets by simply changing the recognition element and has the prospect of developing a universal detection strategy., 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.)

Traditional DNA walkers face enormous challenges due to limited biostability and reaction kinetics. Herein, we designed a self-driven close-looped DNAzyme walker (cl-DW) with high structural biostability and catalytic activity that enabled rapid electrochemiluminescence (ECL) detection of pesticide residue acetamiprid. Specifically, cl-DW exhibited increasing ability to resist nuclease degradation with a 570-fold longer half-degradation time than that of the single-stranded DNAzyme walker (ss-DW) due to the protected DNA terminal. Furthermore, cl-DW achieved high catalytic activity with a 4.3-fold faster reaction kinetic than that of ss-DW due to the circularized nanostructure of an available catalytic domain. Consequently, we utilized cl-DW as a signal amplifier and tin-based sulfide (SnS 2 ) nanoflowers as ECL emitters to construct an ECL aptasensor, which realized the sensitive detection of acetamiprid with a limit of detection of 0.85 nM. This work provides a reliable approach to exploring DNA walkers with high catalytic activity and better biostability for molecular 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 © 2023. Published by Elsevier Ltd.)

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.

Autocatalysis, a self-sustained replication process where at least one of the products functions as a catalyst, plays a pivotal role in life's evolution, from genome duplication to the emergence of autocatalytic subnetworks in cell division and metabolism. Leveraging their programmability, controllability, and rich functionalities, DNA molecules have become a cornerstone for engineering autocatalytic circuits, driving diverse technological applications. In this tutorial review, we offer a comprehensive survey of recent advances in engineering autocatalytic DNA circuits and their practical implementations. We delve into the fundamental principles underlying the construction of these circuits, highlighting their reliance on DNAzyme biocatalysis, enzymatic catalysis, and dynamic hybridization assembly. The discussed autocatalytic DNA circuitry techniques have revolutionized ultrasensitive sensing of biologically significant molecules, encompassing genomic DNAs, RNAs, viruses, and proteins. Furthermore, the amplicons produced by these circuits serve as building blocks for higher-order DNA nanostructures, facilitating biomimetic behaviors such as high-performance intracellular bioimaging and precise algorithmic assembly. We summarize these applications and extensively address the current challenges, potential solutions, and future trajectories of autocatalytic DNA circuits. This review promises novel insights into the advancement and practical utilization of autocatalytic DNA circuits across bioanalysis, biomedicine, and biomimetics.

Dynamic DNA nanodevices, particularly DNA walkers, have proven to be versatile tools for target recognition, signal conversion, and amplification in biosensing. However, their ability to detect low-abundance analytes in complex biological samples is often compromised by limited amplification depth and severe signal leakage. To address these challenges, we developed a simple yet highly efficient strategy to engineer a self-replicating bipedal DNAzyme (SEDY) walker for sensitive and selective electrochemiluminescence (ECL) bioanalysis. Unlike conventional DNA walkers that are typically constructed by catalytic DNA assembly in a single direction, the SEDY walker integrates a self-replicating feedback mechanism that greatly enhances both the selectivity and sensitivity of bioanalysis. First, the SEDY walker is assembled through a target-triggered, enzyme-free, self-replicating catalytic approach, minimizing the risk of undesired side reactions and signal leakage by simplifying reactant complexity. Furthermore, the SEDY walker features newly exposed trigger sequences that facilitate its autonomous replication, leading to a robust and exponential amplification of its products. Our experiments demonstrate that the SEDY walker can sensitively and selectively detect acetamiprid by navigating specific probes within cross-shaped DNA orbits. The ECL biosensor offers a linear detection range from 1 × 10 -15 M to 1 × 10 -9 M, with a limit of detection as low as 5.8 × 10 -16 M. We anticipate that the SEDY walker will be a powerful tool for detecting various analytes in biological applications.

Methods for modifying intact peptides are useful but can be unselective with regard to amino acid position and sequence context. In this work, we used in vitro selection to identify DNAzymes that acylate a Lys residue of a short peptide in sequence-dependent fashion. The DNAzymes do not acylate Lys when placed at other residues in the peptide, and the acylation activity depends on the Lys sequence context. A high acylation yield is observed on the preparative nanomole scale. These findings are promising for further development of DNAzymes for broader application to top-down Lys acylation of peptide and protein substrates., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

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.

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.)

Background: Rapid industrial development has generated serious pollution, including the presence of toxic and harmful heavy metal ions. Among them, trivalent chromium ion (Cr 3+ ) is a very important element that poses a threat to life and health in our industrial wastewater pollution. Thus, it is important to develop efficient fluorescence methods for Cr 3+ detection. In this study, an upconversion luminescence biosensor for detecting Cr 3+ was constructed based on a DNAzyme, strand displacement reaction (SDR), and DNA-functionalized upconversion nanoparticles (UCNPs)., Results: The sulfonate-rich poly (sodium 4-styrene sulfonate) (PSS) was modified onto the surface of UCNPs, forming UCNPs@PSS. Then, NH 2 -Capture probe DNA (NH 2 -Cp) was further modified onto the UCNPs@PSS surface through sulfonylation, resulting in UCNPs@PSS@NH 2 -Cp. The DNAzyme activated by Cr 3+ triggered the release of the primer probe (Pp), which initiated the SDR system cycle, thereby releasing a tetramethylrhodamine (TAMRA)-modified signal probe (TAMRA-Sp). Finally, UCNPs@PSS@NH 2 -Cp bound to TAMRA-Sp through complementary base pairing, causing UCNPs and TAMRA to approach each other. Because of the luminescence resonance energy transfer (LRET) mechanism, the upconversion luminescence (UCL) signal of the UCNPs was quenched by TAMRA, enabling the detection of Cr 3+ by the change of I 585 /I 545 ratio. This biosensor has good stability, selectivity, and sensitivity, with a linear range of 0.5-75 nM and a detection limit of 0.135 nM for Cr 3+ ., Significance and Novelty: Firstly, based on LRET between UCNPs and TAMRA, the quantitative analysis of Cr 3+ is achieved through the changes of ratio fluorescence. Secondly, the specificity of the biosensor is improved by utilizing the specific recognition of DNA enzymes. Thirdly, the signal amplification technology of the SDR cycle greatly improves the sensitivity of biosensor. This biosensor will be useful for future environmental safety monitoring and biopsy of biological fluids., 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 kind of mycotoxin, aflatoxin B1 (AFB1), which is often found in agricultural products, poses a threat to human health. Developing a simple sensitive method for AFB1 detection is in great demand. Here, we reported an aptamer-based fluorescence assay for AFB1 detection by using DNAzyme to generate and amplify a signal. We redesigned a pair of DNA sequences, which originated from the anti-AFB1 aptamer and RNA-cleaving DNAzyme 10-23. In the absence of AFB1, the aptamer hybridized with the region of the substrate-binding arm of the DNAzyme, inhibiting the activity of the DNAzyme. In the presence of AFB1, the binding of AFB1 to the aptamer led to the displacement of the DNAzyme from the aptamer. The substrate-binding arm was unblocked, and the activity of the DNAzyme was restored for the hydrolysis of the fluorophore and quencher-labeled substrate, causing a significant fluorescence increase. This assay could detect AFB1 in the dynamic range from 0.98 to 2000 nmol/L with high selectivity, and the detection limit was 0.98 nmol/L. Moreover, the assay was able to detect AFB1 in a complex sample matrix. This work provides a useful tool for the analysis of AFB1., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Patulin (PAT) is a mycotoxin-produced secondary metabolite that can contaminate foods, causing toxic effects on animal and human health. Therefore, for the first time, we have constructed a "turn-on" dual-mode aptamer sensor for PAT using oleic acid-coated upconversion nanomaterials (OA-UCNPs) and G-Quadruplex-hemin DNAzyme (G4-DNAzyme) as fluorescent and colorimetry probes. The sensor employs aptamers binding to PAT as recognition elements for specific molecule detection. Mxene-Au can be used as a biological inducer to assist OA-UCNPs in controlling fluorescence intensity. In addition, colorimetric signal amplification was performed using the trivalent G4-DNAzyme to increase detection sensitivity and reduce false positives. Under optimal conditions, the dual-mode aptasensor has a detection limit of 5.3 pg mL -1 in fluorescence and 2.4 pg mL -1 in colorimetric methods, respectively, with the wider linear range and limit of detection (LOD) of the colorimetric assay. The combination aptasensor can detect PAT with high sensitivity and high specificity and has broad application prospects in the field of food safety detection., 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.)

The biomimetic enzyme cascade system plays a key role in biosensing as a sophisticated signal transduction and amplification strategy. However, constructing a regulated enzyme cascade sensing system remains challenging due to the mismatch of multiple enzyme activities and poor stability. Herein, we design an efficient dual-enhanced enzyme cascade hybrid system (UFD-DEC) containing DNA-controlled nanozymes (Fe-cdDNA) and enzyme (urease) via combining the electrostatic contact effect with the hydrogel-directed confinement effect. Precise modulation of Fe-cdDNA nanozyme by DNA offers a means to control its catalytic efficiency. This regulated UFD-DEC system accelerates the reaction rate and provides remarkable stability compared with the free enzyme system. Benefiting from the plasticity properties of hydrogels, a "lab-in-a-tube" platform was constructed by encapsulating UFD-DEC in a microcentrifuge tube. Such a UFD-DEC-based hydrogel tube exhibits sufficient adaptability to profile urea when used in conjunction with a smartphone-assisted image processing algorithm, which on-site delivers urea information with a detection limit of 0.12 mmol L -1 . This customizable and inexpensive miniaturized biosensor platform for monitoring urea may facilitate point-of-care testing 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.)

In this work, a platinum-nickel based nanozyme is prepared and used as a coreaction accelerator in the luminol-H 2 O 2 electrochemiluminescence (ECL) system to construct an ECL biosensor for dimethyl phthalate (DMP) detection. The PtNi/NC nanozyme possesses dispersed metal active sites, and the synergistic effect of Pt and Ni endows it with excellent catalytic performance, which effectively converts H 2 O 2 into more superoxide anions, and then significantly enhances the ECL intensity of the luminol system. The ECL mechanism is investigated by combining cyclic voltammetry and ECL with different types of free radical scavengers. Simultaneously, an "off-on" biosensor is constructed by integrating 3D DNA walker with enzyme-free recycling amplification for ultrasensitive detection of DMP. The biosensor based on PtNi/NC nanozyme mediated luminol-H 2 O 2 system and 3D DNA walker exhibits a linear range of 1 × 10 -16 to 1 × 10 -6  M with a detection limit of 4.3 × 10 -17  M (S/N = 3), and displays good stability and specificity. This study demonstrates the advantages of PtNi/NC nanozyme in enhancing the luminol-H 2 O 2 ECL system, providing new strategy for designing efficient ECL emitter and offering a new method for detecting phthalate esters., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The CRISPR/Cas12a system is increasingly used in biosensor development. However, high background signal and low sensitivity for the non-nucleic acid targets detection is challenging. Here, a padlock activator which could inhibit the trans-cleavage activity of CRISPR/Cas12a system in the intact form by steric hindrance effect (PAIT effect) was designed for non-nucleic acid targets detection. The PAIT effect disappeared when padlock activator was separated into two split activators. To verify the feasibility of padlock activator, a Ca 2+ sensor was developed based on PAIT effect with the assistance of DNAzyme, activity of which was Ca 2+ dependent. In the presence of Ca 2+ , DNAzyme was activated to cleave its substrate, a padlock activator modified with adenine ribonucleotide, into split padlock activators which would trigger the trans-cleavage activity of Cas12a to generate fluorescence. There was a mathematical relationship between the fluorescence intensity and the logarithm of Ca 2+ concentration ranging from 10 pM to 1 nM, with a limit of detection of 3.98 pM. The little interference of Mg 2+ , Mn 2+ , Cd 2+ , Cu 2+ , Na + , Al 3+ , K + , Fe 2+ , and Fe 3+ indicated high selectivity. Recovery ranged from 93.32% to 103.28% with RSDs from 1.87% to 12.74% showed a good accuracy and reliability. Furthermore, the proposed sensor could be applied to detect Ca 2+ in mineral water, milk powder and urine. The results were consistent with that of flame atomic absorption spectroscopy. Thus, PAIT effect is valuable for expanding the application boundary of CRISPR/Cas12a system., 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.)

Catalytic DNA has gained significant attention in recent decades as a highly efficient and tunable catalyst, thanks to its flexible structures, exceptional specificity, and ease of optimization. Despite being composed of just four monomers, DNA's complex conformational intricacies enable a wide range of nuanced functions, including scaffolding, electrocatalysis, enantioselectivity, and mechano-electro spin coupling. DNA catalysts, ranging from traditional DNAzymes to innovative DNAzyme hybrids, highlight the remarkable potential of DNA in catalysis. Recent advancements in spectroscopic techniques have deepened our mechanistic understanding of catalytic DNA, paving the way for rational structural optimization. This review will summarize the latest studies on the performance and optimization of traditional DNAzymes and provide an in-depth analysis of DNAzyme hybrid catalysts and their unique and promising properties.

This study constructed an electrochemiluminescence (ECL) biosensor for ultrasensitive detection of Pb 2+ in a ternary system by employing DNAzyme. The ternary system is composed of a potassium-neutralized perylene derivative (K 4 PTC) as the ECL emitter, K 2 S 2 O 8 as the coreactant, and neodymium metal-organic frameworks (Nd-MOFs) as the coreaction accelerators. Nd-MOFs immobilize DNAzymes and enhance the luminescence intensity of the K 4 PTC/K 2 S 2 O 8 system. As part of this system, K 4 PTC enhances the ECL signal in solution and supports Pb 2+ detection. The sequence of ferrocene (Fc)-linked DNA (DNA-Fc) is catalytically cleaved by DNAzymes in the presence of Pb 2+ . This causes the removal of DNA1-Fc from the electrode surface to recover the ECL signal. As a result, the as-prepared ECL biosensor can quantify Pb 2+ with a detection limit (LOD) of 4.1 fM in the range of 1 μM to 10 fM. The ECL biosensor displays high specificity, good stability, excellent reproducibility, and desirable practicality for Pb 2+ detection in tap water. Moreover, by simply changing the sequence of the DNAzyme, new biosensors can be designed for ultrasensitive detection of different heavy metal ions, offering an excellent approach for monitoring water quality safety.

Dipeptidyl peptidase IV (DPPIV) is an enzyme belonging to the type II transmembrane serine protease family that has gained wide interest in the fields of hematology, immunology, and cancer biology. Moreover, DPPIV has emerged as a promising target for therapeutic intervention in type II diabetes. Due to its biological limitations, traditional strategies cannot meet the requirements of low abundance DPPIV analysis in complex environments. In this work, combining the high programmability of DNA and the chemical diversity of peptides, we designed DNA-peptide conjugates that can be specifically recognized, polypeptides as specific substrates for target DPPIV and DNA probes as primers for catalytic hairpin assembly (CHA), recycling a large amount of DNAzymes by triggering CHA amplification. The DNAzyme substrate modified with the FAM fluorescent group was immobilized on the surface of gold nanoparticles by S-Au chemical bonds to form a signal output probe. The DNAzymes enzyme cleaved the substrate of the signal outputs probe, yielding a double-amplified fluorescence signal. This method has a detection limit as low as 0.18 mU mL -1 and a linear range of 0-5 mU mL -1 in serum samples, showing high stability and good potential for practical applications.

The development of highly sensitive and precise imaging techniques capable of visualizing crucial molecules at the subcellular level is essential for elucidating mitochondrial functions and uncovering novel mechanisms in biological processes. However, traditional molecular imaging strategies are still limited by off-mitochondria signal leakage because of the "always-active" sensing mode. To address this limitation, we have developed a light-triggered activation sequence activated plasmonic DNAzyme walker (PDW) for accurate subcellular molecular imaging by the combination of an organelle localized strategy, upconversion nanotechnology, and a plasmon enhanced fluorescence (PEF) technique. Exploiting the advantage of light activation enables precise control over when and where to activate the probe's sensing function, effectively reducing off-mitochondria signal leakage as validated by the dynamic monitoring of changes in off-mitochondria signals during the mitochondrial entry process. Furthermore, by leveraging the PEF capability of triangular gold nanoprisms (Au NPRs), the fluorescence intensity can be enhanced by approximately 11.9 times, ensuring highly sensitive and accurate subcellular molecular imaging.

DNA walkers have attracted considerable attention in biosensing and bioimaging. Compared with the conventional single leg-based DNA walker, the bipedal DNA walker has remarkable advantages, with improved sensitivity and fast kinetics, and can work efficiently in a crowded cellular environment. However, most reported bipedal DNA walkers are powered by exogenous supplementation, and elaborate DNA sequence designs, auxiliary additives or extra carriers are often needed. A highly integrated bipedal DNA walker that can address robustness, sensitivity and consistency issues in a single system is highly desirable but remains a great challenge. We herein report a novel bipedal DNA nanowalker system through simple assembly of a DNA substrate, hairpin functionalized-AuNPs (AuNPs-H2), and a blocked Mn 2+ -dependent DNAzyme hairpin (H1) on degradable MnO 2 nanosheets, which holds great potential for living cell operation. Highly integrated features enable the simultaneous delivery of core components of the bipedal DNA walker, including a walking track (AuNPs-H2), a walking strand (H1 cleaved by APE1), and a driving force (Mn 2+ -dependent DNAzyme cleavage) as a whole, thereby enhancing the control of the spatiotemporal distribution of these components at the intracellular target sites. The redox reaction between the MnO 2 nanosheets and GSH inside the cells not only consumed the intracellular GSH to improve the biostability of the walking track but also generated abundant Mn 2+ as a cofactor of the DNAzyme. As a proof of concept, the developed nanowalker was demonstrated to work efficiently for monitoring base excision repair (BER)-related human apurinic/apyrimidinic endonuclease 1 (APE1) in living cells, highlighting the great potential of the bipedal DNA nanowalker in biological systems., (© 2024. The Author(s).)

CRISPR/Cas12a fluorimetry has been extensively developed in the biosensing arena, on account of its high selectivity, simplicity, and rapidness. However, typical CRISPR/Cas12a fluorimetry suffers from low sensitivity due to the limited trans-cleavage efficiency of Cas12a, necessitating the integration of other preamplification techniques. Herein, we develop an enhanced CRISPR/Cas12a fluorimetry via a DNAzyme-embedded framework nucleic acid (FNAzyme) substrate, which was designed by embedding four CLICK-17 DNAzymes into a rigid tetrahedral scaffold. FNAzyme can not only enhance the trans-cleavage efficiency of CRISPR/Cas12a by facilitating the exposure of trans-substrate to Cas12a but also result in an exceptionally high signal-to-noise ratio by mediating enzymatic click reaction. Combined with a functional nucleic acid recognition module, this method can profile methicillin-resistant Staphylococcus aureus as low as 18 CFU/mL, whose sensitivity is approximately 54-fold higher than that of TaqMan probe-mediated CRISPR/Cas12a fluorimetry. Meanwhile, the method exhibited satisfactory recoveries in food matrices ranging from 80% to 101%. The DNA extraction- and preamplification-free detection format as well as the potent detection performance highlight its tremendous potential as a next-generation analysis tool.

The 10-23 DNAzyme is one of the most active DNA-based enzymes, and in theory, can be designed to target any purine-pyrimidine junction within an RNA sequence for cleavage. However, purine-pyrimidine junctions within a large, structured RNA (lsRNA) molecule of biological origin are not always accessible to 10-23, negating its general utility as an RNA-cutting molecular scissor. Herein, we report a generalizable strategy that allows 10-23 to access any purine-pyrimidine junction within an lsRNA. Using three large SARS-CoV-2 mRNA sequences of 566, 584 and 831 nucleotides in length as model systems, we show that the use of antisense DNA oligonucleotides (ASOs) that target the upstream and downstream regions flanking the cleavage site can restore the activity (kobs) of previously poorly active 10-23 DNAzyme systems by up to 2000-fold. We corroborated these findings mechanistically using in-line probing to demonstrate that ASOs reduced 10-23 DNAzyme target site structure within the lsRNA substrates. This approach represents a simple, efficient, cost-effective, and generalizable way to improve the accessibility of 10-23 to a chosen target site within an lsRNA molecule, especially where direct access to the genomic RNA target is necessary., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We have systematically investigated and found surprising superior catalytic activities of very short DNAzymes for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), both in solution and on surface. As a key reaction of the "click chemistry" class, CuAAC is a highly efficient and specific covalent conjugation tool with demonstrated applications in organic synthesis, bioconjugation, and surface functionalization; however, it requires the presence of the Cu(I) catalyst, which is an unstable species in aqueous solutions. We show here that one ultrashort, 14-nucleotide-truncated fragment of an earlier in vitro selected DNAzyme (CLICK-17) shows a striking and superior catalytic activity toward the in trans CuAAC reaction in solution and on surface in the presence of either Cu(I) or Cu(II), at significantly lowered concentrations. These results obviate the need for long-sequence DNAzymes, selected out of the homogeneous solution phase, for application in complex surface environments.

Misuse of chloramphenicol (CAP) can lead to severe food safety issues. Therefore, the accurate and sensitive detection of CAP residues is important for public health. Herein, a convenient and reliable interfacial self-assembly technique was used to form a uniform Au@Ag nanobipyramids (NBPs) film on an ordered SiO 2 nanosphere array (SiO 2 NS), which served as a Raman-enhanced substrate. In conjunction with a deoxyribonucleic acid enzyme-induced signal amplification strategy, we developed a novel surface-enhanced Raman scattering (SERS) biosensor for the selective and sensitive detection of CAP. The biosensor exhibited a detection limit of 6.42 × 10 -13  mol·L -1 and a detection range of 1.0 × 10 -12 -1.0 × 10 -6  mol·L -1 . The biosensor could detect CAP in spiked milk samples with a high accuracy, and its recovery rates ranged from 97.88% to 107.86%. The as-developed biosensor with the advantages of high sensitivity and high selectivity offers a new strategy for the rapid, reliable and sensitive detection of CAP, rendering it applicable to food safety control., 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. Neither the entire manuscript nor any part of its content has been published or has been accepted elsewhere and this manuscript has not been submitted to any other journal. No portion of the text has been copied from other material in the literature. All of the authors in this manuscript have read and approved the final version submitted, and there are no conflicts involved in this submission., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Different types of pathogenic viruses that have common transmission path can be co-infected, inducing distinct disease procession in comparison to that infection of one. Also, in the post COVID-19 time, more types of respiratory infectious virus are becoming prevalent and are concurrent. Those bring an urgent need for detection of co-existing viruses. Here, we propose a visualized lateral flow assay for logic determination of co-existing viral RNA fragments. In the presence of specific viral RNA inputs, DNAzyme is de-blocked according to defined logic, and catalyzes the hydrolysis of hairpin-structural substrate. One of cleaved substrates contains DNAzyme domain to realize dual signal amplification, which obtains copious of the other cleaved substrates. The cleaved substrates act as linking strands for bridging DNA-modified gold nanoparticles onto lateral flow strip to induce coloration on test line. "AND", "OR" and "INHIBIT" controlled lateral flow assays are respectively demonstrated for co-existing viral RNA detection, and the visual results can be obtained by the same kind of prepared strip, without need of re-fabricating strips according to logic systems. The work provides a flexible, convenient, visual and logic-processing strategy for simultaneous analysis of co-existing viruses., 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 circulating tumor cells (CTCs) have demonstrated considerable importance in liquid biopsy, their detection is limited by low concentrations and complex sample components. Herein, we developed a homogeneous, simple, and high-sensitivity strategy targeting breast cancer cells. This method was based on a non-immunological stepwise centrifugation preprocessing approach to isolate CTCs from whole blood. Precise quantification is achieved through the specific binding of aptamers to the overexpressed mucin 1 (MUC1) and human epidermal growth factor receptor 2 (HER2) proteins of breast cancer cells. Subsequently, DNAzyme cleavage and parallel catalytic hairpin assembly (CHA) reactions on the cholesterol-stacking DNA machine were initiated, which opened the hairpin structures T-Hg 2+ -T and C-Ag + -C, enabling multiple amplifications. This leads to the fluorescence signal reduction from Hg 2+ -specific carbon dots (CDs) and CdTe quantum dots (QDs) by released ions. This strategy demonstrated a detection performance with a limit of detection (LOD) of 3 cells/mL and a linear range of 5-100 cells/mL. 42 clinical samples have been validated, confirming their consistency with clinical imaging, pathology findings and the folate receptor (FR)-PCR kit results, exhibiting desirable specificity of 100% and sensitivity of 80.6%. These results highlight the promising applicability of our method for diagnosing and monitoring breast 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 © 2024 Elsevier B.V. All rights reserved.)

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.)

Multiplex detection can enhance diagnostic precision and improve diagnostic efficiency, providing important assistance for epidemiological investigation and epidemic prevention. There is a great need for multi-detection sensing platforms to accurately diagnose diseases. Herein, we reported a μPAD-based chemiluminescence (CL) assay for ultrasensitive multiplex detection of AIV biomarkers, based on three DNAzyme/Lum/PEI/CaCO 3 . Three time-resolved CL signals were sequentially generated with detection limits of 0.32, 0.34, and 0.29 pM for H1N1, H7N9, and H5N1, respectively, and with excellent selectivity against interfering DNA. The recovery test in human serum displayed satisfactory analysis capabilities for complex biological samples. The μPAD-based CL assay achieved multiplex detection within 70 s, with a high time resolution of 20 s. The proposed strategy has the advantages of low cost, high sensitivity, good selectivity, and wide time resolution, the μPAD-based CL assay has shown great potential in the early and accurate diagnosis of 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 Inc. All rights reserved.)

Developing synergistic targeted therapeutics to improve treatment efficacy while reducing side effects has proven promising for anticancer therapies, but how to conveniently modulate multidrug cooperation remains a challenge. Here, a novel synergistic strategy using a G-quadruplex-programmed versatile nanorobot (G4VN) containing two subunits of DNAzyme (DzG4) and ligand-drug conjugates (LDCs) is proposed to precisely target tumors and then execute both gene silencing and chemotherapy. As the core module of this nanorobot, a well-designed G4 responding to a high level of K + in tumor microenvironment smartly kills three birds with one stone, which makes two TfR aptamers proximate to improve their efficiency of targeting tumor cells, and in situ activates a split 10-23 DNAzyme to downregulate target mRNA expression, meanwhile promotes the cell uptake of a GSH-responsive LDCs to enhance drug efficacy. Such a design enables a potently synergistic anticancer therapy with low side effects in vivo, showing great promise for broad applications in precision disease treatment., (© 2024 Wiley‐VCH GmbH.)

DNAzymes, a class of single-stranded catalytic DNA with good stability, high catalytic activity, and easy synthesis, functionalization and modification properties, have garnered significant interest in the realm of biosensing and bioimaging. Their integration with fluorescent dyes or chemiluminescent moieties has led to remarkable bioimaging outcomes, while DNAzyme-based biosensors have demonstrated robust sensitivity and selectivity in detecting metal ions, nucleic acids, proteins, enzyme activities, exosomes, bacteria and microorganisms. In addition, by delivering DNAzymes into tumor cells, the mRNA therein can be cleaved to regulate the expression of corresponding proteins, which has further propelled the application of DNAzymes in cancer gene therapy and synergistic therapy. This paper reviews the strategies for screening attractive DNAzymes such as SELEX and high-throughput sequencing, and briefly describes the amplification strategies of DNAzymes, which mainly include catalytic hairpin assembly (CHA), DNA walker, hybridization chain reaction (HCR), DNA origami, CRISPR-Cas12a, rolling circle amplification (RCA), and aptamers. In addition, applications of DNAzymes in bioimaging, biosensing, and cancer therapy are also highlighted. Subsequently, the possible challenges of these DNAzymes in practical applications are further pointed out, and future research directions are suggested.

Background: Rapid and sensitive colorimetric detection methods are crucial for diseases diagnosis, particularly those involving proteases like furin, which are implicated in various conditions, including cancer. Traditional detection methods for furin suffer from limitations in sensitivity and practicality for on-site detection, motivating the development of novel detection strategies. Therefore, developing a simple, enzyme-free, and rapid colorimetric analysis method with high sensitivity for furin detection is imperative., Results: Herein, we have proposed a colorimetric method in this work for the first time to detect furin, leveraging the assembly of G-quadruplex/hemin DNAzyme with enhanced catalytic activity. Specifically, a peptide-DNA conjugate (PDC) comprising a furin-recognition peptide and flanking DNA sequences for signal amplification is designed to facilitate the DNAzyme assembly. Upon furin treatment, PDC cleavage triggers a cyclic catalytic hairpin assembly reaction to form the complementary double-stranded structures by hairpin 1 (HP1) and hairpin 2 (HP2), bringing the G-quadruplex sequence in HP1 closer to hemin on HP2. Moreover, the resulting G-quadruplex/hemin DNAzymes exhibit robust peroxidase-like activity, enabling the catalysis of the colorimetric reaction of ABTS 2- for furin detection. Our method demonstrates high sensitivity, rapid response, and compatibility with complex sample matrices, achieving a detection limit as low as 1.1 pM., Significance: The DNAzyme reported in this work exhibits robust catalytic activity, enabling high sensitivity and good efficiency for the detection. By eliminating the requirement for exogenous enzymes, our approach enables visual furin detection without expensive instrumentation and reagents, promising significant utility in biomedical and clinical diagnostic applications. Given the various design of peptide sequence and the programmability of DNA, it can be readily applied to analyzing other useful tumor 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 Elsevier B.V. All rights reserved.)

The dynamic landscape of non-canonical DNA G-quadruplex (G4) folding into G-triplex intermediates has led to the study of G-triplex structures and their ability to serve as peroxidase-mimetic DNAzymes. Here we report the formation, stability, and catalytic activity of a 5'-truncated c-MYC promoter region G-triplex, c-MYC-G3. Through circular dichroism, we demonstrated that c-MYC-G3 adopts a stable, parallel-stranded G-triplex conformation. The chemiluminescent oxidation of luminol by the peroxidase mimicking DNAzyme activity of c-MYC-G3 was increased in the presence of Ca 2+ ions. We utilized surface plasmon resonance to characterize both c-MYC-G3 G-triplex formation and its interaction with hemin. The detailed study of c-MYC-G3 and its ability to form a G-triplex structure and its DNAzyme activity identifies issues that can be addressed in future G-triplex DNAzyme designs.

Lead ions (Pb 2+ ) are a widely distributed and highly toxic heavy metal pollutant, which seriously threatens the environment, economy and human safety. Here, a label-free ratiometric fluorescent biosensor was constructed for Pb 2+ detection using DNAzyme-driven target cycling and exonuclease III (Exo III)-mediated DNA cycling as a dual signal amplification strategy. The SYBR Green I (SGI) and N -methyl mesoporphyrin IX (NMM) used in this study are characterized by low cost, storage resistance, and short preparation time compared with conventional signaling probes labeled with fluorescent groups. Unlike the single-emission fluorescence strategy, monitoring the fluorescence intensity ratio of SGI and NMM can effectively reduce external interference to achieve accurate detection of Pb 2+ . DNAzyme structures on the surface of magnetic beads (MBs) can recognize Pb 2+ and activate the target circulatory system to cleave single-stranded DNA (ssDNA). The ssDNA further initiated the Exo III-assisted DNA circulatory system to digest double-stranded DNA (dsDNA) and release guanine-rich G1. Finally, the fluorescence signals of SGI and NMM were weakened and enhanced, respectively. The sensing strategy achieved a wide linear range from 0.5 to 500 nM and a low limit of detection (LOD) of 26.4 pM. Furthermore, its anti-interference ability and potential applicability for Pb 2+ detection in actual samples were verified. This work ingeniously combines the dual signal amplification strategy with the ratiometric sensing strategy constructed by structure-specific fluorescent dyes, which provides a promising method for constructing sensitive and accurate fluorescent biosensors.

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.

Binary (also known as split) nucleic acid enzymes have emerged as novel tools in biosensors. We report a new split strategy to split the DNAzyme kinase into two independent and non-functional fragments, denoted Dk1sub and Dk1enz. In the presence of the specific target, their free ends are brought sufficiently close to interact with each other without the formation of Watson-Crick base pairings between Dk1sub and Dk1enz, thus allowing the DNA phosphorylation reaction. We term this approach proximity-dependent activation of split DNAzyme kinase (ProxSDK). The utility of ProxSDK is demonstrated by engineering a biosensing system that is capable of measuring specific DNA-protein interactions. We envision that the approach described herein will find useful applications in biosensing, imaging, and clinical diagnosis., (© 2024 Wiley-VCH GmbH.)

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.)

Developing highly sensitive and specific on-site tests is imperative to strengthen preparedness against future emerging infectious diseases. Here, we describe the construction of a Cas12a-mediated DNAzyme actuator capable of converting the recognition of a specific DNA sequence into an amplified colorimetric signal. To address viral RNA extraction challenges for on-site applications, we developed a rapid and efficient method capable of lysing the viral particles, preserving the released viral RNA, and concentrating the viral RNA. Integration of the DNAzyme actuator with the viral RNA extraction method and loop-mediated isothermal amplification enables a streamlined colorimetric assay for highly sensitive colorimetric detection of respiratory RNA viruses in gargle and saliva. This assay can detect as few as 83 viral particles/100 μL in gargle and 166 viral particles/100 μL in saliva. The entire assay, from sample processing to visual detection, was completed within 1 h at a single controlled temperature. We validated the assay by detecting SARS-CoV-2 in 207 gargle and saliva samples, achieving a clinical sensitivity of 96.3 % and specificity of 100%. The assay is adaptable for detecting specific nucleic acid sequences in other pathogens and is suitable for resource-limited settings., 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.)

Hemin/G-quadruplex (hG4) complexes are frequently used as artificial peroxidase-like enzymatic systems (termed G4 DNAzymes) in many biosensing applications, in spite of a rather low efficiency, notably in terms of detection limits. To tackle this issue, we report herein a strategy in which hemin is chemically modified with the amino acids found in the active site of parent horseradish peroxidase (HRP), with the aim of recreating an environment conducive to high catalytic activity. When hemin is conjugated with a single arginine, it associates with G4 to create an arginine-hemin/G4 (R-hG4) DNAzyme that exhibits improved catalytic performances, characterized by kinetic analysis and DFT calculations. The practical relevance of this system was demonstrated with the implementation of biosensing assays enabling the chemiluminescent detection of G4-containing DNA and colorimetry detection of the flap endonuclease 1 (FEN1) enzyme with a high efficiency and sensitivity. Our results thus provide a guide for future enzyme engineering campaigns to create ever more efficient peroxidase-mimicking DNA-based systems.

A label-free fluorescent sensing strategy for the rapid and highly sensitive detection of Pb 2+ was developed by integrating Pb 2+ DNAzyme-specific cleavage activity and a tetrahedral DNA nanostructure (TDN)-enhanced hyperbranched hybridization chain reaction (hHCR). This strategy provides accelerated reaction rates because of the highly effective collision probability and enriched local concentrations from the spatial confinement of the TDN, thus showing a higher detection sensitivity and a more rapid detection process. Moreover, a hairpin probe based on a G-triplex instead of a G-quadruplex or chemical modification makes hybridization chain reaction more controlled and flexible, greatly improving signal amplification capacities and eliminating labeled DNA probes. The enhanced reaction rates and improved signal amplification efficiency endowed the biosensors with high sensitivity and a rapid response. The label-free detection of Pb 2+ based on G-triplex combined with thioflavin T can be achieved with a detection limit as low as 1.8 pM in 25 min. The proposed Pb 2+ -sensing platform was also demonstrated to be applicable for Pb 2+ detection in tap water, river water, shrimp, rice, and soil samples, thus showing great potential for food safety and environmental 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.)

Developing sensors with high selectivity and sensitivity is of great significance for pesticide analysis in environmental assessment. Herein, a versatile three-way sensor array was designed for the detection of the pesticide atrazine, based on the integration of catalytic hairpin assembly (CHA) amplification and three-mode signal transducers. With atrazine, CHA was triggered to generate abundant G-quadruplex. The produced G-quadruplex hybrid could assemble with thioflavin T (TFT) or hemin to mimic enzyme and induce the fluorescence enhancement by TFT, or the colorimetric increase by the oxidized chromogenic substrate and the naked-eye color change by inhibiting the L-cysteine-mediated aggregation of gold nanoparticles. A distinctive three-mode array was successfully constructed with convenience, on-site accessibility and high sensitivity for enzyme-free practical analysis of atrazine. It is also effective and reliable for analyzing real samples including paddy water, paddy soil and polished rice. The detection limits for atrazine were as low as 7.4 pg/mL by colorimetric observation and 0.25 pg/mL by fluorescent detection. Furthermore, the array was exploited to monitor the residue, distribution and bioaccumulation of atrazine in maize and rice for food security and environmental assessment. Hence, this work presented a versatile example for sensitive and on-site all-in-one pesticide analysis arrays with multiple signal report modes., 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 substantial development in nanoscale materials possessing catalytic activities comparable with natural enzymes has been accomplished. Their advantages were owing to the excellent sturdiness in an extreme environment, possibilities of their large-scale production resulting in higher profitability, and easy manipulation for modification. Despite these advantages, the main challenge for artificial enzyme mimetics is the lack of substrate selectivity where natural enzymes flourish. This review addresses this vital problem by introducing substrate selectivity strategies to three classes of artificial enzymes: molecularly imprinted polymers, nanozymes (NZs), and DNAzymes. These rationally designed strategies enhance the substrate selectivity and are discussed and exemplified throughout the review. Various functional mechanisms associated with applying enzyme mimetics in biosensing and bioassays are also given. Eventually, future directives toward enhancing the substrate selectivity of biomimetics and related challenges are discussed and evaluated based on their efficiency and convenience in biosensing and bioassays., 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.)

Metallothionein (MT) has shown to be an important biomarker for environmental monitoring and various diseases, due to its significant binding ability to heavy metal ions. On the basis of such a characteristic and the Hg 2+ -stabilized DNA duplex (Hg 2+ -dsDNA) probe, as well as a new autocatalytic hairpin assembly (aCHA)/DNAzyme cascaded signal enhancement strategy, the construction of a highly sensitive and label-free electrochemical MT biosensor is described. Target MT molecules bind Hg 2+ in Hg 2+ -dsDNA to disrupt the duplex structure and to release ssDNA sequences, which trigger subsequent aCHA for efficient production of mimic aCHA triggering strands and many bivalent DNAzymes. The signal hairpins on the electrode are then cyclically cleaved by DNAzyme amplification cascade to liberate plenty G-quadruplex sequences, which bind hemin and yield largely enhanced currents for sensitive assay of MT with a detection limit of 0.217 nM in a label-free approach. Such sensor also shows selective discrimination capability to MT against other interfering proteins and assay of MT in normal serums with dilution has also been verified, indicating its potential for highly sensitive detection of different heavy metal ion binding molecules for various application scenarios., 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.)

Modern cryptography based on computational complexity theory is mainly constructed with silicon-based circuits. As DNA nanotechnology penetrates the molecular domain, utilizing molecular cryptography for data access protection in the biomolecular domain becomes a unique approach to information security. However, building security devices and strategies with robust security and compatibility is still challenging. Here, this study reports a time-controlled molecular authentication strategy using DNAzyme and DNA strand displacement as the basic framework. A time limit exists for authorization and access, and this spontaneous shutdown design further protects secure access. Multiple hierarchical authentications, temporal Boolean logic authentication, and enzyme authentication strategies are constructed based on DNA networks'good compatibility and programmability. This study gives proof of concept for the detection and protection of bioinformation about single nucleotide variants and miRNA, highlighting their potential in biosensing and security protection., (© 2024 Wiley‐VCH GmbH.)

The photocurrent-polarity-switching signal transduction strategy has provided an effective approach to improve the performance of photoelectrochemical (PEC) biosensors. Herein, we prepared a MXene/CdS nanocomposite based PEC biosensor and combined it with the polymerization and isomerization cyclic amplification (PICA)-assisted capture of a Fe 3+ and Cu 2+ co-coordinating 5,10,15,20-tetrakis(4-aminophenyl)porphyrin (TFC) based signal probe to develop a novel photocurrent-polarity-switching PEC biosensing method for the detection of kanamycin antibiotic. The PICA arose from the aptamer recognition-triggered release of an Mg 2+ -dependent DNAzyme (MNAzyme) strand to cleave its substrate. This reaction also triggered the assembly of a DNA nanowire decorated with abundant MNAzymes through hybridization chain reaction. Due to the DNA nanowire-enhanced PICA to extremely increase the probe capture at the biosensor and the good energy level matching between TFC and MXene/CdS, a strong polarity-switching based cathodic photocurrent response was produced to construct the signal transduction strategy. This not only excluded the possible false positive or negative signal interferences but also endowed a seven-order magnitude wide linear range and a very low detection limit of 0.03 fg mL -1 to the method. In addition, it also has convenient assay manipulation, high selectivity, outstanding repeatability, and excellent reliability, which determine its high potential promising for practical 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.)

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.)