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Antibiotic-induced inflammation involves the release of myeloperoxidase (MPO), an enzyme whose expression in tissues is associated with the inflammatory pathway. However, existing methods for detecting MPO in cells are limited. In this study, a DNAzyme nanorobot was developed using a scaffold of gold nanoparticles (AuNPs) decorated with functional DNAzyme strands and their fluorophore-labeled substrate strands. The DNAzyme remains inactive due to a self-assembled hairpin structure, with a phosphorothioate (PT) modification inserted into the stem domain. When MPO is present, it triggers a halogenation process that generates hypochlorous acid (HClO). HClO specifically catalyzes the cleavage of the PT-site, releasing free DNAzyme strands to cleave their substrates and generating an increasing fluorescent signal. The detection limit for MPO and its primary product, HClO, were determined to be 0.038 μg/mL and 0.013 μM, respectively. The DNAzyme nanorobot can be readily introduced into cells and function autonomously to differentiate increased MPO/HClO levels caused by antibiotics. This approach was applied to image RAW264.7 cells exposed to four prevalent antibiotics found in the environment (phorbol 12-myristate 13-acetate, erythromycin, penicillin, and tetracycline) as well as antibiotic production wastewater. This nanorobot offers novel strategies for monitoring inflammation to evaluate the health impacts of antibiotic exposure.

The development of an advanced analytical platform with regard to SARS-CoV-2 is crucial for public health. Herein, we present a machine learning platform based on paper-assisted ratiometric fluorescent sensors for highly sensitive detection of the SARS-CoV-2 RdRp gene. The assay involves target-induced rolling circle amplification to generate magnetic DNAzyme, which is then detectable using the paper-assisted ratiometric fluorescent sensor. This sensor detects the SARS-CoV-2 RdRp gene with a visible-fluorescence color response. Moreover, leveraging different fluorescence responses, the ResNet algorithm of machine learning assists in accurately identifying fluorescence images and differentiating the concentration of the SARS-CoV-2 RdRp gene with over 99% recognition accuracy. The machine learning platform exhibits exceptional sensitivity and color responsiveness, achieving a limit of detection of 30 fM for the SARS-CoV-2 RdRp gene. The integration of intelligent artificial vision with the paper-assisted ratiometric fluorescent sensor presents a novel approach for the on-site detection of COVID-19 and holds potential for broader use in disease diagnostics 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 B.V. All rights reserved.)

Background: Theranostic nanoplatforms with integrated diagnostic imaging and multiple therapeutic functions play a vital role in precise diagnosis and efficient treatment for breast cancer, but unfortunately, these nanoplatforms are usually stuck in single-site imaging and single mode of treatment, causing unsatisfactory diagnostic and therapeutic efficiency. Herein, a dual biomarkers-activatable facile hollow mesoporous MnO 2 (H-MnO 2 )-based theranostic nanoplatform, DNAzyme@H-MnO 2 -MUC1 aptamer (DHMM), was constructed for the simultaneous multi-site diagnosis and multiple treatment of breast cancer., Results: The DHMM acted as an integrated diagnostic and therapeutic nanoplatform that realizes multi-site fluorescence imaging-guided high-efficient photothermal/chemodynamic/gene synergistic therapy (PTT/CDT/GT) for breast cancer. The H-MnO 2 exhibits high loading capacity for Cy5-MUC1 aptamer (3.05 pmoL μg -1 ) and FAM-DNAzyme (3.37 pmoL μg -1 ), and excellent quenching for the probes. In the presence of MUC1 on the cell membrane and GSH in the cytoplasm, Cy5-MUC1 aptamer and FAM-DNAzyme was activated triggering dual-channel fluorescence imaging at different sites. Moreover, the self-supplied Mn 2+ was further supplied as DNAzyme cofactors to catalytic cleavage intracellular EGR-1 mRNA for high-efficient GT and stimulated the Fenton-like reaction for CDT. The H-MnO 2 also showcases a favorable photothermal performance with a photothermal conversion efficiency of 44.16%, which ultimately contributes to multi-site fluorescence imaging-guided synergistic treatment with an apoptosis rate of 71.82%., Significance: This dual biomarker-activatable multiple therapeutic nanoplatform was realized multi-site fluorescence imaging-guided PTT/CDT/GT combination therapy for breast cancer with higher specificity and efficiency, which provides a promising theranostic nanoplatform for the precision and efficiency of breast cancer treatment., 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.)

Benefiting from specific target recognition and trans-cleavage capabilities, the CRISPR/Cas12a system has great application prospects in the design of highly sensitive and rapid fluorescence biosensors. The CRISPR/Cas12a-based fluorophore-quencher molecular beacons exhibit single-color emission and are easily exposed to interference from environmental factors. Herein, we design a CRISPR/Cas12a-derived ratiometric fluorescence sensor for Pb 2+ detection based on embedded carbon dots@zeolitic imidazolate framework-8 (CDs@ZIF-8) composites and DNAzyme. The functions of ZIF-8 about encapsulating red emissive CDs in the inner cavity and adsorbing DNA on the outer surface are integrated to establish dual fluorescence signals, thereby reducing the possibility of interference and improving sensing accuracy. The presence of Pb 2+ is converted into the change of activator by the GR5 DNAzyme to activate the CRISPR/Cas12a system, which provides signal amplification through multiple turnovers of side branch cutting, achieving highly sensitive detection of Pb 2+ with a low detection limit of 18 pM. This method has the advantages of simplicity, universality, and excellent quantitative ability, and has broad prospects in sensing 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.)

An electrochemiluminescence (ECL) sensing platform for ultrasensitive and highly selective detection of kanamycin (KANA) was developed based on the prepared Ru(bpy) 3 2+ -functionalized MOF (Ru@MOF) composites by hydrothermal synthesis and Ag + -dependent DNAzyme. In this sensor, the stem-loop DNA (HP) with the ferrocene (Fc) was used as substrate chain to quench the ECL emission generated by the Ru@MOF. Using the specific recognition effect between KANA and the KANA aptamer (Apt) and the DNAzyme dependence on Ag + , the KANA aptamer as the pendant strand of the DNAzyme was assembled on Ru@MOF/GCE with the aptamer. When both Ag + and KANA were present simultaneously, KANA specifically was binded to KANA aptamer as a pendant chain. Subsequently, Ag + -dependent DNAzyme walker continuously cleaved the HP chain and released the modified end of Fc to restore the ECL signal of Ru@MOF composites, thus achieving selective and ultrasensitive detection of KANA. The constructed KANA biosensor exhibits a wide detection range (30 pM to 300 μM) accompanied by a low detection limit (13.7 pM). The KANA in seawater and milk samples are determined to evalute the practical application results of the sensor. This ECL detection strategy could be used for detecting other similar analytes and has broad potential application in biological 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. Published by Elsevier Inc.)

Inappropriate use of veterinary drugs can result in the presence of antibiotic residues in animal-derived foods, which is a threat to human health. A simple yet efficient antibiotic-sensing method is highly desirable. Programmable DNA amplification circuits have supplemented robust toolkits for food contaminants monitoring. However, they currently face limitations in terms of their intricate design and low signal gain. Herein, we have engineered a robust reciprocal catalytic DNA (RCD) circuit for highly efficient bioanalysis. The trigger initiates the cascade hybridization reaction (CHR) to yield plenty of repeated initiators for activating the rolling circle amplification (RCA) circuit. Then the RCA-generated numerous reconstituted triggers can reversely stimulate the CHR circuit. This results in a self-sufficient supply of numerous initiators and triggers for the successive cross-invasion of CHR and RCA amplifiers, thus leading to exponential signal amplification for the highly efficient detection of analytes. With its flexible programmability and modular features, the RCD amplifier can serve as a universal toolbox for the high-performance and accurate sensing of kanamycin in buffer and food samples including milk, honey, and fish, highlighting its enormous promise for low-abundance contaminant analysis in foodstuffs.

Herein, novel europium metal-organic gels (Eu-MOGs) with excellent cathode electrochemiluminescence (ECL) emission are first used to construct biosensors for the ultrasensitive detection of miRNA-222. Impressively, N and O elements of organic ligand 2,2':6,2″-terpyridine 4,4',4″-tricarboxylic acid (H3-tctpy) can perfectly coordinate with Eu 3+ to form Eu-MOGs, which not only reduce nonradiative transition caused by the intramolecular free rotation of phenyl rings in other MOGs to enhance the ECL signal with extraordinary ECL efficiency as high as 37.2% (vs the [Ru(bpy) 3 ] 2+ /S 2 O 8 2- ECL system) but also reinforce ligand-to-metal charge transfer (LMCT) by the strong affinity between Eu 3+ and N and O elements to greatly improve the stability of ECL signals. Besides, an improved nucleic acid cascade amplification reaction is developed to greatly raise the conversion efficiency from target miRNA-222 to a DNAzyme-mediated dual-drive DNA walker as output DNA, which can simultaneously shear the specific recognition sites from two directions. In that way, the proposed biosensor can further enhance the detection sensitivity of miRNA-222 with a linear range of 10 aM-1 nM and a detection limit (LOD) of 8.5 aM, which can also achieve an accurate response in cancer cell lysates of MHCC-97L and HeLa. Additionally, the biosensor can be self-regenerated by the folding/unfolding of related triplets with pH changes to simplify experimental operations and reduce the cost. Hence, this work proposed novel MOGs with stable and intense ECL signals for the construction of a renewable ECL biosensor, supplying a reliable detection method in biomarker analysis and disease diagnosis.

DNA assemblies are commonly used in biosensing, particularly for the detection and imaging of microRNAs (miRNAs), which are biomarkers associated with tumor progression. However, the difficulty lies in the exploration of high-sensitivity analytical techniques for miRNA due to its limited presence in living cells. In this study, we introduced a DNA nanosphere (DS) enhanced catalytic hairpin assembly (CHA) system for the detection and imaging of intracellular miR-21. The single-stranded DNA with four palindromic portions and extending sequences at the terminal was annealed for assembling DS, which avoided the complex sequence design and high cost of long DNA strands. Benefiting from the multiple modification sites of DS, functional hairpins H1 (modified with Cy3 and BHQ2) and H2 were grafted onto the surface of DS for assembling DS-H1-H2 using a hybridization reaction. The DS-H1-H2 system utilized spatial confinement and the CHA reaction to amplify fluorescence signals of Cy3. This enabled highly sensitive and rapid detection of miR-21 in the range from 0.05 to 3.5 nM. The system achieved a limit of determination (LOD) of 2.0 pM, which was 56 times lower than that of the control CHA circuit with freedom hairpins. Additionally, the sensitivity was improved by 8 times. Moreover, DS-H1-H2 also showed an excellent imaging capability for endogenous miR-21 in tumor cells. This was due to enhanced cell internalization efficiency, accelerated reaction kinetics, and improved biostability. The imaging strategy was shown to effectively monitor the dynamic content of miR-21 in live cancer cells and differentiate various cells. In general, the simple nanostructure DS not only enhanced the detection and imaging capability of the conventional probe but also could be easily integrated with the reported DNA-free probe, indicating a wide range of potential applications.

Due to the threat of lead pollution to health, environmental and food safety, developing simple and fast detection methods is highly required. Whereas, traditional single-mode probe suffers from limited application scenario. In this study, a colorimetric and fluorometric dual-mode probe for Pb 2+ determination was constructed by using bifunctional G-quadruplex-hemin complex. In this dual-mode probe, enzyme strand and substrate strand of 8-17 DNAzyme are labeled with G-quadruplex-hemin complex and fluorophore, respectively. In the absence of Pb 2+ , the self-assembly of enzyme strand and substrate strand inhibits intrinsic mimic peroxidase of G-quadruplex-hemin complex by base-pairing, which also quench the fluorescence via in proximity effect. When the DNAzyme is activated by Pb 2+ , the quenched fluorescence is restored as well as the inherent peroxidase mimetic activity, leading to dual signal output. Under optimal conditions, this dual-mode probe exhibit a good linear relationship between logarithm of Pb 2+ concentration and signal difference within the range from 1.5 nM to 20 nM and 0.5 nM to 10 nM for colorimetric and fluorescence mode, respectively. The detection limits for the corresponding mode were estimated to be 1.29 nM and 0.16 nM, respectively. This dual-mode probe also successfully applied for the spiked river water assay with satisfactory recovery in the range of 93.2 %-115.3 %. This work paves a new way for DNAzyme based dual-mode probe construction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Metastasis is the leading cause of death in patients with breast cancer. Detecting high-risk breast cancer, including micrometastasis, at an early stage is vital for customizing the right and efficient therapies. In this study, we propose an enzyme-free isothermal cascade amplification-based DNA logic circuit in situ biomineralization nanosensor, HDNAzyme@ZIF-8, for simultaneous imaging of multidimensional biomarkers in live cells. Taking miR-21 and Ki-67 mRNA as the dual detection targets achieved sensitive logic operations and molecular recognition through the cascade hybridization chain reaction and DNAzyme. The HDNAzyme@ZIF-8 nanosensor has the ability to accurately differentiate breast cancer cells and their subtypes by comparing their relative fluorescence intensities. Of note, our nanosensor can also achieve visualization within breast cancer organoids, faithfully recapitulating the functional characteristics of parental tumor. Overall, the combination of these techniques offers a universal strategy for detecting cancers with high sensitivity and holds vast potential in clinical cancer diagnosis.

Retinoblastoma (RB) is an aggressive tumor of the infant retina. However, the ineffective targeting of its theranostic agents results in poor imaging and therapeutic efficacy, which makes it difficult to identify and treat RB at an early stage. In order to improve the imaging and therapeutic efficacy, we constructed an RB-targeted artificial vesicle composite nanoparticle. In this study, the MnO 2 nanosponge (hMNs) was used as the core to absorb two fluorophore-modified DNAzymes to form the Dual/hMNs nanoparticle; after loaded with the artificial vesicle derived from human red blood cells, the RB-targeted DNA aptamers were modified on the surface, thus forming the Apt-EG@Dual/hMNs complex nanoparticle. The DNA aptamer endows this nanoparticle to target the nucleolin-overexpressed RB cell membrane specifically and enters cells via endocytosis. The nanoparticle could release fluorophore-modified DNAzymes and supplies Mn 2+ as a DNAzyme cofactor and a magnetic resonance imaging (MRI) agent. Subsequently, the DNAzymes can target two different mRNAs, thereby realizing fluorescence/MR bimodal imaging and dual-gene therapy. This study is expected to provide a reliable and valuable basis for ocular tumor theranostics.

Transmembrane channels play a vital role in regulating the permeation process, and have inspired recent development of biomimetic channels. Herein, we report a class of artificial biomimetic nanochannels based on DNAzyme-functionalized glass nanopipettes to realize delicate control of channel permeability, whereby the surface wettability and charge can be tuned by metal ions and DNAzyme-substrates, allowing reversible conversion between different permeability states. We demonstrate that the nanochannels can be reversibly switched between four different permeability states showing distinct permeability to various functional molecules. By embedding the artificial nanochannels into the plasma membrane of single living cells, we achieve selective transport of dye molecules across the cell membrane. Finally, we report on the advanced functions including gene silencing of miR-21 in single cancer cells and selective transport of Ca 2+ into single PC-12 cells. In this work, we provide a versatile tool for the design of rectifying artificial nanochannels with on-demand functions., (© 2024. The Author(s).)

MicroRNA detection is crucial for early infectious disease diagnosis and rapid cancer screening. However, conventional techniques like reverse transcription-quantitative polymerase chain reaction, requiring specialized training and intricate procedures, are less suitable for point-of-care analyses. To address this, we've developed a straightforward amplifier based on an exonuclease III (exo III)-propelled DNAzyme walker for sensitive and selective microRNA detection. This amplifier employs a specially designed hairpin probe with two exposed segments for strand recognition. Once the target microRNA is identified by the hairpin's extended single-strand DNA, exo III initiates its digestion, allowing microRNA regeneration and subsequent hairpin probe digestion cycles. This cyclical process produces a significant amount of DNAzyme, leading to a marked reduction in electrochemical signals. The biosensor exhibits a detection range from 10 fM to 100 pM and achieves a detection limit of 5 fM (3σ criterion). Importantly, by integrating an "And logic gate," our system gains the capacity for simultaneous diagnosis of multiple microRNAs, enhancing its applicability in RNA-based disease diagnostics., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

As a highly toxic and refractory heavy metal contaminant, Pb 2+ seriously endangers human health. The problems of low sensitivity and high cost of signal labeling widely exist in common electrochemical biosensors. Herein, a Pb 2+ electrochemical biosensor was constructed using a DNAzyme-driven bipedal DNA Walker and catalytic hairpin assembly as the multistage signal amplification strategy. Compared with Zr-MOF, AuNPs@Zr-MOF has a larger porosity and specific surface area, which can effectively load MB to amplify the current signal. Pb 2+ can trigger a dual signal amplification reaction to gradually accumulate the signal of methylene blue/gold nanoparticle @ zirconium-based metal organic frameworks (MB/AuNPs@Zr-MOF) on the electrode. The ingeniously designed sensing strategy realized the analysis of Pb 2+ with a wide linear range from 0.05 to 1000 nmol/L and a lower limit of detection (LOD) of 4.65 pmol/L. In addition, the sensor has strong anti-interference ability and can accurately detect Pb 2+ in various food 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 © 2023 Elsevier Ltd. All rights reserved.)

A dual-mode sensing platform for Pb 2+ was constructed based on the dual optical channels of Au NPs system with the amplification of DNAzyme, and it was successfully applied for Pb 2+ determination in preserved egg with satisfactory results. The presence of Pb 2+ activated the DNAzyme and induced the dispersion change of Au NPs in high salt concentration. The sequent absorption change of Au NPs was translated to the fluorescence change of carbon dots through FRET, and the scattering change was transferred to grey value of images involving the Tyndall effect. Thus, a sensing platform based on fluorescence and colorimetric dual-technique was achieved for Pb 2+ detection, under the optimized conditions. With the assistance of DNAzyme, the linear range of fluorometric and colorimetric method were 2.0 × 10 -14  ∼ 8.0 × 10 -10 mol/L and 2.4 × 10 -13  ∼ 9.5 × 10 -9 mol/L, respectively. The dual-mode sensing platform demonstrated its promising application in the environmental monitoring and food safety field., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Ratiometric imaging of tumor-related mRNA is significant, yet spatiotemporally resolved regulation on the ratiometric signals to avoid non-specific activation in the living cells remains challenging. Herein, orthogonally sequential activation of concatenated DNAzyme circuits is, first, developed for Spatio Temporally regulated Amplified and Ratiometric (STAR) imaging of TK1 mRNA inside living cells with enhanced reliability and accuracy. By virtue of the synthesized CuO/MnO 2 nanosheets, orthogonally regulated self-powered DNAzyme circuits are operated precisely in living cells, sequentially activating two-layered DNAzyme cleavage reactions to achieve the two ratiometric signal readouts successively for reliable monitoring of low-abundance mRNA in living cells. It is found that the ratiometric signals can only be derived from mRNA over-expressed tumor cells, also irrespective of probes' delivery concentration. The presented approach could provide new insight into orthogonally regulated ratiometric systems for reliable imaging of specific biomarkers in living cells, benefiting disease precision diagnostics., (© 2024 Wiley‐VCH GmbH.)

Escherichia coli O157: H7 (E. coli O157: H7) is one of the most common foodborne pathogens and is widespread in food and the environment. Thus, it is significant for rapidly detecting E. coli O157: H7. In this study, a colorimetric aptasensor based on aptamer-functionalized magnetic beads, exonuclease III (Exo III), and G-triplex/hemin was proposed for the detection of E. coli O157: H7. The functional hairpin HP was designed in the system, which includes two parts of a stem containing the G-triplex sequence and a tail complementary to cDNA. E. coli O157: H7 competed to bind the aptamer (Apt) in the Apt-cDNA complex to obtain cDNA. The cDNA then bound to the tail of HP to trigger Exo III digestion and release the single-stranded DNA containing the G-triplex sequence. G-triplex/hemin DNAzyme could catalyze TMB to produce visible color changes and detectable absorbance signals in the presence of H 2 O 2 . Based on the optimal conditions, E. coli O157: H7 could be detected down to 1.3 × 10 3  CFU/mL, with a wide linear range from 1.3 × 10 3 to 1.3 × 10 7  CFU/mL. This method had a distinguished ability to non-target bacteria, which showed good specificity. In addition, the system was successfully applied to detect E. coli O157: H7 in milk samples., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yujun Jiang reports financial support was provided by the Joint Funds of the National Natural Science Foundation of China., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) (CRISPR/Cas) system enables sensitive and specific detection of biomolecules, thanks to its programmability, high fidelity, and powerful signal amplification capabilities. Herein, a universal smartphone-assisted label-free G-quadruplex (G4) DNAzyme-based chemiluminescence CRISPR/Cas12a biosensing platform (G4CLCas) is firstly described that achieves on-site, ultrasensitive visual detection of nucleic acid and non-nucleic acid targets. The G4CLCas-based sensing platform relies on Cas12a trans-cleavage activation that triggers the cleavage of the G4 DNAzyme, resulting in chemiluminescence signals off/on compared to that of the control. Chemiluminescence signals are captured as images that are quantitatively analyzed and visualized using a smartphone-assisted imaging cartridge. Under optimal conditions, G4CLCas achieves a low limit of detection (LOD) of 8.6 aM (∼5.2 copies/μL) for monkeypox virus (MPXV) DNA within the linear concentration range of 10-300 aM and can accurately quantify viral DNA in spiked samples. G4CLCas can also detect non-nucleic acid targets, whereby it achieves a low LOD value of 84.3 nM for adenosine triphosphate (ATP) within the linear concentration range of 2-2000 μM. Here, a label-free, portable, on-site CRISPR/Cas12a chemiluminescence biosensing platform based on the G4 DNAzyme substrates is proposed with potential applications in clinical detection and bioanalytical chemistry research., Competing Interests: Declaration of competing interest All authors have given approval of this manuscript. The authors declared that they have no financial or other contractual agreements that might cause conflicts of interest., (Copyright © 2023. Published by Elsevier B.V.)

Rapid and accurate detection of the hepatitis C virus (HCV) is essential for early diagnosis and prevention of virus transmission. This study presents a novel approach that combines the three-dimensional (3D)-DNA walking nanomachine with catalytic hairpin assembly (CHA) and copper nanoclusters (CuNCs). By integrating CHA with the 3D DNA walking nanomachine, efficient target amplification on 3D surfaces was achieved, leading to improved reaction speed and detection performance. Terminal deoxynucleotidyl transferase (TdT) was utilized to generate T-rich DNA sequences. These sequences served as templates for the formation of CuNCs, which functioned as the readout signal. The optimized 3D-DNA walking nanomachine exhibited excellent sensitivity in detecting HCV, with a detection limit of 42.4 pM and a linear range of 100 pM to 2 nM. The biosensor demonstrated excellent selectivity and reproducibility, with a recovery rate ranging from 94% to 108% for the detection of real samples. This design holds great potential for sensitive, label-free, and reliable detection of HCV in clinical settings. Furthermore, the versatility of this approach allows for the customization of target sequences, thereby facilitating the detection of various nucleic acid targets. Therefore, this method has the potential to advance personalized medicine, disease management, and genetic analysis in the field of molecular 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 © 2023 Elsevier B.V. All rights reserved.)

Circulating tumor DNA (ctDNA) is a highly promising biomarker for the early diagnosis and treatment of gastric cancer (GC). However, there is still a lack of effective and practical ctDNA detection methods. In this work, a simple and economical capillary non-gel sieving electrophoresis-LED induced fluorescence detection (NGCE-LEDIF) platform coupled with catalytic hairpin assembly (CHA) as the signal amplification strategy is proposed for quantitative detection of PIK3CA E542K and TP53 (two types of ctDNA associated with GC). We have reasonably designed two pairs of programmable oligonucleotide hairpin probes for PIK3CA E542K and TP53. Using a one-pot reaction, the presence of ctDNA triggers the cyclic amplification of CHA, forming numerous thermodynamically stable H1/H2 double-strands. The H1/H2 double-stranded DNA catalyzed by PIK3CA E542K and TP53 can be easily separated by NGCE due to their different lengths, enabling simultaneous detection of both ctDNAs. Under optimal experimental conditions, the detection limits of this strategy for detecting GC-related biomarkers PIK3CA E542K and TP53 are 20.35 pM and 19.61 pM, respectively, and can achieve 730-fold signal amplification. This strategy has a good recovery in the serum matrix. The results of this study show that this strategy has significant advantages such as high selectivity, a simple process, no special instruments and equipment, no need for fluorescence modification of hairpin probes in advance, high automation, low cost, and minimal sample consumption. This provides a powerful method for the detection of trace cancer biomarkers in the serum matrix with good application prospects.

Herein, pyrenecarboxaldehyde@graphene oxide (Pyc@GO) sheets with highly efficient electrochemiluminescence (ECL) as emitters were prepared by a noncovalent combination to develop a neoteric ECL biosensing platform for the ultrasensitive assessment of human apurinic/apyrimidinic endonuclease1 (APE1) activity. Impressively, the pyrenecarboxaldehyde (Pyc) molecules were able to form stable polar functional groups on the surface of GO sheets through the noncovalent π-π stacking mechanism to prevent intermolecular restacking and simultaneously generate Pyc@GO sheets. Compared with the tightly packed PAH nanocrystals, the Pyc@GO sheets significantly reduced internal filtering effects and diminished nonactivated emitters to enhance ECL intensity and achieve strong ECL emission. Furthermore, the APE1-activated initiators could trigger the recyclable cascade amplified system based on the synergistic cross-activation between catalytic hairpin assembly (CHA) and DNAzyme, which improved the signal amplification and transduction ability. Consequently, the developed ECL platform for the detection of APE1 activity displayed exceptional sensitivity with a low detection limit of 4.6 × 10 -9 U·mL -1 ranging from 10 -8 to 10 -2 U·mL -1 . Therefore, the proposed strategy holds great promise for the future development of sensitive and reliable biosensing platforms for the detection of various biomarkers.

An advanced nanoplatform was developed by integrating catalytic hairpin assembly (CHA) with glutathione-responsive nanocarriers, enabling superior imaging of dual cancer-related miRNAs. Two distinct CHA circuits for the sensing of miRNA-21 and miRNA-155 were functionalized on biodegraded MnO 2 . In the presence of GSH and the corresponding miRNAs, the degraded MnO 2 released the DNA cargos, activating the CHA circuits and recovering the fluorescence. This approach offers a reliable sensing performance with highly selective cell-identification capacity, positioning it as a pivotal tool for imaging multiple biomarkers in living cells.

Background: Lead (Pb) is one of the most toxic heavy-metal pollutants. Additionally, lead ions (Pb 2+ ) can accumulate in the human body through the food chain, causing irreversible damage through organ damage and system disorders. In the past few years, the detection of Pb 2+ has mainly relied on instrumental methods such as atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). Nonetheless, these techniques are complicated in terms of equipment and procedures, along with being time-intensive and expensive in terms of detection. These drawbacks have limited their wide application. Hence, there is a pressing need to develop detection techniques for Pb 2+ that are not only cost-efficient but also highly sensitive and specific., Results: A novel "on-off-on" electrochemiluminescence (ECL) sensor for detecting Pb 2+ was developed based on the resonance energy transfer (RET) effect between AuNPs and boron nitride quantum dots (BN QDs) and the recognition of Pb 2+ by DNAzyme along with the cleavage reaction of the substrate chain. Poly(6-carboxyindole)/stannic sulfide (P6ICA/SnS 2 ) nanocomposite was employed as a co-reaction accelerator to consequently facilitate the production of intermediate SO 4 •- . This effective enhancement of the reaction led to an improved ECL intensity of BN QDs and enabled the sensor platform to exhibit a higher original ECL response. Benefiting from the combination of the DNAzyme signal amplification strategy with the "on-off-on" design, the ECL sensor showed satisfactory selectivity, good stability, and high sensitivity. This ECL sensor exhibited a linear detection range (LDR) of 10 -12 -10 -5  M and a limit of detection (LOD) of 2.6 × 10 -13  M., Significance: In the present work, an "on-off-on" ECL sensor is constructed based on RET effect for ultrasensitive detection of Pb 2+ . P6ICA/SnS 2 was investigated as the co-reaction accelerator in this sensor. Moreover, this ECL sensor exhibited excellent analytical capability for detecting Pb 2+ in actual water samples, providing a method for detecting other heavy metal ions as well., 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.)

Freshwater lead pollution has posed severe threat to the environment and human health, underscoring the urgent necessity for accurate and user-friendly detection methods. Herein, we introduce a novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas) sensor for highly sensitive Pb 2+ detection. To accomplish this, we designed a dual-functional deoxyribozyme (df-DNAzyme) probe that functions as an activator for the CRISPR-Cas12a system while also recognizing Pb 2+ . The df-DNAzyme probe was subsequently combined with gold nanoparticles (AuNPs) to fabricate a DNAzyme/AuNP nanoprobe, facilitating the activation of CRISPR-Cas12a in a one-to-multiple manner. Upon exposure to Pb 2+ , the df-DNAzyme is cleaved, causing disintegration of the DNAzyme/AuNP nanoprobe from magnetic beads. The degraded DNAzyme/AuNP containing multiple double-stranded DNA activators efficiently triggers CRISPR-Cas12a activity, initiating cleavage of fluorescence-quenched reporter DNA and generating amplified signals accordingly. The amplified fluorescence signal is accurately quantified using a quantitative polymerase chain reaction (qPCR) instrument capable of measuring 96 or 384 samples simultaneously at the microliter scale. This technique demonstrates ultra-sensitive detection capability for Pb 2+ at concentrations as low as 1 pg/L within a range from 1 pg/L to 10 μg/L, surpassing limits set by World Health Organization (WHO) and United States Environmental Protection Agency (US EPA) guidelines. This study offers an ultrasensitive and high-throughput method for the detection of Pb 2+ in freshwater, thereby advancing a novel approach towards the development of precise and convenient techniques for detecting harmful contaminants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided by a double-stranded DNA fragment, a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application in nucleic acid detection assays.

Despite the progress that has been made in diverse DNA-based nanodevices to in situ monitor the activity of the DNA repair enzymes in living cells, the significance of improving both the sensitivity and specificity has remained largely neglected and understudied. Herein, we propose a regulatable DNA nanodevice to specifically monitor the activity of DNA repair enzymes for early evaluation of cancer mediated by genomic instability. Concretely, an AND logic gate-regulated DNAzyme nanoflower was rationally designed by the self-assembly of the DNA duplex modified with both apurinic/apyrimidinic (AP) site and methyl lesion site. The DNAzyme nanoflower could be reconfigured under the repair of AP sites and O 6 -methylguanine sites by apurinic/apyrimidinic endonuclease 1 (APE1) and O 6 -methylguanine methyltransferase (MGMT) to produce a fluorescent signal, realizing the sensitive monitoring of the activity of APE1 and MGMT. Compared to the free DNAzyme duplex, the fluorescent response of the DNAzyme nanoflower increased by 60%, due to the effective enrichment of the DNA probes by the nanoflower structure. More importantly, we have demonstrated that the dual-enzyme activated strategy allows imaging of specific cancer cells in the AND logic gate manner using MCF-7 as a cancer cell model, improving the specificity of cancer cell imaging. This AND logic gate-regulated multifunctional DNAzyme nanoflower provides a simple tool for simultaneously visualizing multiple DNA repair enzymes, holding great potential in early clinical diagnosis and drug discovery.

Apolipoprotein A4 (Apo-A4) is considered as a prospective molecular biomarker for diagnosis of depression due to its neurosynaptic toxicity. We develop a proximity hybridization-induced DNAzyme-driven bipedal DNA walker strategy for Apo-A4 quantification based on rolling circle amplification (RCA) triggered by poly adenine binding to Ag nanoparticles (AgNPs). With the help of DNAzyme, the free-running bipedal DNA walker can quickly and sequentially shear a molecular beacon that acts as a primer to initiate the RCA process, producing a large number of long DNA strands containing numerous adenines. The long repetitive adenine strands then absorb large amounts of AgNPs on the electrode interface, which is then electrochemically stripped of the AgNPs. The method has a linear detection range of 0.001 ∼ 100 ng mL -1 and a detection limit of 0.46 pg mL -1 . The presented detection strategy is label-free, which allows high sensitivity and selectivity for detection of a wide range of protein targets by corresponding DNA-based affinity probes, which have potential applications in bioanalysis., 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 B.V.)

Stimulus-responsive mode is highly desirable for improving the precise monitoring and physiological efficacy of endogenous biomarkers (EB). However, its integrated application for visual detection and therapy is limited by inappropriate use of responsive triggers and poor delivery of EB signal-transducing agents, which remain challenging in simultaneous monitoring and noninvasive therapy of EB and EB-mediated pathological events. Target microRNA (miRNA) as controllable reaction triggers and DNAzyme as signal-transducing agent are proposed to develop target-stimulated multifunctional nanocabinets (MFNCs) for the visual tracking of both miRNA and miRNA-mediated anticancer events. The MFNCs, equipped with a target-discriminating sequence-incorporated DNAzyme motif, can specifically release therapeutic molecules through target-triggered conformational switches, accompanied by transduction signal output. Target detection and molecule release performance are recorded in parallel via reverse dual-signal feedback at the single-molecule level. In addition, the intrinsic thermal-replenishing of the MFNCs leads to tumor ablation without invasive exogenous aids. The system achieves visual target quantification, anticancer molecule real-time tracking, and tumor suppression in vivo and in vitro. This work proposes a new paradigm for precise visual exploration of EB or EB-mediated bio-events and provides a demonstration of efficacious all-in-one detection and therapy based on the target-triggered multifunctional nanosystem., (© 2023 Wiley-VCH GmbH.)

Emerging research suggests that mitochondrial DNA is a potential target for cancer treatment. However, achieving precise delivery of deoxyribozymes (DNAzymes) and combining photodynamic therapy (PDT) and DNAzyme-based gene silencing together for enhancing mitochondrial gene-photodynamic synergistic therapy remains challenging. Accordingly, herein, intelligent supramolecular nanomicelles are constructed by encapsulating a DNAzyme into a photodynamic O 2 economizer for mitochondrial NO gas-enhanced synergistic gene-photodynamic therapy. The designed nanomicelles demonstrate sensitive acid- and red-light sequence-activated behaviors. After entering the cancer cells and targeting the mitochondria, these micelles will disintegrate and release the DNAzyme and Mn (II) porphyrin in the tumor microenvironment. Mn (II) porphyrin acts as a DNAzyme cofactor to activate the DNAzyme for the cleavage reaction. Subsequently, the NO-carrying donor is decomposed under red light irradiation to generate NO that inhibits cellular respiration, facilitating the conversion of more O 2 into singlet oxygen ( 1 O 2 ) in the tumor cells, thereby significantly enhancing the efficacy of PDT. In vitro and in vivo experiments reveal that the proposed system can efficiently target mitochondria and exhibits considerable antitumor effects with negligible systemic toxicity. Thus, this study provides a useful conditional platform for the precise delivery of DNAzymes and a novel strategy for activatable NO gas-enhanced mitochondrial gene-photodynamic therapy., (© 2023 Wiley-VCH GmbH.)

Background: MicroRNAs play an important role in regulating cell function and gene expression. Early prevention and clinical diagnosis of diseases have high requirements for high-sensitivity detection of microRNAs. Due to the limitations of tedious operation and large sample size, miRNA with small molecular weight and low expression abundance cannot be accurately detected in traditional miRNA detection. To improve the sensitivity and accuracy of detection, we established a novel biosensor based on nucleic acid circuit of signal amplification, which converted miRNA recognition into a fluorescence signal for amplification., Results: We designed a biosensor based on an exponential amplification reaction with cascaded HCR and DNAzyme nucleic acid circuit (named E-NOF biosensor) by amplicon sub-fragments to trigger the construction of fluorescence nano-orbitals (NOF), which could be used to detect miRNA ultrasensitively. By modifying two fluorophores (Cy3 and Cy5) on the chain of constructing nano-orbitals, when the amplicon triggered the construction of nano-orbitals, fluorescence resonance energy transfer (FRET) occurred between Cy3 and Cy5, and then two fluorescence signals with different trends could be observed. Therefore, through the ratio of the two signals, we could quantitatively and quickly detect the miRNA from 1 fM to 100 nM, and the E-NOF biosensor detection limit was as low as 0.129 fM. Furthermore, the HCR nucleic acid circuit cascaded with DNAzyme could enrich the fluorophores on the nano-orbitals and significantly enhance the fluorescence signal by accelerating the reaction rate., Significance: According to our understanding, the E-NOF biosensor is the first strategy to cascade EXPAR with HCR and DNAzyme nucleic acid circuit for miRNA-1246 detection. Accurate results can be obtained in only 120 min. Compared with the traditional HCR system, the sensitivity of the new E-NOF biosensor is increased by 1 × 10 9 times. Furthermore, the biosensor can also detect biomarkers in human serum samples. It has great potential in miRNA detection and identification., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Herein, magnetic electrochemiluminescence (ECL) nanoparticle Fe 3 O 4 @PtPd/Ru(bpy) 3 2+ had been synthesized then been coupled with CRISPR/Cas13a system and Zn 2+ dependent DNAzyme to design a novel ECL biosensor for specific detection of microRNA-145 (miRNA). The synthesized multifunctional magnetic nanoluminescent materials Fe 3 O 4 @PtPd/Ru(bpy) 3 2+ not only load Ru(bpy) 3 2+ to provide ECL signals, but also can quickly achieve separation and enrichment from complex matrices. In addition, ferrocene (Fc) was used as a quencher in the Ru(bpy) 3 2+ /tripropylamine (TPA) system. Fc was modified on DNA bound to Fe 3 O 4 @PtPd. Benefited from the highly specific recognition ability of CRISPR/Cas13a, the target miRNA induces CRISPR/Cas13a trans-cleavage to trigger the Zn 2+ -dependent DNAzyme cyclic cleavage to realize the dual signal amplification. DNA modified by Fc was split by target miRNA-induced cleaving, and then magnetic separation was performed to keep Fc away from the surface of the nanoparticles. Thus, the enhanced ECL signal was obtained to detect miRNA-145. Under optimized conditions, the prepared sensor showed a wide linear range (1 fM to 1 nM) and a low limit of detection (LOD) down to 0.41 fM. Furthermore, it shows excellent selectivity and good reproducibility. The proposed ECL platform has huge potential applications in the development of various sensitive sensors for detecting the other miRNA., 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 B.V.)

Systematic management of infected wounds requires simultaneous antiinfection and wound healing, which has become the current treatment dilemma. Recently, a multifunctional silver nanoclusters (AgNCs)-based hydrogel dressing to meet these demands is developed. Here a diblock DNA with a cytosine-rich fragment (as AgNCs template) and a guanine-rich fragment (to form G-quadruplex/hemin DNAzyme, termed G4/hemin) is designed, for G4/hemin functionalization of AgNCs. Inside bacteria, G4/hemin can not only accelerate the oxidative release of Ag + from AgNCs but also generate reactive oxygen species (ROS) via catalase- and peroxidase-mimic activities, which enhance the antibacterial effect. On the other hand, the AgNCs exhibit robust anti-inflammatory and antioxidative activities to switch M1 macrophages into M2 phenotype, which promotes wound healing. Moreover, the hemin is released to upregulate the heme oxygenase-1, an intracellular enzyme that can relieve oxidative stress, which significantly alleviates the cytotoxicity of silver. As a result, such silver-based dressing achieves potent therapeutic efficacy on infected wounds with excellent biosafety., (© 2023 Wiley-VCH GmbH.)