DNA Walker Research Articles

3D Walking Nanomachine for Monitoring of miRNA in Living Cells.

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

Multispatially Localized DNA Walker Coupling Covalent Organic Framework for Dual-Mode Detection of Nucleocapsid Protein Using the Walking-Recycling-Conversion Strategy.

DNA walkers have emerged as a powerful tool in bioanalysis; however, many existing approaches are still restricted by low reaction kinetics and inaccurate single-mode detection. Herein, a fluorescence (FL) and electrochemical (EC) dual-mode biosensor was proposed based on a multispatially localized DNA walker (m-DNA walker) coupling covalent organic framework (COF) using the walking-recycling-conversion strategy. Specifically, the functionalized COF not only served as a three-dimensional nanocarrier but also acted as an effective quencher of the walking tracks. In the presence of the target, the activated m-DNA walker moved fast along the numerous quenching tracks, leading to the cleavage of Cy3-H1 and the recovery of the FL signal. To further improve the detection sensitivity, the Cy3-H1 fragments' recycling process was implemented with the generation of a large amount of S1 and S2, which caused the assembly of DNA-Fe3+-polydopamine network amplifiers on the electrode. The rapid electrochemical conversion was introduced to convert DNA-Fe3+-polydopamine into electroactive Prussian Blue, providing a significant EC signal output. Using nucleocapsid protein (N-protein) as the model target, the designed biosensing platform produced a FL/EC dual-mode readout with the detection limits of 65.0 fg/mL for FL mode and 2.3 fg/mL for EC mode, which could eliminate the interference from different reactive pathways and improve the detection accuracy, holding potential application in early disease diagnosis and treatment.

An inverted tetrahedron-mediated DNA walker for sulfadimethoxine detection.

Aninverted DNA tetrahedron-mediated modular DNA walker was developed for the determination of sulfadimethoxine. The inverted DNA tetrahedron scaffold raises several advantages of recognition module including appropriate lateral space, multiple recognition domains, and cost-effectiveness. The proposed inverted DNA tetrahedron-based recognition module exhibited better binding affinity and kinetics toward target antibiotic than that of other DNA tetrahedron counterparts. Upon specific binding with target, the released bipedal DNA walking strand hops to the signal amplification module and moves stochastically with assistant of nicking enzyme. By coupling these two modules, a good linear relationship between the fluorescence intensity of supernatant and the concentration of sulfadimethoxine was achieved in the range 0.1-100nM, and the limit of detection was 64.7pM. Furthermore, this modular DNA walker had also successfully applied tospiked honey and milk samples with satisfactory recoveries from 91.5 to 108.8%, demonstrating its practical sensing capability.

Electrochemical biosensor based on Au5Ir@graphene quantum dot nanocomposite and DNA walker for detection of atrazine in environmental water with extremely high sensitivity and selectivity

Existing electrochemical sensors are inadequate for detecting pesticides at extremely low level. This study presents an electrochemical biosensor based on gold-iridium alloy (Au5Ir) and DNA walker for detection of atrazine. Au5Ir@RFBP-GQD nanocomposite was synthesized via the reduction of chloroauric acid and iridium trichloride using arginine and folic acid-functionalized boron and phosphorus-doped graphene quantum dot (RFBP-GQD). The synergy between Au and Ir, along with the formation of Schottky heterojunction, enhances the catalytic activity. The Au5Ir was covalently conjugated with hairpin DNA and thionine forming a redox probe that was used to construct the electrochemical biosensor for atrazine detection, coupled with a DNA walker mechanism. Atrazine triggers the DNA walker, leading to the introduction of multiple redox probes onto the gold electrode surface. The oxidation and reduction of thionine molecules within these redox probes elicit highly sensitive electrochemical response. The integration of Au5Ir@RFBP-GQD catalysis with the DNA walker results in significant signal amplification. The biosensor exhibits superior sensitivity and selectivity compared to other atrazine sensors, with a linear detection range from 1×10−18 to 1×10−12 M and a low detection limit of 3.4×10−19 M (S/N=3). The proposed analytical method was successfully used for detection of atrazine in environmental water.

Early Diagnosis of Triple-Negative Breast Cancer Based on Dual microRNA Detection Using a Well-Defined DNA Crown-Carbon Dots Structure as an Electrochemiluminescence Sensing Platform.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC). Thus, early detection and accurate diagnosis of this cancer are crucial for improving the survival rate of patients. Specific microRNAs (miRNAs) have been implicated in the occurrence, proliferation, and metastasis of TNBC. Addressing this need, our study developed a biosensor platform for early and accurate TNBC diagnosis by integrating electrochemiluminescence (ECL) technology with a DNA sensing strategy. Specifically, synthesized positively charged carbon dots (CDs) were used to neutralize the electrostatic repulsion between DNA strands and facilitate the assembly of DNA triangular prisms (DNA TP-CDs). Hairpins were then incorporated into the DNA TP-CDs to form the final DNA crown structure. The early TNBC biomarker, microRNA-93-3p (miR-93-3p), allowed for the binding between the DNA Crown and the DNA track on the electrode and initiated the ECL signal. Subsequently, microRNA-210 (miR-210) unlocked the DNA tripedal walker, and its movement on the DNA Crown eventually quenched the ECL signal, enabling accurate TNBC diagnosis and tumor stage assessment. Our proposed biosensor had satisfactory sensing efficiency due to the ordered DNA track and rapid-moving DNA walker. The data revealed a good linear relationship between the ECL signals and the logarithm of miRNA concentrations, with miR-93-3p having a detection limit of 31.04 aM and miR-210 having a detection limit of 7.69 aM. The biosensor also showed satisfactory performance in serum samples and cells. Taken together, this study hopes to provide ideas and applications for clinical diagnosis as well as the personalized treatment of TNBC.

Self-Replicating Catalytic Hybridization Assembly of Bipedal DNAzyme Walkers for Enhanced Electrochemiluminescence Bioanalysis.

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.

Dual quenching ECL strategy based on AgInZnS quantum dots and a new co-reaction promoter oxygen vacancy-modified P5AIn/TiO2 for sensitive CEA detection

A novel electrochemiluminescence (ECL) biosensor for the sensitive detection of carcinoembryonic antigen (CEA) was constructed, utilizing environmentally friendly AgInZnS quantum dots (AgInZnS QDs) and a new co-reaction promoter enriched with oxygen vacancies, namely titanium dioxide/poly(5-aminoindole) (Ov-TiO2/P5AIn). The composite of P5AIn and TiO2 significantly enhanced the reaction rate of AgInZnS QDs with tripropylamine (TPrA). In addition, the presence of oxygen vacancies significantly enhances the electron migration rate of the electrode material, leading to more efficient catalysis of TPrA+ generation and a significant enhancement of ECL signals. Furthermore, a dual-quenching sensing strategy was employed using cuprous oxide-triaminophenol (Cu2O@APF) as the quencher, enabling the biosensor to switch. Through dual quenching of the ECL emission from AgInZnS QDs by Cu2O and APF, the biosensor achieves an extremely low “off” state of the ECL signal. Furthermore, a signal amplification strategy driven by the synergistic action of exonuclease III (ExoIII) and restriction endonuclease (Nt.BbvCI) was designed for a dual-site DNA walker, achieving cyclic amplification of the target signal and ECL signal, thereby turning the signal “on” again. The above mechanism significantly improved the specificity and sensitivity of the biosensor for CEA detection. The prepared biosensor exhibits good stability, selectivity, and sensitivity. The linear range for CEA detection is 5 × 10−14 to 10−8 M, with a detection limit of 16 fM. Additionally, the biosensor demonstrates satisfactory analytical capability for CEA in real blood samples. This strategy provides a reliable new approach for the sensitive detection of CEA and other tumor markers.

Uracil base PCR implemented for reliable DNA walking

PCR-based DNA walking is of efficacy for capturing unknown flanking genomic sequences. Here, an uracil base PCR (UB-PCR) with satisfying specificity has been devised for DNA walking. Primary UB-PCR replaces thymine base with uracil base, resulting in a primary PCR product composed of U-DNAs. A single-primer (primary nested sequence-specific primer) single-cycle amplification, using the four normal bases (adenine, thymine, cytosine, and guanine) as substrate, is then performed on the primary PCR product. Clearly, only those U-DNAs, ended by the primary nested sequence-specific primer at least at one side, will produce the corresponding normal single strands. Next, the single-cycle product undergoes uracil-DNA glycosylase treatment to destroy the U-DNAs, while the normal single strands are unaffected. Afterward, secondary even tertiary PCR is performed to exclusively enrich the target product. The feasibility of UB-PCR has been checked by obtaining unknown sequences bordering the three selected genetic sites.

Highly integrated, self-powered and activatable bipedal DNA nanowalker for imaging of base excision repair in living cells

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 Mn2+-dependent DNAzyme hairpin (H1) on degradable MnO2 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 (Mn2+-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 MnO2 nanosheets and GSH inside the cells not only consumed the intracellular GSH to improve the biostability of the walking track but also generated abundant Mn2+ 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.Graphical abstract

Multipedal DNA Walker: Engineering Strategy, Biosensing Application and Future Perspectives.

With the continuous development of DNA nanotechnology, DNA walkers have attracted increased attention because of their autonomous and progressive walking along predesigned tracks. Compared with the traditional DNA walkers, the emerged multipedal DNA walkers showed their special charm with sustainable walking capability, higher reaction efficiency, expanded walking region, and improved amplification capability. Consequently, multipedal DNA walkers have developed rapidly and shown potential in biosensing applications. Hence, in this review, we make a comprehensive representation of the engineering strategy of multipedal DNA walkers, which focused on the design of multiple walking strands as well as the construction of tracks and driving forces. Meanwhile, the application of multipedal DNA walkers in biosensors has been thoroughly described according to the type of biosensing signal readout. By illustrating some representative works, we also summarized the merits and challenges of multipedal DNA walker-based biosensors and offered a deep discussion of the latest progress and future.

Ultrasensitive detection of circulating tumor DNA using a CRISPR/Cas9 nickase-driven 3D DNA walker based on a COF-AuNPs sensing platform.

Aelectrochemical biosensor was designedutilizing a CRISPR Cas9n-driven DNA walker combined with gold-nanosphere-like covalent organic frameworks (COFs-AuNPs) to detect breast cancer markers (PIK3CA E545K ctDNA). The DNA walker probe is activated only in the presence of circulating tumor deoxyribonucleic acid(ctDNA), binding to a support probe to form a double strand that is then specifically cleaved by the Cas9n/sgRNA complex. This cleavage produces numerous DNA fragments for signal amplification. The COF-AuNPs as electrode materials facilitate electronic transfer and provide additional active sites for the immobilization of nucleic acid probes. This setup achieves a detection limit of 1.76 aM, demonstrating high sensitivity. Additionally, Cas9n improves the specificity of the sensor, accurately distinguishing a pair of base-mismatched sequences, and reducing the occurrence of false positives. Overall, the sensor exhibits excellent selectivity, reproducibility, and potential for early diagnosis of breast cancer.

Asymmetric Nanopore Sensor for Logic Detection of Dam and M.SssI Methyltransferases in Combination of DNA Walker and Autocatalytic Hybridization Reaction.

The detection of DNA methyltransferase (MTase) was crucial for understanding gene expression regulation, cancer mechanisms, and various biological processes, contributing significantly to disease diagnosis and drug development. Herein, a nanopore sensor based on cascaded signal amplification of DNA walker and autocatalytic hybridization reaction (AHR) was developed for the ultrasensitive determination of various MTases. In the presence of Dam MTase, the hairpin structure HD underwent methylation and cleavage by DpnI endonuclease, forming T-DNA fragments. These T-DNA fragments were used to activate the DNA walker, which moved across the surface of magnetic beads step by step, generating a large quantity of initiator I by cleaving the substrate. The initiator I subsequently activated the AHR. The AHR included a hybridization chain reaction (HCR) amplifier and a catalytic hairpin assembly (CHA) convertor. The HCR amplifier generated multiple novel CHA triggers, which activated the CHA convertor. This, in turn, stimulated the HCR amplifier, creating an AHR circuit that resulted in the formation of numerous DNA nanowires. These DNA nanowires were adsorbed onto the G4-PAMAM-modified nanopore surface under the influence of an electric field, thereby altering the surface charge of the nanopore and changing the ionic rectification curve. The detection limit of the Dam MTase nanopore sensor reached 0.0002 U/mL. By modification of the recognition sites of the probes, this nanopore system could also be used for the detection of M.SssI MTase. Moreover, a four-input parallel concatenated logic circuit (AND//INHIBIT-OR) had been constructed and applied for the multivariate detection of Dam MTase and M.SssI MTase, presenting a novel conceptual model for advancing the construction of nanopore logic gate systems and their applications in biosensing.

A gold nanoparticle conjugated single-legged DNA walker driven by catalytic hairpin assembly biosensor to detect a prokaryotic pathogen

Catalytic hairpin assembly (CHA)-DNA walker allows nanostructures to spontaneously hybridize to the nucleic acids. The localized surface plasmon resonance provides the ability of color-shift for Au nanoparticles (AuNPs) to design a colorimetric biosensor by implementing CHA-DNA walker reaction on AuNPs. A target gene in Klebsiella pneumoniae as the reaction cascade trigger, was selected. H1 and H2 oligonucleotides as the components of the system were designed and verified by NUPACK. The AuNPs were conjugated to H1. The conjugation of the probes to the AuNPs was evaluated using FT-IR. The signal amplification process was conducted at 25℃. TEM imaging, zeta potential, spectroscopy, and gel-electrophoresis were used to examine the conduction of the reaction cascade and specificity. The sensitivity of the method was analyzed using serial dilution of the target. The formation of over-52 bp intermediate secondary structures (which only exist when the reaction happens) was confirmed by gel-electrophoresis. The color distinction between positive (0.08 to 0.058) and negative samples (0.098 to 0.05) was evidenced instantly and in a period of 90 min of the reaction as a drop change of 520 nm intensity absorbance. TEM imaging confirmed the further distance of AuNPs in the positive sample in comparison to that of the negative sample which reveals effective detection of the pathogen. The LOD of the technique was measured as 2.5 nM of the target sequence. The diagnostic approach is a label-free, enzyme-independent approach and can be executed in a single step. It has been designed by employing the CHA-DNA walker system along with the colorimetric properties of AuNPs for the first time, thereby paving the way for more rapid and accurate diagnostic kits.

Bisphenol a monitoring by commercial pregnancy test strips transformed by DNA walking machine

Frequently occurred food contamination issues bring about a tremendous global demand for point-of-care testing (POCT) devices. Pregnancy test strip (PTS) retains the highest POCT market share and a low cost of $0.10 per test, but challenged by limited analytical sensitivity and non-autonomous signal transduction for drink contaminant monitoring. Herein, we developed a DNA walking machine-assisted commercial PTS method for on-site bisphenol A (BPA) analysis in drink. DNAzyme was operated as the efficient autonomous catalytic walker chain to drive the DNA machine for the amplified signal output of BPA in drink samples. A detection limit of 16.8 pg/mL was obtained, which is three orders of magnitude lower than the regulatory limit of European Union (Commission Regulation (EU) 2020/1245). The POCT strategy constructed in this work has the characteristics of low cost, convenient operation and high analytical sensitivity, represents an attractive option for biosensing and on-site drink safety monitoring.

PCR-free and visual detection of antibiotic resistance gene in feces genome of aquatic products by coupling CuO nanoparticles concatemers-Au@Fe3O4 and 3D DNA walker based on a portable blue light gel imager

Antibiotic resistance genes (ARGs) present a significant health issue, and the sensitive detection of ARGs is essential for preventing antibiotic resistance. Herein, a novel highly sensitive and visual biosensor was developed by combining gold nanoparticle (AuNP)-coated Fe3O4 (Au@Fe3O4), CuO nanoparticles (CuONPs) concatemers, Cu2+-triggered 3D DNA walker (DW, which included DNAzyme-capped AuNPs (DNAzyme@AuNPs) and AuNPs capped by substrate DNA modified with fluorescein (FAM-Sub@AuNPs), namely CuONPs-Au@Fe3O4-DW method. When the target ARG was present in the sample, it acted as a bridge between the complementary strand DNA (cDNA) on the surface of Au@Fe3O4 and H1-CuONP which paired with repeatedly H2-CuONP by a hybridization chain reaction, resulting in an ARG coupling multiple CuONPs (CuONPs concatemers) effects. After magnetic separation, hydrochloric acid was added to dissolve the CuONPs concatemers to release plenty of Cu2+ ions, which activated the DNAzyme@AuNPs of the DW. Subsequently, the FAM-Sub@AuNP of the DW was sheared by its DNAzyme, resulting that the FAM molecules kept away from AuNPs, and generated high fluorescence signals. The fluorescence signals can be measured by a 96-well microplate reader with a limit of detection (LOD) of 0.89 pM and visualized using a portable blue light gel imager with a LOD of 8.1 pM. Remarkably, the results of the qPCR method are high consistent with those of the microplate reader (r = −0.935) and the blue light gel imager (r = −0.994) in detecting tetA of aquatic faecal samples. Furthermore, our PCR free, high sensitivity and visual method is also suitable for the identification of other ARGs by replacing the corresponding gene sequence, and enables on-site detection of the ARGs in aquatic products by integrating the blue light gel imager.

One-step cascade amplification system based on entropy-driven catalysis and DNAzyme triggered DNA walker for label-free detection of acetamiprid

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 Mg2+. 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.

Self-reinforced D-MOF and autocatalytic AuPd@SnS2 dual-wavelength electrochemiluminescence biosensor for detection of ctDNA and CEA

Trace detection of circulating tumor DNA (ctDNA) and CEA is of great significance for early prevention and diagnosis of cancer. In this work, the well electrochemiluminescence (ECL) of the dual-ligand metal-organic frameworks (D-MOFs) and AuPd@SnS2 QDs nuclear-satellite conjugate was newly explored, and a dual-wavelength biosensing platform based on ECL energy transfer between D-MOFs and AuPd NPs coupled with catalytic effect of AuPd NPs on ECL of SnS2 QDs was developed for detection of trace ctDNA and CEA. Here, thanks to the ordered heterogeneous arrangement and network charge transfer of the luminescent ligand, the prepared D-MOF exhibits highly enhanced ECL without exogenous co-reactants. Target ctDNA was exponentially amplified using an entropy-driven DNA cycle amplification strategy to obtain bandage DNA, which triggered the DNA walker to connect the AuPd@SnS2 core-satellite probe to the electrode. Therefore, the ECL ratio of D-MOF and sensitized SnS2 at 535 nm and 650 nm was used to achieve ultrasensitive detection of trace ctDNA. Moreover, the specific recognition of target CEA to T1 DNA induced changes in dual-wavelength signals to achieve further detection of CEA, and ensure the accuracy of cancer analysis. The work opened up two novel ECL materials and developed a novel ECL biosensor for rapid detection of double tumor markers, providing a new way for early prevention and diagnosis of actual cancer.

Ingenious entropy-driven DNA circuit intercommunicating with DNAzyme-powered DNA walker for dual-mode biosensing

Conventional DNA motor-based cascaded amplification methods have relative low DNA utilization efficiency and lack of self-feedback. Herein, a novel dual-mode biosensor of miRNA-155 was constructed based on a waste-free entropy-driven DNA circuit (EDC) cascaded with a self-feedback DNAzyme-powered DNA walker (DNAzyme Walker). The target (T)-triggered EDC generates two same single-stranded DNA (ssDNA) labelled with CdTe QDs as the signal probes (CdTe-O) and one double-stranded DNA (dsDNA) S/F to unlock the downstream blocked DNAzyme (D), which could then be activated in the presence of Mn2+ ions cofactor, and stochastically walk on the surfaces of SiO2 nanospheres, producing a lot of target analogue (T*) for self-feedback. With the assistance of Fe3O4@SiO2-capture DNA, the released CdTe-O can be rapidly extracted for the following self-validating dual-mode biosensing. The autocatalytic EDC module possesses a high operation efficiency and atomic economy, since it is not only hairpin- and leak-free, but also enzyme- and waste-free. The interactive EDC-DNAzyme Walker network-based fluorescent and colorimetric biosensor has a limit of detection low to 0.35 amol L−1 and 1.87 fmol L−1 at 3σ/S, respectively. The as-designed biosensor not only enables reliable and robust detection of miRNA expression levels, but also provides a remarkable signal cascaded amplification platform with self-feedback and high atom economy.

A target-triggered colorimetric sensor for ultrasensitive detection of miRNAs based on self-powered three-dimensional DNA walker

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.

Catalytic hairpin assembly-driven DNA walker to develop a label-free electrochemical aptasensor for antibiotic detection.

Anelectrochemical aptasensor was developed by utilizing a DNA walker driven by catalytic hairpin assembly (CHA) with kanamycin as the model analyte. Kanamycin bound to the aptamer, causesthe release of DNA walker, triggers the CHA reaction, leadsto the cyclic movement of the walker's long arm, and resultsin cascade amplification of thesignal. The guanine-rich sequences of the double-stranded products produced by CHA were folded to form G-quadruplex structures, with electrochemical active molecules Hemin embedded, formsG-quadruplex/Hemin complexes in situ on the electrode surface, thereby achieving sensitive, efficient, and label-free detection of kanamycin with a limit of detection (LOD) of 0.27pM (S/N = 3). Meaningfully, the aptasensor demonstrated high sensitivity and reliability in the detection of kanamycin in milk and livestock wastewater samples, suggesting that it has great potential for application in detecting antibiotics in food products and water samples from the environment.