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

A electrochemical biosensor was designed utilizing 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.

A Self-Serviced-Track 3D DNA Walker for Ultrasensitive Detection of Tumor Exosomes by Glycoprotein Profiling.

Sensitive and accurate analysis of low-concentration of tumor-derived exosomes (Exos) in biofluids is essential for noninvasive cancer diagnosis but is still challenging due to the lack of high-sensitive methods with low-cost and easy-operation. Herein, exploiting target Exos as a three-dimensional (3D) track for the first time, we developed a self-serviced-track DNA walker (STDW) for wash-free detection of tumor Exos using exosomal glycoprotein, which was enabled by split aptamer-recognition-initiated autonomous running powered by a catalytic hairpin assembly (CHA). Benefiting from high selectivity and sensitivity of the STDW assay, direct detection of tumor Exos in cell culture medium and serum could also be realized. Furthermore, this method exhibited high accuracy in clinical sample analysis, offering the potential for early cancer diagnosis and postoperative response prediction.

Ratiometric fluorescent 3D DNA walker and catalyzed hairpin assembly for determination of microRNA.

Using 6-carboxyfluorescein (FAM) and tetramethyl rhodamine (TAMRA) as fluorescent signals a ratiometric fluorescent three-dimensional (3D) DNA walker based on a catalytic hairpin assembly (CHA) reaction for microRNA-122 detection was constructed. This method uses CHA reaction triggered indirectly by the target to mediate the 3D DNA walker operation to amplify the signal. The dual emission ratio fluorescent signal with a single excitation wavelength was used as the signal output. This strategy combines DNA walker with CHA reaction and proportional fluorescence signal output methods, which can effectively reduce the background fluorescence signal and the risk of generating false-positive signals. Thus, the impact of environmental factors on the experiment is reduced, thereby obtaining reliable and stable experimental results. It uses the fluorescence excitation wavelength of 488 nm and the maximum fluorescence emission wavelength of 520 nm and 580 nm, respectively. It has a good linear response at a microRNA concentration range of 156.0 pM ~ 7.00 nM and a detection limit of 42.94 pM. This strategy has been successfully applied to detect microRNAs in spiked serum samples. Graphical abstract Schematic representation of three-dimensional (3D) DNA walker constructed using catalytic hairpin self-assembly reaction (CHA)-assisted amplification and ratiometric fluorescence signal output for the detection of miRNA-122 closely related to hepatitis.

Enhanced luminol electrochemiluminescence biosensing system based on highly dispersed bimetallic nanozyme coreaction accelerator and 3D DNA walker signal amplifier.

In this work, a platinum-nickel based nanozyme is prepared and used as a coreaction accelerator in the luminol-H2O2 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 H2O2 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-H2O2 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-H2O2 ECL system, providing new strategy for designing efficient ECL emitter and offering a new method for detecting phthalate esters.

Enhancing 3D DNA Walker-Induced CRISPR/Cas12a Technology for Highly Sensitive Detection of ExomicroRNA Associated with Osteoporosis.

Exosomal microRNAs (exomiRNAs) have emerged as promising biomarkers for the early clinical diagnosis of osteoporosis. However, their limited abundance and short length in peripheral blood present significant challenges for the accurate detection of exomiRNAs. Herein, we have designed and implemented an efficacious fluorescence-based biosensor for the highly sensitive detection of exomiRNA associated with osteoporosis, leveraging the enhancing 3D DNA walker-induced CRISPR/Cas12a technology. The engineered DNA walker is capable of efficiently transforming target exomiRNA into amplifying DNA strands, thereby enhancing the sensitivity of the developed biosensor. Concurrently, the liberated DNA strands serve as activators to trigger Cas12a trans-cleavage activity, culminating in a significantly amplified fluorescent signal for the highly sensitive detection of exomiRNA-214. Under optimal conditions, the devised technology demonstrated the capacity to detect target exomiRNA-214 at concentrations as low as 20.42 fM, encompassing a wide linear range extending from 50.0 fM to 10.0 nM. Moreover, the fluorescence-based biosensor could accurately differentiate between healthy individuals and osteoporosis patients via the detection of exomiRNA-214, which was in agreement with RT-qPCR results. As such, this biosensing technology offers promise as a valuable tool for the early diagnosis of osteoporosis.

A dual-core 3D DNA nanomachine based on DNAzyme positive feedback loop for highly sensitive MicroRNA imaging in living cells.

A double 3D DNA walker nanomachine by DNAzyme self-driven positive feedback loop amplification for the detection of miRNA was constructed. This method uses two gold nanoparticles as the reaction core, and because of the spatial confinement effect the local concentration of the reactants increase the collision efficiency was greatly improved. Meanwhile, the introduction of positive feedback loop promotes the conversion efficiency. In presence of miRNA-21, a large amount of DNAzyme was released and hydrolyze the reporter probe, resulting the recovery of fluorescence signal. The linear range for miRNA-21 is 0.5-60 pmol/L, and the detection limit is 0.41 pmol/L (S/N = 3). This nanomachine has been successfully used for accurate detection of miRNA-21 expression levels in cell lysates. At the same time, it can enter cells for intracellular miRNA-21 fluorescence imaging, distinguishing tumor cells from normal cells. This combination of in vitro detection and imaging analysis of living cells can achieve the goal of jointly detecting cancer markers through multiple pathways, providing new ideas for early diagnosis and screening of diseases.

Superior graphdiyne self-powered biosensing platform with highly sensitivity and reliability for dual-mode detection of MicroRNA by integrating T7 Exonuclease and 3D DNA walker induced rolling circle amplification.

A highly sensitivity self-powered biosensor is developed based on T7 exonuclease (T7 Exo) and 3D DNA walker induced rolling circle amplification (RCA) for electrochemical/colorimetric dual-mode detection of microRNA-21 (miRNA-21) with improved reliability. Taking its advantage of fascinating properties, such as high structure defects and good conductivity, graphdiyne is prepared and used to prepare high-performance enzyme biofuel cell. T7 Exo-assisted 3D DNA walker target recognition triggers RCA reaction to obtain a significantly amplified signal response. A capacitor is integrated to the enzyme biofuel cell to further amplify the electrochemical output signal of the self-powered biosensor. In detection system, glucose oxidase catalyzes glucose oxidation to produce hydrogen peroxide, and 3,3',5,5'-tetramethylbenzidine (TMB) is then catalyzed to generate colored products, so as to achieve the colorimetric detection of the target. Analysis signals of diverse modes are recorded independently. Consequently, detection of microRNA with improved reliability and wider signal response range are achieved by electrochemical/colorimetric dual-mode with detection limits of 0.15 and 33 fM (S/N = 3) respectively. In addition, the proposed self-powered biosensor successfully applied for the detection of miRNA-21 in human serum samples, confirming its practical applicability in clinical diagnosis. It is powerfully anticipated the proposed self-powered biosensor possesses great potential to be applied to other biomedical domains.

Cu2+@NMOFs-to-bimetallic CuFe PBA transformation: An instant catalyst with oxidase-mimicking activity for highly sensitive impedimetric biosensor.

In this work, a facile impedance biosensor was constructed for sensitive assaying of miRNA-10b based on the Cu2+ modified NH2-metal organic frameworks (NMOF@Cu2+) coupling with a three-dimensional (3D) DNA walker signal amplification strategy. Specifically, abundant Cu2+ can adhere to the MOF via the coordination reaction between NH2 and Cu2+, which can be applied as a skeleton to produce CuFe Prussian blue analogue@NMOF (CuFe PBA@NMOF) just in time. Meanwhile, the carboxyl group, which is rich in the organic ligands of the NMOF, can be used to assemble DNA strands (complementary strand, CS) (CS-NMOF@Cu2+) for biorecognition reaction. With the introduction of the target, a 3D DNA walker was triggered to shear out large amounts of assistant strands (AS), which were then anchored on the surface of GCE. Afterward, CS-NMOF@Cu2+ can be assembled on the GCE by hybridization with AS. Eventually, abundant CuFe PBA@NMOF were generated in situ on the electrode with the help of K3[Fe(CN)6], which can catalyze the 4-chloro-1-naphthol (4-CN) precipitation without H2O2, thereby increasing the resistance of the platform. Under the optimal conditions, the EIS biosensor presents reliable analytical performance in a wide linear range from 0.8 pM to 250 pM with a low detection limit of 0.5 pM.

A novel nanoparticle surface-constrained CRISPR-Cas12a 3D DNA walker-like nanomachines for sensitive and stable miRNAs detection.

The CRISPR-Cas system has broad prospects as a new type of nucleic acid signal amplification technology based on the trans-cleavage activity of Cas12a to single-stranded DNA, but the trans-cleavage reaction efficiency is relatively low in solution. In order to overcome this negative factor, a new 3D DNA nanomachine whose CRISPR-Cas12a is limited to the surface of nanoparticles is used for sensitive and stable detection of miRNA. By loading Cas12a activator onto spherical nucleic acid (SNA), the CRISPR-Cas12a activator system on the surface of Au nanoparticles (AuNPs) acts as a walker to carry out continuous recognition-walking-cutting reaction on the surface of AuNPs, which enhances the trans-cleavage activity of Cas12a to SNAs. Benefiting from the confinement effect of spherical nucleic acids surface, a 3D DNA nanomachine has been developed for the detection of miRNA-21, which has achieved high sensitivity and accuracy, and the detection limit is able to reach 8.0 pM. This new 3D DNA walker-like nanomachine provided another insight for future bioanalysis and early clinical diagnoses of disease and liquid biopsy.

A triple-cycle amplification biosensor for colorimetric detection of mutant PIK3CAE545K based on cascade-driven DNA walker and branched hybridization strand reaction.

Circulating tumor DNA (ctDNA) is an ideal candidate for liquid biopsy biomarkers. Therefore, detecting a low abundance of ctDNA is essential for early cancer diagnosis. Here, we developed a novel triple circulation amplification system integrating entropy and enzyme cascade-driven three-dimensional (3D) DNA walker and branched hybridization strand reaction (B-HCR) for ultrasensitive detection of breast cancer-related ctDNA. In this study, the 3D DNA walker was constructed by inner track probes (NH) and complex S on a microsphere. Once the DNA walker was triggered by the target, the strand replacement reaction ran first and kept circulating to rapidly displace the DNA walker containing 8-17 DNAzyme. Secondly, the DNA walker could repeatedly cleave NH autonomously along the inner track, generating numerous initiators, and then promoting B-HCR to activate the third cycle. Subsequently, the split G-rich fragments were brought in close to form the G-quadruplex/hemin DNAzyme by adding hemin, with the addition of H2O2 and ABTS, the target could be observed. Benefiting from triplex cycles, the PIK3CAE545K mutation detection possesses a good linear range from 1-103 fM, and the limit of detection was 0.65 fM. Due to the low cost and high sensitivity, the proposed strategy has great potential in early diagnosis of breast cancer.

Highly sensitive α-hemolysin nanopore detection of MUC1 based on 3D DNA walker.

Mucin 1(MUC1) is an effective marker of breast cancer, so it is of great significance to develop a simple, sensitive and highly selective MUC1 detection sensor. Herein, we constructed a label-free nanopore biosensor for rapid and highly sensitive detection of MUC1. The presence of MUC1 triggered the modification of the DNAzyme walking chain on the surface of Fe3O4 nanoparticles and separation from the aptamer. In the presence of Zn2+, DNAzyme catalyzed hydrolytic cleavage of the hairpin substrate at the scissile rA. The DNAzyme was divided into two fragments and ssDNA was released. ssDNA products from the hairpin substrate can generate a current blocking signal during α-hemolysin nanopore testing. The frequency of signature events showed a linear response toward the concentration of MUC1 in the range of 0.01 nM-100 nM. The sensing system also exhibited high selectivity against other protein and can be used for the detection of real sample.

AuNPs/CdS QDs/CeO2 ternary nanocomposite coupled with scrollable three-dimensional DNA walker mediated cycling amplification for sensitive photoelectrochemical miRNA assay.

Herein, a novel ternary nanocomposite (AuNPs/CdS QDs/CeO2) with excellent photoelectrochemical (PEC) performance was synthesized as signal probe to construct a near-zero background biosensor for sensitive miRNA-182-5p detection, by integrating with a scrollable three-dimensional (3D) DNA walker mediated cleavage cycling amplification. Impressively, the formation and rolling of scrollable 3D DNA walker triggered by target could realize dynamic, rapid and specific digestion of hairpin DNA on electrode with the aid of Exonuclease III (Exo III), which thus exposed abundant binding sites for assembling stable DNA labeled AuNPs/CdS QDs/CeO2 nanoprobes. Thanks to the formation of type-II heterojunction (between CeO2 and CdS QDs) and Schottky junction (generated by CeO2 and AuNPs), an ideal photoelectric conversion efficiency accompanied with stunningly improved photocurrent was thus acquired for significantly improving the detection sensitivity. It turned out that the detection limit (LOD) of biosensor was ultralow (31 aM). Significantly, the proposed PEC biosensor would exhibit great potential for the composite as a splendid indicator and provide an avenue for constructing the sensing platform with excellent sensitivity and ultralow background.

The fluorescence amplification strategy based on 3D DNA walker and CRISPR/Cas12a for the rapid detection of BRAF V600E.

Circulating tumor DNA (ctDNA) is a fragment of single- or double-stranded DNA originating from tumor or circulating tumor cells and provides accurate information regarding the molecular characteristics of tumors. Therefore, sensitive detection of ctDNA is of great significance to mutation analysis and clinical diagnosis. Among various ctDNAs, the BRAF V600E is related to aggressive behavior, disease recurrence, and disease-specific mortality in papillary thyroid carcinoma. Herein, we selected the BRAF V600E gene sequence as an in vitro biomarker, and established a fluorescence detection strategy combined 3D DNA walker with CRISPR/Cas12a. In the presence of the target ctDNA, 3D DNA walker could identify and bind it, and thus released a large amount of output DNAs through cyclic cleavage with the assistance of specific endonuclease (Nb.BbvCI). The output DNAs were specifically bound to crRNA and activated the non-specific trans-cleavage activity of Cas12a. Finally, the fluorescence signal was significantly enhanced. Notably, this method can detect the BRAF V600E in a range of 1 fM ~ 20 nM with a detection limit of 0.37 fM without DNA polymerase. Due to the powerful amplification capability of 3D DNA walker and high specificity and programmability of CRISPR/Cas12a, the entire process took only a maximum of 70 min. Furthermore, it can be potentially used for the detection of ctDNA in human serum. In summary, this method not only provides a platform for the rapid detection of ctDNA, but also shows good potential for early clinical diagnosis and biomedical research.

Cu(I)-Catalyzed Click Reaction-Triggered 3D DNA Walker for Constructing an "OFF-ON" Fluorescent Biosensor for Cu2+ Detection.

Herein, a highly selective and sensitive "OFF-ON" fluorescent biosensor was designed for intracellular Cu2+ detection. Compared to the fluorescent Cu2+ biosensors reported so far, this work tackled the tricky issue of reliability of Cu2+, which mainly depends on the integration of the high selectivity of the Cu(I)-catalyzed click reaction with the ultrahigh sensitivity of a spherical nucleic acid-based 3D DNA walker. Typically, DNA track is carried out by coconjugating N3-S1 and Cy3-HP onto gold nanoparticles (AuNPs). On this state, fluorophore (Cy3) was close to the surface of AuNPs (as a nanoquencher), generating a quenched fluorescence and thus causing the initial "OFF" state. In the presence of Cu2+ and H2C2-swing arm, Cu+ was in situ generated quickly from the reduction of Cu2+ with the assistance of ascorbic acid, which could promptly and selectively trigger the Cu(I)-catalyzed click reaction-based 3D DNA walker between azide on N3-S1 and alkyne on the H2C2-swing arm. Sequentially, the activated H2C2-swing arm was able to hybridize with adjacent Cy3-HP and the 3D DNA walker was automatically driven by N.BstNBI to produce multiple Cy3-labeled DNA fragments away from the AuNP surface for signal amplification, performing a recovered fluorescence response (turning into the "ON" state). Accordingly, the ingenious integration of an efficient click reaction and smart 3D DNA walker endows the constructed fluorescent biosensor with superior selectivity and ultrahigh sensitivity. We further apply this platform for Cu2+ sensing in biological systems; this assay will provide a signal transduction strategy for evaluating intracellular Cu2+ at picomolar levels.

A simple, one-pot and ultrasensitive DNA sensor via Exo III-Assisted target recycling and 3D DNA walker cascade amplification.

Rapid, sensitive, and user-friendly nucleic acid detection is of growing importance in early clinical diagnosis. Here, we construct a simple, one-pot and ultrasensitive DNA sensor via exonuclease III (Exo III)-assisted target recycling amplification (ERA) combined with 3D DNA walker cascade amplification. In the presence of single-stranded DNA target, the ERA process is activated to generate numerous walker strands (WS). Thereafter, Exo III-powered WSs autonomously move along magnetic bead (MB)-based 3D track to release numerous AgNCs into the supernatant as an amplified signal output. This biosensor had a low detection limit of 18 fM and an analytical range of 40 fM to 1 pM. Furthermore, the practical application potential of this biosensor was also confirmed by the spiking experiments of p53 into human serum and urine samples.

Nanoscale assembly line composed of dual DNA-machines enabling sensitive microRNA detection using upconversion nanoparticles probes.

DNA machines are smart artificial devices that perform well-organized DNA hybridization reactions or nanoscale mechanical movements. Herein, a nanoscale assembly line composing of dual DNA machines is meticulously designed by coupling a catalytic hairpin assembly (CHA)-based machine with a 3D DNA walker machine. Equipped with upconversion nanoparticles (UCNPs) as signal tags, the dual DNA machines-based assembly line (DDMAL) can efficiently amplify the fluorescent signal of target recognition event, enabling sensitive detection of microRNA (miRNA). In detail, once activated by target miRNA-21, the CHA machine is initiated to constantly produce a single-stranded DNA (named binding DNA) via the strand displacement reaction. The binding DNA as a trigger factor can initiate the DNA walker machine by linking a walking strand DNA with an anchor strand DNA immobilized on the surface of magnetic beads (MBs). The movement of walking strand on the surface of MBs is then driven by Mn2+-dependent DNAzyme formed through the hybridization of walking strand with a UCNPs-linked substrate strand. The DNAzyme-catalyzed cleavage of substrate strand is accompanied by the release of numerous UCNPs from MBs. By measuring the fluorescent signal of released UCNPs after the magnetic separation, target miRNA-21 can be detected by the DDMAL system in a linear range from 1.0 fM to 10 nM, with a limit of detection (LOD) of 0.62 fM (3σ). Moreover, the practicability of DDMAL system was demonstrated by using it to evaluate the expression levels of miRNA-21 in cell lines and assay miRNA-21 in human serum.

Two-layer three-dimensional DNA walker with highly integrated entropy-driven and enzyme-powered reactions for HIV detection.

Here, we propose a new two-layer three-dimensional (3-D) DNA walker sensor with highly integrated entropy-driven and enzyme-powered reactions for the first time. The 3-D DNA walker sensor is constructed by assembling densely carboxyfluorescein-labeled single strand oligonucleotides (inner-layer tracks) and nucleic acid complex S (outer-layer tracks) on a microparticle. In the presence of the target, outer and inner tracks are activated in turn, thereby releasing a great deal of the signal reporters for signal reading. As a result, our 3-D DNA walker sensor can realize the target detection in the range from 2 pM to 5 nM within one hour. Besides, the specific walker sensor can clearly distinguish even one-base mismatched target analogue. More importantly, our walker sensor can also test the target in human serum samples in the concentrations as low as 0.1 nM, which provides a bridge between real sample detection and clinical application. Certainly, this smart strategy could also be generalized to any target of interest by proper design.