Walking Strand Research Articles

Advanced Pt hollow nanospheres/rubrene nanoleaves coupled with M-shaped DNA walker for ultrasensitive electrochemiluminescence bioassay

Herein, an intense electrochemiluminescence (ECL) was achieved based on Pt hollow nanospheres/rubrene nanoleaves (Pt HNSs/Rub NLs) without the addition of any coreactant, which was employed for ultrasensitive detection of carcinoembryonic antigen (CEA) coupled with an M-shaped DNA walker (M-DNA walker) as signal switch. Specifically, in comparison with platinum nanoparticles (Pt NPs), Pt HNSs revealed excellent catalytic performance and pore confinement-enhanced ECL, which could significantly amplify ECL intensity of Rub NLs/dissolved O2 (DO) binary system. Then, the tracks and M-DNA walker were confined on the Pt HNSs simultaneously to promote the reaction efficiency, whose M-structure boosted the interaction sites between walking strands and tracks and reduced the rigidity of their recognition. Once the CEA approached the sensing interface, the M-DNA walker was activated based on highly specific aptamer recognition to recover ECL intensity with the assistance of exonuclease Ⅲ (Exo Ⅲ). As proof of concept, the “on-off-on” switch aptasensor was constructed for CEA detection with a low detection limit of 0.20 fg/mL. The principle of the constructed ECL aptasensor also enables a universal platform for sensitive detection of other tumor markers.

Aptamer/organometallic receptor-based and label-free sensitive electrochemical detection of amino acid via multi-pedal DNA walker/DNAzyme amplifications

Sensitive and accurate monitoring of amino acid concentration variations plays an important role for diagnosis of diseases at early stage. Here, the aptamer/organometallic receptor-based methodology for label-free and amplified electrochemical determination of phenylalanine, an amino acid, through multi-pedal DNA walker and DNAzyme dual signal enhancement is demonstrated. The binding of the phenylalanine/organometallic receptor complex with the aptamer results in the release of active DNAzyme walking strands to trigger the multi-pedal DNA walkers to move on the surface of electrode. The substrate hairpins are then cyclically cleaved by the DNA walkers to liberate a large number of free G-quadruplexes, which interact with and confine hemin on the electrode via the G-quadruplex/hemin structures. Significantly amplified current signals can therefore be generated by electrochemical reduction of hemin for the sensitive measurement of phenylalanine down to 1.03 μM. Such an aptasensor also has a high selectivity and can monitor phenylalanine in diluted serums, making it a promising alternative for the development of versatile and robust aptamer-based platforms for detection of various amino acids.

Colorimetric detection of viral RNA fragments based on an integrated logic-operated three-dimensional DNA walker

Timely and accurate detection of virus is crucial for preventing spread of disease and early treatment of the infected cases. Herein we design an integrated logic-operated three-dimensional DNA walker for colorimetric detection of viral RNA fragments, by taking SARS-CoV-2 as an example. The DNA walker is composed of small amounts of dually-blocked walking strands and large amounts of dual-stem-loop track strands on gold nanoparticles. The walking strand contains a swing arm domain and a DNAzyme domain blocked at both sides of catalytic core, while the track strand contains a substrate domain located at the peripheral larger loop. Only the presence of both ORF1ab and N RNA fragments can fully de-block the walking strand, which then continuously hybridizes with track strands and cleaves them by DNAzyme-catalyzed hydrolysis. As the cleavage of track strands from long-stranded, double stem-loop structure to short-stranded, linear sequence, the DNA walker shows much lowered stability due to decreased negative charge density and diminished steric repulsion, which then gets aggregated at high salt concentration, accompanied by a visible color change. The colorimetric DNA walker detects RNA fragments down to 1 nM, responds dual viral genes in a “AND” logic way, and shows high specificity to target sequence. It can further detect large nucleic acids containing ORF1ab and N sequences, and reach 200 copies/mL detection limit by coupling a simple upstream amplification of sample. The method may provide a convenient way for reliable detection of viral RNA.

Wireframe Orbit-Accelerated Bipedal DNA Walker for Electrochemiluminescence Detection of Methyltransferase Activity.

In spite of the DNA walkers executing the signal accumulation task in the process of moving along the predetermined paths, the enhancement of walking dynamics and walking path controllability are still challenging due to the unprogrammed arrangements of DNA orbits. Taking these dilemmas into account, a bipedal DNA walker was designed skillfully by the virtue of wireframe orbits assembled by DNA cubes in order, which improved the efficiency and the continuity of walking. It could be attributed to the fact that both the contact chance and the dynamic interaction between walking strands and designated orbits were beneficial to minimize the possibility of derailment and improve the accumulation of signal. In addition, the hollow titanium dioxide nanospheres coated with rubrene (Rub@TiO2 NSs) were prepared by the etching of inner silicon dioxide nanoparticles (SiO2 NPs) to regulate the distribution pattern of rubrene (Rub) molecules and expose more electrochemically active sites for high-efficient electrochemiluminescence (ECL). Benefiting by the pore confinement-enhanced ECL, the electron and mass transfer was significantly accelerated because of the hollow structure of Rub@TiO2 NSs. Subsequently, endogenous dissolved oxygen as the coreactant and palladium nanoparticles (Pd NPs) as the coreaction accelerator were employed to constitute a ternary ECL system with explosive signal response. Combining with this ECL platform, the bipedal walker activated by the target can autonomously and directionally move on the DNA wireframe orbits to release the quenching probes continuously. In this way, the biosensor displayed a low detection limit (2.30 × 10-8 U·mL-1) and a wide linear range (1.0 × 10-7 to 1.0 × 10-1 U·mL-1) for the sensitive detection of Dam methyltransferase (Dam MTase) activity. Therefore, a novel strategy for the accurate quantification of epigenetic targets was developed by virtue of improving the walking dynamics of DNA walker and amplifying the ECL of Rub molecules.

Split aptamer remodeling-initiated target-self-service 3D-DNA walker for ultrasensitive detection of 17β-estradiol

DNA walker machines, as one of the dynamic DNA nanodevices, have attracted extensive interest in the field of analysis due to their inherent superiority. Herein, we reported a split aptamer remodeling-initiated target-self-service 3D-DNA walker for ultrasensitive, specific, and high-signal-background ratio determination of 17β-estradiol (E2) in food samples. Two split probes (STWS-a and STWS-b) were rationally designed that can undergo structural reassembled to serve as walking strands (STWS) under the induction of the target. Meanwhile, an intact E6-DNAzyme region was formed and activated at the tail of STWS. The activated E6-DNAzyme then continuously drives the 3D-DNA walker for signal amplification and specific detection of E2. Under optimal conditions, the proposed DNA walker-based biosensor exhibited excellent linearity in the range of 1 pM to 50 nM with a low limit of detection (LOD) of 0.28 pM, and good precision (2.7%) for 11 replicate determinations of 1 nM of E2. Furthermore, the developed DNA walker-based biosensor achieved excellent sensitive analysis of E2 in the complex food matrix with recoveries of 95.6–106.5%. This newly proposed split aptamer-based strategy has the advantages of ultrasensitive, high signal-to-background ratio, and high stability. Noteworthy, the successful operation of the DNA walker initiated by the split aptamer expands the principles of DNA walker design and provides a universal signal amplification platform for trace analysis.

An electrochemical DNA sensor based on an integrated and automated DNA walker

As an artificial nanomachine, a DNA walker demonstrates the potential for biosensing. In this study, a highly integrated, biostable, and autonomous electrochemical DNA walker sensor was rationally designed by a simple assembly of a Mn2+-dependent DNAzyme-powered DNA walker with nanoscale Mn2+ @MOFs containing free carboxylic acid groups UiO-66(Zr)-(COOH)2. In this study, the release of Mn2+ from Mn2+@MOFs was exploited to drive the autonomous and progressive operation of the DNA walker, and the DNAzyme-driven DNA walker was constructed by the co-modification of walking strands and track strands onto the gold electrode (GE) surface. The walking strand was a single-stranded DNA containing a DNAzyme sequence, which was pre-silenced by the locking strand. The track strand was a specially designed DNA sequence that the target can hybridize with the locking strand; hence, the walking strand is unlocked, and the liberated DNAzyme catalyzes the cleavage of track strands to drive the DNA walker operation, shifting tetraferrocene away from the electrode and producing a significant signal change. A detection limit of 38 fM was obtained with our new system, exhibiting a wide linear range from 1.5625 × 10−9 M to 1 × 10−13 M. The proposed approach provided a novel means for constructing an highly integrated, automated, and DNAzyme-driven DNA walker for bioanalysis.

Advances in Electrochemiluminescence Biosensors Based on DNA Walkers.

Mediated by Watson-Crick base-pairing principle, DNA can be used to construct multi-functional molecular machines, such as DNA walkers, tweezers, logic gates and motors. It is noteworthy that DNA walkers with the advantages of programmability and diverse structures within the micro-nano scale have attracted intense attention in the field of biosensing, bioimaging, drug delivery, and genetic diagnosis. DNA walkers are comprised of driving power, walking strands and the tracks. The driving power acts as an external stimulus and the tracks as a platform for the walking strands to move autonomously. Under the specific external stimulus as driving power, such as strand displacement strategies, enzymatic reactions and environmental condition stimulus, DNA walkers could realize the generation and amplification of signals. Electrochemiluminescence (ECL) biosensors, combining ECL technology and bio-identification strategies, exhibit the virtue of high sensitivity, wide linear response range and low background interference. Recently, the construction of DNA walker-based ECL biosensors can amplify the targets and signals via multiple identification and recycles, achieving ultrasensitive detection for diverse targets. Herein, this review systematically summarizes the construction of different types of DNA walkers and their applications in ECL biosensors. Ultimately, this review summarizes and discusses the prospects for DNA walkers.

A primer extension activating 3D DNAzyme walker for in situ imaging and sensitive detection of telomerase activity.

Acquiring information on telomerase activity at multiple levels contributes to a better understanding of its role in various physiological and pathological processes. Herein, a primer extension activating 3D DNAzyme walker is developed for in situ imaging and sensitive detection of telomerase activity. This walker is constructed via co-modifying specially designed hairpin structured walking strands and track strands on a gold nanoparticle (AuNP). The walking strand contains a pre-blocked DNAzyme sequence and a telomerase primer hybridized to its root. The track strand embeds at an RNA cleavage site and is labeled with the FAM group. After this walker is taken up by cells, the telomerase primer is extended under the action of endogenous telomerase to liberate DNAzyme. The liberated DNAzyme cuts track strands in the presence of the cofactor Mn2+ to drive the walker's processive operation, resulting in an enhanced fluorescence recovery of the AuNP-quenched FAM fluorophore. In situ imaging of telomerase activity in three different cell lines (MCF-7 cells, HeLa cells and HL-7702 cells) was well implemented. The discrimination of cancer cells from normal cells and the screening of telomerase inhibitors have been achieved. The sensitive detection of telomerase activity in HeLa cell lysate has also been realized with a detection limit of 10 cells. This walker performed a new approach for monitoring telomerase activity from different levels, providing a potential tool for clinical diagnosis, prognostic evaluation and drug screening.

A potent fluorescent biosensor integrating 3D DNA walker with localized catalytic hairpin assembly for highly sensitive and enzyme-free Zika virus detection

Zika virus (ZIKV) with worldwide distribution, high incidence and great harm, has emerged as a serious global health threat in the past few years. There is an urgent need for fast, low-cost and accurate diagnosis of ZIKV. Herein, a DNA walker integrated with localized catalytic hairpin assembly (LCHA) cascade amplification strategy is proposed as a new ZIKV assay platform satisfying the above requirements. In this strategy, the DNA walker is constructed by functionating AuNPs with locked walking strands and hairpin DNA track strands; LCHA is simply designed by fixed hairpin DNA probes H1 and H2 on a DNA tetrahedron. The target ZIKV RNA sequence initiates the DNA walker process, which generates numerous trigger sequences for activating the subsequent LCHA, generating an amplified fluorescence signal. This method allows the limit of ZIKV sequence detection down to 20 pM without enzymatic DNA amplification. Moreover, the assay can detect ZIKV from serum sample without RNA extraction, proving its practical application in biological analysis.

A mismatch-suppressed, duplex-specific nuclease powered nanowalker for multiplexed sensing of microRNA

DNA molecular machines have attracted immense interest for their potential in biosensing, drug delivery, and cellular imaging. Herein, we report a duplex-specific nuclease (DSN) powered nanowalker that can autonomously and progressively move on a spherical three-dimensional track, which is constructed by functionalizing a 13 nm diameter gold nanoparticle (AuNP) with densely mismatched DNA duplexes. The motion is initiated by an RNA walking strand, and in its absence, the walker is suppressed because the DSN is inactive toward the mismatched DNA duplexes. Once the walking strand is added, perfectly matched DNA-RNA hybrid is formed via a toehold-mediated displacement reaction between the walking strand and mismatched duplex. Thereafter, the DNA-RNA hybrid is simultaneously cleaved by DSN, by releasing the walking strand, which autonomously moves on the track with the aid of DSN. The present study provides a novel energy input and power mechanism for the operation of 3-D nanowalker with high efficiency. Moreover, the proposed nanowalker can be designed in a target microRNA (miRNA)-specific manner by altering the mismatched duplexes, and it exhibits femtomole level sensitivity in both singleplexed and multiplexed sensing of three miRNA targets. In addition, multiplexed quantification of the three miRNAs in biological samples is achieved, further suggesting that the proposed nanowalker has immense potential in biomedical research and early diagnosis of clinical disorders.

Flap Endonuclease 1-Assisted DNA Walkers for Sensitively and Specifically Sensing ctDNAs.

DNA walkers have shown superior performance in biosensing due to their programmability to design molecular walking behaviors with specific responses to different biological targets. However, it is still challenging to make DNA walkers capable of distinguishing DNA targets with single-base differences, so that DNA walkers that can be used for circulating tumor DNA sensing are rarely reported. Herein, a flap endonuclease 1 (FEN 1)-assisted DNA walker has been proposed to achieve mutant biosensing. The target DNA is captured on a gold nanoparticle (AuNP) as a walking strand to walk by hybridizing to the track strands on the surface of the AuNP. FEN 1 is employed to report the walking events by cleaving the track strands that must form a three-base overlapping structure recognized by FEN 1 after hybridizing with the captured target DNA. Owing to the high specificity of FEN 1 for structure recognition, the one-base mutant DNA target can be discriminated from wild-type DNA. By constructing a sensitivity-enhanced DNA walker system, as low as 1 fM DNA targets and 0.1% mutation abundance can be sensed, and the theoretical detection limits for detecting the DNA target and mutation abundance achieve 0.22 fM and 0.01%, respectively. The results of epidermal growth factor receptor (EGFR) L858R mutation detection on cell-free DNA samples from 15 patients with nonsmall cell lung cancer were completely consistent with that of next-generation sequencing, indicating that our DNA walker has potential for liquid biopsy.

Ultrasensitive colorimetric miRNA detection based on magnetic 3D DNA walker and unmodified AuNPs

Abstract Gold nanoparticles (AuNPs) based colorimetric assay is a promising platform for simple and low-cost point-of-care (POC) testing, but tends to have low sensitivity, which limits their clinical applications for trace nucleic acid assays. Herein, a simple colorimetric assay was developed by using unmodified AuNPs and magnetic 3D DNA walker, which could detect as few as 16.7 fM of miRNA-155, thus easily solves the main technical challenge of AuNPs-based colorimetric nucleic acid assays. In this platform, hundreds of substrate sing-stranded DNA (DNA tracks) and dozens of blocked walking strands were attached on an Au-Fe3O4 nanocomposite, applied as a magnetic 3D DNA walker. The target could activate this DNA walker powered by DNAzyme cleavage mechanism, generating a great deal of single-stranded product (displaced ssDNA). After magnetic separation, the generated displaced ssDNA (supernatant) was incubated with AuNPs to protect the unmodified nanoparticles from aggregation in high-salt solutions. Based on this, the concentration of miRNA-155 could be visualized through a color change from grayish black to purple and quantitatively determined by UV–vis spectroscopy. Furthermore, we demonstrated that our approach could be used to detect target miRNA-155 in real human serum samples without complicated sample pretreatment. Thus, the magnetic 3D DNA walker-AuNPs colorimetric method is a facile, sensitive and specific detection platform that shows great potential for development of next-generation POC nucleic acids diagnostics.

A strand-elongation initiated DNAzyme walker for terminal deoxynucleotidyl transferase activity detection

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase found to be overactive in most leukemia cells and is recognized as a biomarker of leukemia. Here, we develop a strand-elongation initiated DNAzyme walker to detect TdT activity. The walker is constructed by modifying specially designed track strand (TS) and incomplete walking strand (WS) onto the surface of a mesoporous silica nanoparticle (MSN) loaded with large amounts of rhodamine 6 G (Rh6 G) in its pores. The TS is a hairpin DNA with an RNA cleavage site, serving as substrate for DNAzyme and encapsulating the Rh6 G. The incomplete WS contains one side of binding arm and partial catalytic core of DNAzyme. Under the action of TdT, the incomplete WS is elongated with polyA, completing the lacking part of catalytic core and the other side of binding arm of DNAzyme. Then the completed DNAzyme cleave the TSs, unblocking and releasing the Rh6 G to indicate the TdT activity. This walker is completed and initiated only after the action of TdT, ensuring its strict specificity for TdT activity. Besides, each WS cleave multiple TSs, and each cleavage leads massive Rh6 G to be released. Such double-amplified signal accumulation endows the walker with good sensitivity. This walker specifically detected TdT activity down to 0.093 U mL−1, and successfully detected TdT activity in human serum. The proposed walker demonstrates a novel method for specific and sensitive detection of TdT activity, providing an effective tool for TdT-related biological research and leukemia theranostics.

A zero-background fluorescent aptasensor for ultrasensitive detection of pesticides based on magnetic three-dimensional DNA walker and poly(T) -templated copper nanoparticles

A zero-background fluorescent aptasensor for the ultrasensitive detection of pesticides has been successfully constructed, using poly(T) stabilized copper nanoparticles (CuNPs) as a fluorescent signal and three-dimensional (3-D) DNA walker as a signal amplifier. To fabricate the aptasensor, one strand of dsDNA and one strand of ssDNA are immobilized onto magnetic Fe2O3 beads. The dsDNA is hybridized by the target aptamer contained DNA (Apt-1) and the partial complementary DNA which named as WS since it acts as a walking strand (WS) in DNA walker. Upon the specific binding between omethoate with Apt-1, WS becomes a free walking ssDNA strand. With the aid of nicking endonuclease, the 3-D DNA walker can produce a great quantity of poly(T) DNA during the WS walking. When the supernatant is added into ascorbic acid (AA) and Cu2+, poly(T) templated CuNPs forms and strong fluorescence generates. The developed aptasensor has a zero-background, and can ultrasensitively detect omethoate in the linear range of 0–200 nM with the detection limit of 0.22 nM. This zero-background aptasensor provides a facile strategy for pesticides determination in the fields of food safety and environmental monitoring.

DNAzyme walker induced DNAzyme working cascade signal amplification strategy for sensitive detection of protein

Abstract Sensitive detection of disease-related proteins is crucial to better illustrate their roles in physiological and pathological processes and to further evaluate their functions in disease diagnosis and clinical research. Herein, we developed a DNAzyme walker induced DNAzyme working cascade signal amplification strategy for sensitive detection of disease-related protein thrombin. In this work, the DNAzyme walker was constructed by co-modifying walking strands and track strands onto gold nanoparticle surface. The walking strand is a single-stranded DNA containing DNAzyme sequence, which is pre-locked by thrombin aptamer sequence. The track strand is specially designed hairpin DNA, which embeds an RNA cleavage site and seals the same DNAzyme sequence. When the thrombin specifically bound to its aptamer sequence, the walking strand was unlocked and then the liberated DNAzyme catalyzed the cleavage of track strands to drive the DNA walker operation, exposing plenty of new DNAzyme buried in track strands. Subsequently, the exposed DNAzyme catalyzed the continuous cleavage of reporter probes labeled with fluorophore and quencher, accompanying by fluorescence signals accumulation. For thrombin detection, a wide linear ranging from 0.02 nM to 15 nM and a detection limit of 4.5 pM were achieved. Benefiting from the cascade amplification effect of DNA walker operation and DNAzyme cleavage reaction, the detection sensitivity was significantly improved. The proposed strategy will provide an alternative method for sensitive analyzing of proteins and hold great application potential in disease diagnosis and clinical research.

A DNAzyme-driven random biped DNA walking nanomachine for sensitive detection of uracil-DNA glycosylase activity.

Highly specific and ultrasensitive detection of uracil-DNA glycosylase (UDG) activity is of great significance for maintaining genomic integrity and medical research of related diseases. Here, we constructed a random DNA walking nanomachine based on a DNAzyme for UDG activity detection on the AuNP (Au nanoparticle) surface. When UDG is present, the U bases in the Y structure are removed, resulting in AP sites, which will be cleaved by Endo-IV to generate a 3' concave end for Exo-III, causing the locking strand of the DNAzyme to be completely hydrolyzed by the Exo-III and release the walking strand to randomly pair with the substrate strand on the AuNP surface; then, the walking strand exerts its cleavage activity with the assistance of Mg2+ to cleave the substrate strand and keep the fluorophore 6-carboxyfluorescein (FAM) away from the surface of the AuNP, which restores the fluorescence signal of this system. In this way, sensitive detection of UDG can be realized, and the detection limit is as low as 3.69 × 10-6 U mL-1. In addition, we found that this method is highly specific to UDG and can be used to detect UDG specifically in complex samples, which has certain application prospects in biomedical research and clinical diagnosis related to UDG.

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.

Simple Tripedal DNA Walker Prepared by Target-Triggered Catalytic Hairpin Assembly for Ultrasensitive Electrochemiluminescence Detection of MicroRNA.

In contrast to common DNA walkers, multipedal DNA walkers exhibit larger walking area and faster walking kinetics and provide increased amplification efficiency. Consequently, they have received a considerable amount of attention in biosensing. However, most of them are synthesized by immobilizing multiple DNA walking strands on the surface of Au nanoparticles, which is tedious and time-consuming. Simple preparation of multipedal DNA walkers remains a challenge. Herein, we adopted a simple enzyme-free target-triggered catalytic hairpin assembly (CHA) circuit to synthesize a tripedal DNA walker. By walking on a DNA track-functionalized electrode, a sensitive electrochemiluminescence DNA nanomachine biosensor was constructed for sensing miRNA-21. The DNA walker was powered by toehold-mediated strand displacement; the whole process did not need the assistance of enzymes, thus avoiding tedious procedures and enzyme degradation under unfavorable environmental conditions. Specifically, a superior detection limit of 4 aM and a broad linear range of 10 aM to 1 pM were achieved. This CHA-tripedal DNA walker biosensor was then applied for the detection of miRNA-21 in human serum and showed high selectivity and excellent reproducibility, demonstrating its practical application in bioanalysis. In particular, the Y-shaped tripedal DNA walker comes from the DNA circuit, which makes the approach ideally suited for biosensing of small nucleic acid targets.

A bipedal-unequivalent three-dimensional DNA walker and its biosensing application

Herein, a bipedal-unequivalent three-dimensional DNA walker is designed and applied to biosensing. It involves three components: (i) DNA three-way junction switch (DTWJS), in which two longer strands containing different sequences are partially complementary serving as bipedal-unequivalent walking strand (B-UWS) and the shortest one serves as blocking strand; (ii) fluorophore-labelled hairpins H1 and H2, co-modified onto gold nanoparticles (AuNPs), serving as track strands; (iii) hairpins H3 and H4, contain sequences complementary to H1 and H2, respectively, serving as fuel strands. By target binding to blocking strand, B-UWS is liberated to hybridize with H1 and H2 in turn. The unfolded H1 and H2 get hybridized with H3 and H4, respectively, accompanying by B-UWS sequential release and fluorescence signal accumulation. Then B-UWS continues to hybridize with another track strands to walk. Compared with unipedal and bipedal-equivalent DNA walkers, this bipedal-unequivalent DNA walker has twice the sustainable operation capability through kinetic and affinity studies. Using let-7a as a model target, it shows a detection limit of 68 pM and satisfactory reproducibility in biological fluids. This DNA walker provides a new sustainable operation mode and will be a potential analytical tool in clinical diagnosis.

Paper-based electrochemiluminescence determination of streptavidin using reticular DNA-functionalized PtCu nanoframes and analyte-triggered DNA walker.

A paper-based electrochemiluminescence (ECL) biosensor characterized by the signal amplification of reticular DNA-functionalized PtCu nanoframes (DNA-PtCuTNFs) and analyte-triggered DNA walker was developed for sensitive streptavidin assay. Silver microflower functionalized paper-based sensing platform was prepared to fix the hairpin strand (S1). With addition of the streptavidin, plenty of DNA walkers consisting of the walking strands (S2) labeled with biotin and streptavidin were established, which protected S2 from digestion via theterminal protection mechanism. The sequential introduction of the DNA walker and capture probe initiated the hairpin structure opening of S1 and strand displacement reaction (SDR) happening, causing the S2 release. Subsequently, S1 hybridized with S3. The free S2 furtherhybridized with adjacent S1 to trigger the next cycle. After multiple cycles, the DNA-PtCuTNFs, the fire-new signal enhancer, with remarkable peroxidase activity, were successfully attached onto the paper electrode via metal-catalyst-free click chemistry. Based on the SDR of the DNA walker and the catalysis of DNA-PtCuTNFs, a significantly boosted ECL signal of luminol was obtained. Under the optimal conditions, the developed sensor for streptavidin assay exhibited a low detection limit of 33.4 fM with a linear range from 0.1 pM to 0.1μM. Graphical abstract.