Advances in DNA walking nanomachine‐based biosensors

The field of DNA nanotechnology has evolved beyond the realm of controllable movements and randomly shaped nanostructures, now encompassing a diverse array of nanomachines, each with unique nanostructures and biofunctional attributes. These DNA nanostructures boast exceptional characteristics such as programmability, integrability, biocompatibility, and universality. Among this variety, DNA walking nanomachines have emerged as one of the most prominent nanomotors, distinguished by their ingenious design and comprehensive functionality. In recent times, these DNA walkers have witnessed remarkable advancements in areas ranging from nanostructural designs to biological applications, including the creation of sophisticated biosensors capable of efficiently detecting tumor‐related biomarkers and bioactive substances. This review delves into the operational mechanisms of DNA walking nanomachines, which are driven by processes such as protease and DNAzyme action as well as strand displacement and photoactivated reactions. It further provides a comprehensive overview of DNA walking nanomachines with different dimensional (1D, 2D, and 3D) walking tracks. A subsequent section introduces the biosensing applications of DNA walking nanomachines including electrochemical, optical, and other biosensors. The review concludes with a forward‐looking perspective on the novel advancements and challenges in developing DNA walking nanomachine‐based biosensors.


| INTRODUCTION
][6] Meanwhile, DNA nanomaterials reveal many advantages including programmability, specificity, stability, and biocompatibility. 7,8Leveraging these benefits, DNA nanotechnology expanded its investigative scope ranging from controllable motions and nanostructures with random shapes towards different nanomachines with various structures and biofunctions.For instance, complex nanostructures with greater molecular mass are meticulously crafted with almost molecular controls, facilitating the integration of dynamic nanoengineering into functional units including novel biosensors, material assembly and synthesis, drug-delivery systems, and biocomputing modules. 9,10Over the past few years, there has been a surge in the development of dynamic DNA nanostructures, which include DNA tweezers, DNA walkers, DNA nanorobots, and DNA nanomotors. 11Furthermore, the signal transduction of these DNA nanomachines can be effectively manipulated.Specifically, upon activation by relevant triggers such as DNA/RNA, enzymes, or other chemical stimuli, DNA nanomachines exhibit complex behaviors or directional movements in specific ways, which have been utilized in various fields including mass spectrometry, highperformance liquid chromatography, fluorescence, and electrochemistry. 12,13he DNA walking nanomachine, as one of the dynamic DNA nanostructures, has recently garnered significant attention in the design and construction of biosensors. 14,15A conventional DNA walking nanomachine primarily consists of three components: driving forces (enzymatic reactions, strand displacement reactions, and other reactions), walking tracks (1D, 2D, and 3D), and walking strands. 16Upon the introduction of driving forces, the DNA walking nanomachine disrupts the original equilibrium and enables the conversion of chemical or light energy into mechanical energy, thereby causing the walking strand to move directionally along the walking track.Subsequently, through the consumption of fuel molecules, the equilibria are re-established, resulting in the generation of output signal.This cycle of breaking and restoring equilibria is repeated, ultimately leading to the amplification of biosensing signals of the nanomachine. 179][20] Prior studies have demonstrated the operation of DNA walking nanomachines that traverse predetermined pathways on 1D, 2D, or 3D terrains.][23] Prior reviews have furnished a comprehensive outline of the evolution and tenets of DNA walkers as well as their multifarious applications across diverse domains.In this review, we accentuate the unique tactics employed in the progression of DNA walkers, while also delving into the analytical efficacy of these biosensors for biological analysis.Initially, we expounded upon the conceptual framework and practical domains of DNA walking nanomachines.Next, we provided an overview of the conventional propelling mechanisms of DNA walking nanomachines, which encompass enzymatic reactions, strand displacement reactions, and optical and chemical stimuli.Following this, we synthesized a summary of the diverse pathways (1D, 2D, and 3D) available for DNA walking nanomachines.Furthermore, we delineated the unique biosensing applications of DNA walking nanomachines in various fields, such as electrochemical, optical, and alternative biosensing modalities.For each scenario, we collated a summary of the attributes of DNA walking nanomachines in biosensing analysis, which encompassed the merits and demerits of the walking systems.Ultimately, we synthesized an overview of the recent breakthroughs and primary obstacles encountered by DNA walking nanomachines in the detection of biomarkers, while also anticipating the future developmental directions of DNA walker-based biosensors.

WALKING NANOMACHINES
Since Seeman's seminal work in constructing the first DNA nanostructures, there has been a growing number of intricately designed DNA nanomachines that have been synthesized. 24In the realm of DNA nanotechnology, remarkable advancements have been made in recent decades, particularly in the domain of DNA nanomachines. 25Among these intriguing developments, DNA walking nanomachines have emerged as autonomous and progressive nanomachines capable of traversing preassembled tracks.Harnessing their potential in biosensing systems, these DNA walking nanomachines can translate biomarker recognition or incorporation events into measurable parameters such as walking efficiency and step count. 26Furthermore, in the field of bioanalysis, DNA walking nanomachines have shown tremendous promise as signal amplification tools offering unparalleled sensitivity. 27Undoubtedly, the performance of DNA walking nanomachines heavily relies on energy drive, which plays a pivotal role in efficiently propelling them along predetermined trajectories.Consequently, the investigation of driving forces for DNA walking nanomachines has garnered significant attention from researchers. 28,29To this end, we summarised the various types of reactions that drive DNA walking nanomachines (Table 1), including enzymatic reactions involving proteases or DNAzymes, strand displacement reactions, photoinitiation reactions, and chemical stimulation reactions.By comprehensively exploring the impact of these driving forces on DNA walking nanomachines, we aim to shed light on their mechanisms and potential applications.

| Enzymatic reactions based-DNA walking nanomachines
Protease or deoxyribozyme reactions, as typical driving forces, have found application in the design of DNA walking nanomachines.1][32] These remarkable nanomachines are primarily propelled by enzymes such as endonucleases, 33,34 exonucleases, 35 and deoxyribonucleases, [36][37][38] which cleave the DNA substrates along a predetermined track.Acting upon the DNA phosphoribose backbone, the energy released from covalent bond breaking propels the movement of the DNA walking nanomachines. 39This section aims to summarize DNA walking nanomachines driven by enzymatic reactions.
1][42] For instance, Yin and co-workers ingeniously developed a DNA walking nanomachine that employed ligases and restriction endonucleases to facilitate its movement. 43However, the approach required manual incorporation of enzymes at each stage, impeding automation and constraining the utilization of DNA walking nanomachines in biosensing applications.Accordingly, through extensive research endeavors, DNA walking nanomachines have been substantially automated to adhere to predetermined trajectories.For instance, Fang and co-workers recently proposed a 3D DNA walking nanomachine for biosensing applications.This nanomachine could move autonomously on a microsphere-based 3D track, powered by the cleavage of hybridized DNA tracks by incision endonucleases.Similarly, Cheng and co-workers introduced a Flap endonuclease 1 (FEN1)-assisted DNA walking nanomachine for constructing mutation biosensors (Figure 1A). 44By enhancing the sensitivity, the system could detect DNA targets as low as 0.22 fM and mutations down to 0.01%.
In contrast, nucleic acid exonucleases bind to nucleic acid ends through substrate recognition and accurately cleave phosphodiester bonds according to their sheared mechanism.Their advantages rely on not requiring specific recognition sequences, making the design of DNA sequences more convenient and flexible. 45Consequently, researchers find widespread applications in various enzyme-assisted amplification reactions.Shen and coworkers developed a 3D walking nanomachine for signal amplification, where T7 exonuclease (T7 exo) served as the driving force (Figure 1B). 16This system consisted of biotin-modified dsDNA decorated on streptavidinmodified magnetic beads, along with target exomiRNA-155 as the walker.The presence of exomiRNA-155 triggered the release of the P2 strands, which then autonomously moved along the predesigned 3D DNA track, leading to the liberation of a substantial amount of DNA strands and achieving signal amplification.The developed biosensor exhibited an impressive LOD as low as 0.27 fM, indicating its potential for early clinical diagnosis.Additionally, Wang and co-workers combined an exonuclease III (Exo III)-mediated 3D walking nanomachine amplification strategy with an electrochemiluminescence (ECL) platform for ultrasensitive analysis of Burkholderia pseudomallei (Figure 1C). 46Compared to ECL alone, the constructed biosensor using the coupled 3D walking nanomachine amplification strategy exhibited a LOD of 60.3 aM, representing two orders of magnitude improvement.DNAzymes, single-stranded DNA molecules with sheared abilities to selectively bind to target molecules, hold promise in biotechnology and medical research. 47y utilizing metal ions, DNAzymes can propel the autonomous movement of DNA walking nanomachines without the need for additional fuel molecules. 48Tian and co-workers designed a Zn 2þ -dependent bipedal DNAzyme walking amplification strategy (Figure 1D). 49he hybridization of H1 with the miRNA-21 led to the formation of an abundant H1-H2 bipodal DNAzyme walker, releasing the target and triggering the production of a significant amount of H1 through recycling.Similarly, Zhao and co-workers constructed a target-triggered DNAzyme motor for multiple DNA glycosylase electrochemical detection. 50This motor utilized two hairpin DNA probes and two DNA-functionalized particles for detecting hAAG and UDG.In the presence of the target, the metal ion-dependent DNAzyme (Mg 2þ /Pb 2þ ) powered the autonomous movement of the DNA walker.This target-triggered DNAzyme motor demonstrated high sensitivity and specificity, enabling simultaneous measurement of multiple DNA glycosylases in HeLa cells and screening for potential inhibitors.

| Strand displacement reactions- based DNA walking nanomachines
Compared to the aforementioned enzyme-driven DNA walking nanomachines, strand displacement-driven DNA walking nanomachines exhibit superior controllability and autonomous propulsion, rendering them highly versatile for diverse scenarios and applications. 51The strand displacement reaction, as a pivotal driving mechanism for DNA walking nanomachines, achieves kinetic and thermodynamic equilibrium by introducing a trigger that complements and pairs with a segment in the DNA walking nanomachines. 52The interaction consequently releases the segment from its original position allowing for precise manipulation and control at the molecular level. 53,54Such a strategy paves the way for groundbreaking possibilities in the realms of biocomputing modules, nanotechnology, and bioscientific research. 55n recent years, numerous signal amplification strategies based on strand displacement-driven DNA walking nanomachines have been successfully employed in biosensing applications.As depicted in Figure 2A, Li and coworkers constructed a switching ECL biosensor for ultrasensitive adenosine triphosphate (ATP) detection, constructing a small-molecule walking nanomachine driven by strand displacement. 56The constructed 3D DNA walker comprised three integral components: the target ATP serving as a walker, the three-stranded DNA complex immobilized on gold nanoparticles (Au NPs) functioning as a 3D walking track (DNA1/DNA2/DNA3), and DNA4 as a drive strand.Accordingly, DNA1 incorporated an aptamer sequence that captured the target ATP.Initially, DNA2 and DNA3 hybridized with DNA1 to enclose the toehold region, with DNA3 acting as the output strand.Upon the presence of the target ATP, it bound to DNA1, causing a conformational change that triggered the release of DNA2 and exposed the toehold domain.Subsequently, DNA4 hybridized with DNA1 from the exposed toehold domain, displacing both ATP and DNA3.The liberated ATP could bind to the next adjacent track, resulting in a biosensor with a remarkable detection limit as low as 0.5 nM.Additionally, as illustrated in Figure 2B, Li and co-workers developed an electrochemical biosensor encompassing a highly integrated multi-legged 2D DNA rolling walker. 57Within this study, an ultrasensitive analysis of circulating tumor DNA (ctDNA) was achieved through rolling ring amplification (RCA) utilizing Adriamycin@Tetrahedron-Au (DOX@TDN-Au) as an electrochemical indicator.Driven by the target-driven RCA, the multi-legged walker adeptly traversed a predesigned track via strand displacement reaction, which enabled the biosensor to detect the target with extraordinary sensitivity, boasting an impressive detection limit as low as 0.29 fM.In F I G U R E 2 DNA walking nanomachines driven by strand displacement reactions.(A) 3D DNA walking nanomachine driven by strand displacement reaction.Reproduced with permission. 56Copyright 2021, Springer Nature.(B) Multi-legged DNA walking nanomachine driven by the target-driven RCA.Reproduced with permission. 57Copyright 2019, Elsevier.(C) Bipedal DNA walking nanomachine driven by strand displacement reaction with "fuel" and "anti-fuel" DNA strands.Reproduced with permission. 58Copyright 2014, Wiley-VCH.(D) Plasmonic nanorod-comprised DNA walking nanomachine driven by strand displacement reaction.Reproduced with permission. 59Copyright 2015, Springer Nature.
comparison to its 2D counterpart, the 3D DNA walker afforded heightened maneuverability and flexibility owing to its multi-dimensional movement capabilities.
In recent years, scholars have made substantial advancements in the cultivation of strand displacementpropelled DNA walkers, utilizing DNA origami technology, and proficiency in executing numerous sequential manipulations.As depicted in Figure 2C, Miran Liber and co-workers achieved a notable feat by constructing a dynamic DNA device composed of two DNA origami building blocks. 58Initially, two distinct DNA origami pieces were prepared and subsequently joined together using a set of DNA strands to form a stable track.A bipedal DNA walker was then attached to one of the origami units and operated through sequential interactions with "fuel" and "anti-fuel" DNA strands, facilitating its movement from one origami block to another.This study effectively demonstrated the reliable movement of parts between origami units, thereby establishing a dynamic DNA nanomachine comprised of multiple origamis building blocks.As such, Zhou and coworkers reported a DNA walker, comprising anisotropic gold nanorods as its "body" and discrete DNA strands as its "fuel" and "anti-fuel" elements (Figure 2D). 59The walker carried optical information and was capable of reporting its walking direction and successive steps with nanometer precision in situ.This was achieved through dynamic coupling to a plasma stator fixed along its walking track.The amalgamation of DNA nanostructures and plasma nanorods presented the possibility of constructing synthetic machines.Artificial DNA walkers demonstrated their in-situ structural dynamics through stabilization and optical methods, thereby bearing farreaching implications across multiple disciplines.

| Other reactions-based DNA walking nanomachines
Apart from classical mechanisms such as enzymatic reactions and strand displacement reactions, DNA walking nanomachines can be driven by environmental stimuli, giving them more advantages for diverse in-situ tests and controllabilities.This section focuses on the utilization of light and chemical stimuli to propel DNA walking nanomachines.Recently, it has been observed that functionalized DNA strands and controlled light sources can achieve light-driven DNA walking nanomachines. 60- 63In a study conducted by Marko Škugor and colleagues in 2019, 64 a fully light-induced DNA walking nanomachine was presented, employing orthogonal light control (Figure 3A).This novel approach employed two azobenzene derivatives, S-DM-Azo and DM-Azo, to precisely coordinate the strand displacement reaction responsible for the movement of the bipedal walker.These derivatives guided the walker along a predefined trajectory in an orchestrated manner.The walker consisted of two legs, denoted as leg A and leg B. Toehold chains, TO-1 and TO-2 were complementary to partial footholds, F1 and F2, respectively, and participated in competing strand displacements with legs A and B at these footholds.This allowed for branching hybridization when the Azo modification was in trans and branching displacements when it was in cis.The photoisomerization of both S-DM-Azo and DM-Azo facilitated the activation of the walking motion of the proteins through irradiation at specific wavelengths, guided by the ratchet effect of the concerted strand displacement reaction.Furthermore, some researchers have designed light-driven DNA walkers for cancer early diagnosis.Chen and co-workers subsequently developed a photoactivatable DNA walking nanomachine based on DNA nanoflares (Figure 3B), enabling light-controlled signal-amplified imaging of cancer-associated microRNAs in individual living cells. 65he activation of the DNA walking nanomachine involved the UV light-induced initiation of a strand displacement reaction between the hairpin DNA on the surface of Au NPs and miRNA-21, leading to the release of the DNA walking strand.This released strand then hybridized with fluorophore quencher-labeled DNA strands in the surrounding DNA track, resulting in the liberation of TAMRA-modified DNA strands with fluorescence activation.Subsequent hydrolysis by Exo III removed the fluorophore quencher-labeled DNA strand, regenerating the DNA walker and triggering a cascade cycle of DNA walker on the surface of the DNA nanoflare, which was capable of efficient and highly sensitive detection of miRNA-21.
Meanwhile, chemical stimuli could also drive the motion of DNA walking nanomachines, as demonstrated by a bipedal walker constructed by Willner et al. (Figure 3C). 66These DNA nanomachines were activated by H þ /OH − and Hg 2þ /cysteine triggers.The bipedal walker operated on a DNA template consisting of four nucleic acid footpoints.Forward walking was initiated by Hg 2þ or H þ ions using either the thymine-Hg 2þ -thymine complex or the i-motif structure, respectively, as the driving force for DNA translocation.Conversely, backward movement occurred upon activation by OH − ions or cysteines, leading to the disruption of the i-matrix or the thymine-Hg 2þ -thymine complex.Additionally, Ellington and co-workers constructed a four-legged DNA walking nanomachine based on toe-exchange reactions (Figure 3D). 67The movement of the DNA walking nanomachine was controlled by alternating pH changes.A well-characterized, pH-responsive CG-C þ triple-stranded DNA was embedded in a tetrameric catalytic hairpin assembly (CHA) walker.The proton-controlled walker autonomously traversed a non-programmed particulate surface, with the speed and number of steps effectively regulated by pH.The walker's initiation, termination, binding, and dissociation from particles were dynamically controlled by pH.The simplicity and programmability of the proton-controlled walker serve as catalysts for the development of various practical nanomachines.

WALKING NANOMACHINES
In addition to the aforementioned fundamental components that constitute DNA walking nanomachines, the walking track plays an integral role in the assembly of the DNA walker.It is noteworthy that the fabrication of this walking track demands meticulous design and regulation.The engineered walking tracks must not only exhibit precision and controllability but also F I G U R E 3 DNA walking nanomachines driven by environmental stimulus.(A) Orthogonally light-driven DNA walking nanomachine.Reproduced with permission. 64Copyright 2019, Wiley-VCH.(B) Photoactivatable DNA walking nanomachine based on DNA nanoflares.Reproduced with permission. 65Copyright 2021, American Chemical Society.(C) H þ /OH − and Hg 2þ /cysteine stimulusactivated DNA walking nanomachine.Reproduced with permission. 66Copyright 2010, American Chemical Society.(D) DNA walking nanomachine driven by pH changes.Reproduced with permission. 67Copyright 2020, American Chemical Society.
accommodate a diversity of movement patterns tailored to different environments and tasks. 68Consequently, depending upon the mode of movement and the extent of the walking chain, the walking tracks can be categorized into three broad classifications: one-dimensional (1D) linear track, two-dimensional (2D) planar track, and three-dimensional (3D) spatial track.These classifications are fundamental to the design and operation of DNA walking nanomachines, as they dictate the degree of precision, controllability, and adaptability to diverse environmental conditions and task requirements.
3.1 | 1D track for DNA walking nanomachines 1D DNA walking nanomachines are nucleic acid molecules that traverse a linear path. 69Pierce and colleagues developed a bipedal DNA walking nanomachine utilizing a strand displacement reaction as the propulsive force, employing a complementary DNA double helix as the short track. 70The walking track comprised six oligonucleotides, each with four distinct single-stranded branches consisting of 20 bases per strand.A scaffolding helix, spanning 15 bases, segregated the branches from the underlying substrate track.Notably, the stability of the short double helix DNA was diminished under thermodynamic conditions in solution.Furthermore, the limited presence of substrate molecules immobilized on the track hampered the efficiency of the DNA walker movement.In a recent breakthrough, Famulok and co-workers introduced a biologically hybridized DNA catenane walker driven by a rotating nano-engine. 32This novel design harnessed long RNA transcripts generated during roll-over transcription (RCT) to enable a linear motion.The DNA pathway encompassed bundles of six-helix DNA origami nanotubes, featuring protruding single-stranded DNA strands deliberately designed for hybridization with the RCTproduced RNA.This orchestrated interaction between the catenane walker, RCT-derived RNA, and the DNA nanotubes facilitated targeted, enduring, and effective traversal up to approximately 240 nm.In addition, Pan and co-workers presented an innovative biosensor capable of simultaneous visible and near-infrared-II (NIR-II) subdiffraction imaging of the DNA walking nanomachine (Figure 4A). 71To visualize the translocation process of the QD-decorated DNAzyme walking nanomachine, superresolution NIR-II images of a RNA-decorated singlewalled carbon nanotube were employed as a 1D track.Along this track, the QD-decorated DNAzyme walker could be observed.When the DNAzyme walking strand hybridized with the fuel strand, it catalytically cleaved the RNA substrates in the presence of divalent cations, thereby reducing the overall free energy of the system.Subsequently, due to thermal fluctuations, a 7-nucleotide fragment P1 dissociated from the DNAzyme/RNA complex (P1/P2).The upper recognition arm then hybridized with the subsequent available fuel strand S2.Gradually, the lower arm displaced from P2 to S2 via strand displacement, culminating in the irreversible association of the entire walking strand with S2.This completed a flip-flop reaction, representing a progressive step forward.

| 2D track for DNA walking nanomachines
Compared to the 1D track, 2D DNA walking nanomachines exhibit enhanced maneuverability within a 2D plane and possess superior path-finding capabilities.By traversing this plane, such nanomachines are able to engage with various molecules or nanostructures situated on the surface, facilitating processes like substance binding and molecular recognition. 72In recent years, considerable attention has been drawn towards electrodes for electrochemical sensors.Utilizing these electrodes as 2D planar trajectories not only augments the surface area available for molecular immobilization but also amalgamates the DNA walking nanomachines with electrochemical sensors, thereby serving as an efficacious signal amplification strategy.Consequently, it enables highly sensitive detection of targets.Gong and co-workers devised a pioneering label-free and signaling electrochemical aptasensor for the detection of soluble α-synuclein (α-syn) oligomers, employing the Mg 2þ -dependent DNAzyme (MNAzyme)-driven tripodal DNA walker strategy. 73Through the utilization of a tripodal DNA walker and MNAzyme as dual signal amplifiers, this study accomplished an exceptional detection limit of 0.46 fM, along with a wide linear range spanning from 1 fM to 10 pM.The electrochemical aptasensor demonstrated remarkable sensitivity, immunity to interference, and reproducibility when applied to detect α-syn oligomers in human serum samples.It showcased immense potential for bioanalysis in complex biological environments.Despite the inherent programmability and flexible biological functionality of DNA walking nanomachines, achieving precise control over their mechanical trajectory within nanoscale spaces remains a daunting task.To address this issue, Liao and co-workers presented an intricate dual-engine-triggered DNA walker incorporating a DNA cube scaffold and a dual pendulum arm engine. 74This design significantly enhanced the operational efficiency and controllability of the DNA walker.Furthermore, a ternary ECL system was proposed based on perylene-doped titanium dioxide (Pe-TiO 2 ) nanorods, SHEN ET AL. with S 2 O 8 2-as a co-reactant and silver nanoparticles (Ag NPs) as a co-reaction promoter.By integrating the walking nanomachine with the ternary ECL system, an efficient ECL biosensor was fabricated for the detection of let 7a, showcasing a linear range from 10 fM to 100 nM, and a lower limit of detection of 7.0 fM.Additionally, to address the challenge of reduced efficiency and prolonged reaction time caused by dissociation of the walking strand from the track, Dou and co-workers constructed a DNA walking nanomachine incorporating wedge-shaped fragments in conjunction with a bimetallic metal-organic framework (MOF) electrocatalyst (Figure 4B). 75In this design, methylated DNA served as a monopodal walker, while an immobilized probe, modified on the electrode, contained a wedge fragment that complemented the structure of the methylated DNA, inhibiting its dissociation from the track.Fuel strands, modified with the bimetallic MOF, facilitated branch migration of the target strand, enabling continuous movement along the track.This biosensor successfully achieved sensitive detection of methylated DNA within 20 min, boasting an astonishingly low detection limit of 200 aM, and effectively discriminated between DNA with different methylation states.Owing to its convenience, Reproduced with permission. 71Copyright 2017, the American Association for the Advancement of Science.(B) Wedged DNA walking nanomachine with 2 D walking track.Reproduced with permission. 75Copyright 2023, American Chemical Society.(C) DNAzyme-based DNA walking nanomachines with a 3D walking track.Reproduced with permission. 78Copyright 2023, Elsevier.(D) Dual-targeting multivalent aptamer regulated DNA walking nanomachines with 3 D walking track.Reproduced with permission. 80Copyright 2023, Wiley-VCH.
rapidity, and signal amplification capabilities, this biosensor holds tremendous potential for early disease diagnosis and molecular biology research.

| 3D track for DNA walking nanomachines
In comparison to the aforementioned 1D and 2D walkers, 3D DNA walking nanomachines offer unprecedented level of freedom and flexibility in movement, enabling them to perform tasks in three dimensions.This enhanced capability opens up a realm of possibilities for their application in various fields, such as biomedicine, nanomechanics, and nanoelectronics. 76,77Exploiting the convenience and stability of gold-sulfur bonding through sulfhydryl modification, DNA strands have been successfully bonded to Au NPs, leading to the construction of numerous DNA walking nanomachines.Liu and coworkers devised a DNA walker strategy based on ZIF-8@DNAzyme for high-precision intracellular miRNA imaging (Figure 4C). 78Not only did this strategy exhibit an exceptionally low background signal during live cell miRNA imaging, but it also distinguished miRNA expression levels between tumor cells and normal cells, showcasing immense potential in disease diagnosis.This advancement further propelled the utilization of DNA walkers in live cell miRNA imaging, while also offering novel concepts for combined applications of MOFs and nucleic acid detection.Additionally, Wang and coworkers capitalized on the advantageous separation capabilities of magnetic nanobeads through simple magnetic separation operations. 79They constructed an ECL biosensor for sensitive detection of miRNA-21, employing 3D DNA nanomachines and a doublestranded body-specific nuclease (DSN)-mediated target cycle amplification strategy.The integration of the 3D DNA nanomachine and DSN-mediated target dual amplification strategy resulted in excellent performance of the developed ECL biosensor for miRNA-21 detection, featuring a wide linear range from 10 fM to 10 nM and an impressively low detection limit of 1.0 fM.This study presented a fresh perspective on the application of DNA walkers in biosensor construction.To further improve the identification and anti-interference capabilities of the sensor, Jia and co-workers designed a biomimetic biosensor, integrating dual-targeted multivalent aptamer/ walker double-stranded functionalized biomimetic magnetic beads with an enzyme-driven DNA walker signal amplification strategy (Figure 4D). 80The magnetic beads were equipped with anti-leukocyte adhesion capability by encapsulating nanomagnetic beads with a leukocyte membrane.This innovative biomimetic biosensor achieved efficient and high-purity enrichment of heterogeneous circulating tumor cells (CTCs), while minimizing leukocyte interference.Furthermore, the capture of target cells triggered the release of walking strands, activating the enzyme-driven DNA walkers and leading to cascade signal amplification for the ultrasensitive and accurate detection of scarce heterogeneous CTCs.It is worth noting that the captured CTCs remained viable and could be successfully recultured in vitro.Overall, this work offered new insights into the effective detection of heterogeneous CTCs through bionic membrane coatings, laying the foundation for early cancer diagnosis.

| APPLICATIONS
2][83] Consequently, the development of biosensors capable of detecting biomarkers with high specificity and low detection limits is of paramount importance. 846][87] Notably, DNA walking nanomachine-based biosensors have exhibited remarkable analytical performance in biosensing applications.DNA walking nanomachines, as an innovative nanotechnology, offer a broad range of applications with a significant area.By combining DNA walking nanomachines with biomolecular recognition sequences, highly sensitive and selective biosensors can be constructed.In this section, we will provide an overview of different types of DNA walking nanomachine-based biosensors, including electrochemical, optical, and other biosensing modalities.

| DNA walking nanomachine-based electrochemical biosensors
An electrochemical biosensor is a device that utilizes electrochemical principles to detect the concentration, activity, or other properties of a target substance through electrochemical reactions.These biosensors offer several advantages, including high sensitivity, fast response time, affordability, and ease of use. 88,89By combining the DNA walking nanomachines with electrochemical biosensors, it is possible to enhance the sensitivity and achieve highly selective detection of various target substances such as biomolecules, pathogens, environmental pollutants, and so on. 90This combination opens up new opportunities in the fields of biomedical research, clinical diagnosis, environmental monitoring, and beyond.In the field of food security, Huang and co-workers developed an autodriven aptasensor based on the DNA walking nanomachines to detect ochratoxin A (Figure 5A). 91A metalorganic frame (MOF) was utilized to load the auxiliary ion Mn 2þ required for the functioning of DNAzyme.By modifying the DNA walking nanomachine and Mn 2þ @MOF on the surface of the gold electrode, this biosensor achieved autonomous movement without the need for external addition of DNAzyme auxiliary ions, enabling immediate ochratoxin A detection.The biosensor demonstrated a sensitivity of 0.289 pg/mL and successfully analyzed real food samples with excellent portability, accuracy, and sensitivity.Furthermore, due to the vulnerability of DNA enzymes to external factors, Wang and co-workers designed a flexible three-legged DNA walker formed by target-initiated CHA, capable of autonomous movement along the DNA double-stranded track on electrodes through toe-mediated DNA strand displacement (TMSD, Figure 5B). 92The design of multi-legged walkers minimized derailment of DNA and reduced movement time on electrodes, ensuring efficient operation.The continuous movement of the three-legged walker was driven by TMSD, eliminating the limitations associated with costly and unstable enzyme-assisted amplification technologies.Consequently, electrochemical biosensors based on three-legged DNA walkers hold great potential for ochratoxin A detection, opening new avenues for food safety analysis and clinical diagnosis.Meanwhile, in the realm of biomedicine, DNA walkers have also received considerable attention from researchers.Guo and co-workers constructed a reproducible electrochemical biosensor for exosome detection using a dual-recognition proximity-binding-induced DNA walker and an "on-off-on" strategy (Figure 5C). 93his study incorporated two proximity probes comprising a Pb 2þ -dependent DNAzyme tail sequence and recognition elements (cholesterol and aptamer).By integrating these proximity probes with the target exosome, a dual F I G U R E 5 DNA walking nanomachine-based electrochemical biosensors.(A) The autonomous driven aptasensor based on DNA walking nanomachine to detect ochratoxin A. Reproduced with permission. 91Copyright 2022, Elsevier.(B) Toehold-mediated DNA strand displacement-driven DNA walking nanomachine to detect ochratoxin A. Reproduced with permission. 92Copyright 2020, Elsevier.(C) A dual-recognition proximity-binding-induced DNA walking nanomachine for exosome detection.Reproduced with permission. 93Copyright 2021, Elsevier.(D) Multiregional linear DNA walking nanomachine-based electrochemical biosensors for ultrasensitive detection of miRNAs.Reproduced with permission. 94Copyright 2022, American Chemical Society.
recognition proximity-binding-induced DNA walker was obtained, converting the exosome into numerous DNA strands.Hybridization induced a conformational change in the hairpin DNA, increasing the distance between the electroactive label and the electrode surface (the biosensor remained in the "off" state).Treatment with nucleic acid exonuclease restored the biosensor to the "on" state.The accurate dual recognition capability, effective amplification of the DNA walker signal, and excellent reproducibility of the "on-off" strategy enabled quantitative detection of exosomes within the range of 5.0 � 10 4 − 1 � 10 8 particles/mL, with a detection limit of 1.6 � 10 4 particles/mL.Overall, this method showed potential in distinguishing exosomes from cancer cells and normal cells, even in complex sample matrices.Besides, in order to increase the walking rate of movement of DNA walkers and ultimately improve their work productivity.Hou and co-workers recently fabricated a multiregional linear DNA walker (MLDW) with a high walking rate and great amplification efficiency for ultrasensitive detection of miRNAs (Figure 5D). 94Notably, the MLDW enrichment of long linear DNAs through RCA mediated by the target miRNAs resulted in a higher localized concentration, collision probability, and significantly accelerated reaction rates.It was noteworthy that the MLDW completed the reaction in less than 30 min, at least four times faster than conventional single-and multi-legged DNA walkers.The electrochemical biosensing platform constructed in this study achieved a detection limit as low as 36 aM.Therefore, the MLDW provided valuable insights into the design of DNA nanodevices and facilitated the study of nucleic acid signaling amplification strategies for potential application in biomolecular detection and clinical disease diagnosis.

| DNA walking nanomachine-based optical biosensors
An optical biosensor is a device that utilizes the interaction and properties of light to detect and quantify diseaserelated substances.Optical biosensors have the capability to identify, localize, and measure target objects by detecting parameters such as light intensity, frequency, scattering, and other pertinent information. 95,96The combination of a DNA walking nanomachine with optical biosensor opens up a plethora of intriguing and innovative applications.Notably, fluorescent optical biosensors are widely employed in this regard.In such cases, the DNA walking nanomachine is labeled with fluorescent molecules that emit discernible signals during its movement.The optical biosensor can effectively capture and record these fluorescent signals, enabling meticulous monitoring and analysis of the position and trajectory of the DNA walking nanomachine.Liang and co-workers developed a 3D DNA walker by employing singlestranded DNA on the surface of DNA nanospheres (DS). 97This unique system, powered by endogenous ATP obtained from living cells, enabled sensitive imaging of miR-21 in the tumor microenvironment (Figure 6A).Upon entering a living cell, the DS walker bound to the intended target, released the walking strand, and initiated an ATP-driven walking response.Consequently, the DS walker emitted a substantial number of Cy3 fluorescent signals that indicated the presence of miR-21, resulting in a significant sensitivity increase (~2.73-fold) and a remarkable reduction in the detection limit (~157-fold).
Importantly, the assembly of the DS walker onto nanoparticles necessitated only a simple hybridization process.Moreover, this endogenous ATP-driven 3D DNA walker allowed real-time in situ imaging of miR-21 in living cells, circumventing signal errors associated with intricate cellular processing and additional cofactors.Furthermore, the DS walker could be employed for intracellular imaging of corresponding biomarkers using complementary aptamers for other biomarkers.As a result, the DS walker proved capable of distinguishing between cancerous and normal cells, presenting an auspicious platform for disease-related biomedical research and clinical applications.Furthermore, in diagnostic oncology studies, tumour exosomes have also been closely correlated with cancer development.Wang and co-workers introduced a self-service track DNA walker (STDW) for the no-wash detection of tumor exosomes. 98y utilizing exosomal glycoproteins as 3D tracks (Figure 6B), the STDW assay achieved direct detection of tumor exosomes in cell culture media and serum owing to its heightened selectivity and sensitivity.Through the self-driven approach, cancer patients could not only be effectively distinguished from healthy individuals, but small differences among clinical samples could also be accurately monitored.This work demonstrated excellent sensitivity, specificity, and accuracy, thereby holding potential for monitoring exosomal markers associated with various diseases.From a specific perspective, ECL biosensors can be categorized as optical biosensors as well.These biosensors utilize an electrochemical reaction to produce optical signals for the detection of biomarkers, providing benefits such as exceptional sensitivity, selectivity, and simplicity. 99Researchers often integrate DNA walking nanomachines into the design of signal amplification strategies in conjunction with ECL technology, further enhancing the sensitivity of these biosensors.In a notable SHEN ET AL. example, Zhang and co-workers utilized aluminiumbased organic nanofibrous gels (AOG) as novel emitters that induced aggregation-induced ECL biosensor (Figure 6C). 100 This biosensor enabled the construction of a rapid and highly sensitive biosensing platform for detecting the influenza A virus DNA, which represented a pivotal biomarker for this virus.Notably, the proposed ECL biosensor leveraged a target-induced pH-responsive rigid DNA walker, based on hydrogen ions generated by loop-mediated isothermal amplification, overcoming limitations encountered by conventional single-or double-stranded DNA walkers in terms of walking trajectory and efficiency due to entanglement.These inverted DNA legs imparted exceptional stability, controllability, and walking efficiency.Consequently, the combination of AOG with its remarkable aggregationinduced ECL performance and nanomachines possessing high walking efficiency and stability resulted in an ECL biosensor with a remarkably low detection limit of 23 ag/μL.Additionally, this strategy provided an invaluable platform for rapid and sensitive monitoring of biomolecules, significantly expanding its potential applications in luminescent molecular devices, clinical diagnostics, and biosensing analyses.Moreover, the DNA walker-based ECL signalling "on-off-on" mode has also attracted significant interest.In the study conducted by Chen et al., biosensors were developed for detecting HBV DNA by merging an ECL signaling switch mode for DNA-specific recognition with a DNA walker-based signaling amplification strategy (Figure 6D). 101This investigation achieved ultrasensitive detection of HBV DNA within a linear range of 100 aM to 10 pM, with a LOD as low as 62.1 aM.Impressively, this innovative approach involved a novel aluminium metal-organic backbone that exhibited excellent ECL properties, characterized by high intensity and exceptional stability even  97 Copyright 2023, American Chemical Society.(B) DNA walking nanomachine-based fluorescent biosensors for tumor exosome detection.Reproduced with permission. 98Copyright 2022, Wiley-VCH.(C) The ECL biosensor with a target-induced pH-responsive DNA walking nanomachine for detecting the influenza A virus DNA.Reproduced with permission. 100Copyright 2022, American Chemical Society.(D) DNA walking nanomachine-based signaling amplification strategy for ultrasensitive detection of HBV DNA.Reproduced with permission. 101Copyright 2023, American Chemical Society.
in assay buffer without the requirement for additional coreactants.Therefore, this work introduced a fresh perspective on co-reactant-free systems within the field of ECL biosensors.

| DNA walking nanomachine-based other biosensors
In addition to the aforementioned biosensors, researchers have devised distinct biosensing strategies based on DNA walking nanomachines.Yang and co-workers engineered a portable aptamer biosensor for rapid detection of aflatoxin B1 (AFB1) in food samples (Figure 7A). 102This biosensor incorporated signal amplification and signal probe release mechanisms through a DNA walker, with results read using a handheld glucose meter (HGM).With a broad detection range from 0.02 to 10 nM and a LOD of 10 pM, this strategy presented portability and costeffectiveness, making it applicable to various analytes and instant detection scenarios.Furthermore, Chen and co-workers developed a portable biosensor for in-situ detection of cardiac troponin I (cTnI) (Figure 7B). 103The biosensor employed an innovative protein-DNA coupling method that offered a time efficiency through the signal amplification function of a DNA walker and the signal reading capability of a low-cost HGM.When the target was present, the DNA walking nanomachine followed a trajectory, liberating short DNA fragments labeled with a convertase enzyme facilitated by a DNA-cleaving nucleic acid endonuclease.The convertase catalyzed the hydrolysis of sucrose into glucose, which was then quantified using the HGM.Under optimal conditions, the LOD for cTnI detection was determined to be 0.001 ng/mL, surpassing the current standard for the detection of acute myocardial infarction (40 pg/mL).This developed biosensor provides a novel approach for cTnI analysis, holding promise for medical diagnosis, biosensors, and other fields requiring on-site and sensitive detection.Moreover, Kang and coworkers designed unique biosensors based on inductively coupled plasma mass spectrometry (ICP-MS) and a cascade 3D DNA walker to achieve quantitative detection of miRNA-21 (Figure 7C). 104In the presence of the target, the cascade DNA walker undergoed catalytic amplification driven by entropy, leading to the release of numerous DNAzyme strands.The DNAzyme autonomously walked along the trajectory on magnetic beads, repeatedly cleaving the DNAzyme substrate.This approach presented a novel concept for constructing highly integrated and efficient cascade DNA walkers for bioanalysis using the ICP-MS strategy.Additionally, Tao and co-workers harnessed the high-resolution capability and efficiency of the DNA walking signal amplification strategy, coupled with the distinct photothermal effects of aggregated and dispersed Au NPs, to fabricate a photothermal biosensor for P53 DNA detection (Figure 7D). 20Upon target recognition, the DNA walking nanomachine was activated, releasing an ample amount of single-stranded DNA that resisted saltinduced aggregation of Au NPs.Leveraging the differential photothermal effects between aggregated and dispersed Au NPs, temperature changes within the reaction system under laser irradiation could be measured using a thermometer, enabling the detection of P53 DNA sequences.This developed photothermal biosensor eliminated the need for sophisticated analytical instruments, exhibiting a low detection limit of 1.4 pM and a linear range of 2.0-120.0pM.Furthermore, owing to the exceptional programmability of the DNA walking nanomachine, this photothermal biosensor design could be adapted to detect various targets by modifying other DNA sequences.

| SUMMARY AND OUTLOOK
In recent years, the progress of DNA walking nanomachines has garnered increasing attention from relevant researchers.Diverse strategies have been employed to fabricate DNA walker-based biosensors, showcasing exceptional capabilities across multiple fields such as electrochemical biosensors, optical biosensors, and other sensing applications.The merits associated with DNA walker-based biosensors can be summarized as follows (Table 2): Firstly, DNA walking nanomachines operate at the nanoscale, allowing precise manipulation and control of substances using DNA molecules.Secondly, DNA walking nanomachines can be constructed through selfassembly, simplifying their design and construction by utilizing complementary base-pairing principles.Thirdly, the behavior of DNA walking nanomachines can be programmatically controlled through the design of DNA sequences, enabling gait control, direction control, and interactions with other molecules.Fourthly, the movement of DNA walking nanomachines can be controlled by external stimuli, providing high controllability for precise manipulation and functionality.Lastly, DNA walking nanomachines can be designed with various functions, making them versatile in areas such as nanotechnology, biomedical sciences, and biosensing analysis.Therefore, DNA walking nanomachines possess nanoscale capabilities, self-assembly, programmability, controllability, and multifunctionality, making them promising molecular nanomachines in the fields of nanotechnology and biological sciences.
The DNA walker, a DNA-based nanomachine capable of movement and performing specific tasks on DNA nanostructures, holds immense potential in the field of nanotechnology.However, the DNA walking nanomachine still faces many challenges as follows: Firstly, Gait control is crucial for the movement and task execution of DNA walking nanomachines, which involves designing primers and auxiliary molecules to ensure precise movement along the desired path under specific conditions.Secondly, the accuracy and reliability of DNA walking nanomachines are important research goals.Accordingly, environmental factors such as temperature, pH, and ion concentration can affect the stability of DNA molecules, impacting the movement and task execution of DNA walking nanomachines.Thirdly, efficacy and efficiency are critical indicators of DNA walker performance.They need to complete tasks within a reasonable timeframe, demonstrating the high efficiency.Enhancing their efficacy and efficiency requires optimizing design and operation to improve moving speed and accuracy.Fourthly, the application and integration of DNA walking nanomachines pose significant challenges.These nanomachines are employed in drug delivery, molecular manipulation, and biosensing platforms.Applying them to practical scenarios requires addressing application-specific issues and integrating them with other nanotechnologies or biotechnologies.Therefore, the research on DNA walking nanomachines necessitates addressing challenges in design and construction, gait control, accuracy and reliability, efficacy and efficiency, as well as applications and integration.Overcoming these challenges requires interdisciplinary collaboration, combining theoretical research and experimental validation, to drive the development and application of DNA walking nanomachines.
In conclusion, leveraging the advancements in selfassembling strategies for DNA nanostructures, a plethora of DNA walking nanomachines have emerged across diverse fields such as electrochemical biosensors, optical biosensors, and other sensing disciplines.Promising prospects lie ahead for pioneering advancements in the design and implementation of DNA walker-based biosensors, offering substantial opportunities for transformative breakthroughs.

F I G U R E 4
DNA walking nanomachines with various walking tracks.(A) DNA walking nanomachine with 1 D walking track.

F I G U R E 6
DNA walking nanomachine-based optical biosensors.(A) DNA walking nanomachine-based fluorescent biosensors for insitu imaging of miR-21 in living cells.Reproduced with permission.

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I G U R E 7 DNA walking nanomachine-based other biosensors.(A) DNA walking nanomachine-based portable aptamer biosensor for rapid detection of aflatoxin B1.Reproduced with permission. 102Copyright 2018, American Chemical Society.(B) DNA walking nanomachine-based biosensor for in-situ detection of cTnI.Reproduced with permission. 103Copyright 2023, Elsevier.(C) The 3D DNA walking nanomachine-based inductively coupled plasma mass spectrometry for the quantitative detection of miRNA-21.Reproduced with permission. 104Copyright 2021, Elsevier.(D) DNA walking nanomachine-based photothermal biosensor for P53 DNA detection.Reproduced with permission.20Copyright 2020, Elsevier.SHEN ET AL.

T A B L E 1 Summary of driving forces for DNA walking nanomachines. Driving process types Driving factor types Walking tracks Applications Ref.
Summary of DNA walker-based biosensors applied in detecting biomarkers.