Relying on Watson-Crick hydrogen bonds, the strand displacement reaction as a key driving force, could enable the walker to govern and control strand hybridization [50]. Through toeholds-mediated strand displacement reaction, the walker could displace the prehybridized strands towards branch migration, initiating the autonomous and progressive walking. Considering that the DNA sequence length and toehold complementary regions can be altered, the strand displacement reaction-based walkers are designed in a variety of models.

It was well-known that Pierce's group first presented the concept of DNA walker in 2004 [35], through designing a synthetic transport device which could mimic the movement of motor proteins. This bipedal DNA walker provided by Pierce's group realized real-time monitoring of walker movement via multiplexed fluorescence quenching. As shown in Fig. 2A, the DNA walker was attached to tracks by the attachment fuel strands (A1 and A2), and moved along tracks under the external control of detachment fuel strands (D). When the D1 hybridized with A1 by the toeholds-mediated strand displacement reaction, the walking leg was released and reattached to the adjacent tracks, resulting in moving along the tracks. Through the progressive movement, this DNA walker achieved the energy conversion by strand displacement reaction with no enzymes. In addition, Xiang's group [51] described a DNA-fueled machine for monitoring low-expressed microRNA (miRNA) in live cells (Fig. 2B). In the presence of target miRNA, the walker could release hairpin sequences from the dsDNA-conjugated gold nanoparticles. Through two cascaded strand displacement reactions, the DNA fuel strands assisted the recycling of the target miRNA sequences, resulting in the signal amplification. Obviously, this persistent walking mode driven by the strand displacement reaction required the addition of specific fuel strands, which leaded to the stable "trap state" formation. To prevent "trap states" formation, a metastable hairpin structure was provided as a fuel strand to propel the walker operation. Ellington's group [12] reported a stochastic DNA walker that traversed a microparticle surface (Fig. 2C). Through catalytic hairpin assembly to a microparticle surface, this novel DNA walker could be triggered by a ssDNA catalyst, due to the fact that the ssDNA could open the hairpin via toehold-mediated strand exchange, then the opened H1 hybridized to a toehold on H2, initiating branch migration. This robust walker could realize more than 30 continuous steps movement. Subsequently, as shown in Fig. 2D, Ellington's group [52] further reported a processive walker relying on a surface-immobilized proximity catalytic hairpin assembly. H1 was modified with biotin for the link to a streptavidin-coated microparticle. Meanwhile, H2 was designed as "fuel" strand that drove the moving. Spontaneous hybridization between H1 and H2 provided the potential of the walker to readily search out adjacent substrates. In conclusion, different from enzymatic process, strand displacement reaction could alter the sequence length and toehold complementarity regions, holding the capability to control and engineer the intelligent DNA walker.

Protein enzyme and DNAzyme are ideal driving forces for the DNA walker [53–56]. With unique specificity and high efficiency, protein enzyme was widely used in biosensors, which could catalyze the breakage of polynucleotide chains by recognizing particular nucleotide sequences [53,54]. Similarly, DNAzyme is a specific DNA sequence related enzymes activity showing strong specificity and high catalytic efficiency for catalyzing the hydrolysis of particular nucleic acid substrates [55,56]. Unlike the protein enzyme denatured by environmental influence, DNAzyme is stable, programmable and modifiable. Overall, either protein enzyme or DNAzyme has its pros and cons. Nowadays, based on the enzymatic hydrolysis reaction, the protein enzyme and DNAzyme were used to be driving forces to power the DNA walker operation. In terms of operation efficiency, DNA walkers powered by the enzyme/DNAzyme presented approximately three orders of magnitude quicker than conventional walkers relying on hybridization reactions.

As shown in Fig. 4A, the Willner's group [43] described a DNA bipedal walker consisted of four nucleic acid footholds, activated by Hg²⁺/cysteine and H⁺/OH⁻ triggers, striding along a 1D track. To trace moving motion, each of the footholds was labelled with fluorescence dyes, F1 (Cy5), F2 (Cy3), F3 (Cy5.5), and F4 (ROX). Meanwhile, the arm of the walker was modified with the quencher units Q (Q1, Q2) at the 5′ and 3′ ends. Under appropriate conditions, through the binding and release between the walker and the tracks, the bipedal walker could realize the mechanistic operation, and monitor the operation of the motion. Mao's group [38] developed a DNAzyme motor, transporting a nanoparticle cargo processively along a 1D RNA-coated carbon nanotube track. By rational design of the DNA walker, we explored the critical parameters which affected the translocation kinetics, including upper and lower recognition arm lengths, DNAzyme catalytic core type, and different divalent metal cations. Compared with the traditional free DNA probe limited by a homogeneous environment, the 1D DNA walker could detect the extremely low targets, by the amplification of detection signal.

Different from 1D tracks only possessing single direction, a 2D DNA walker possessing a planar structure has more extensible directions and rigid backbones, providing a wider range of freedom for movement of the 2D DNA walker on the DNA tracks. To control the movement, the signal molecules were labeled at the selected spots, providing a deeper understanding of intermolecular interactions.

2D DNA origami technology broke the design and construction of the 2D nanomaterial limited by the size and inerratic shape, attracting wide attention. Similar to the Seaman's DNA tiles, the DNA origami is a structure constructed by fixing crosses or junctions between DNA helices. Through many short "staple" chains for assisting the folding of a long "scaffold" DNA strand, the 2D DNA origami could present arbitrarily complex shapes such as triangle, square, rectangle and so on. As shown in Fig. 4B, Nir's team [63] designed a bipedal DNA walker which strode on a 2D DNA origami track with the help of DNA fuel and antifuel strand commands. With accurate speed and direction control, 2D DNA origami could be precise design, and this DNA walker realized 32 walking steps on a DNA origami track. This 2D DNA interface provided potential to reliably, progressively, and reproducibly control DNA walkers. Tan's group [64] applied various DNA origami to directly observe photoinduced movements of single molecules. And the stepwise of the pyrene-modified DNA walker was captured in time-lapse image. Powered by light, this DNA walker could move along a linear track on a single 2D DNA tile. Furthermore, the DNA walker also could be considered as signal amplifier. Tinnefeld's group [40] developed a nicking enzyme-initiated DNA walker moving on a rectangular DNA origami substrate, improving the detection of target molecules. Therefore, this system could be regarded as step counter or fluorescence integrator.

Despite computational, flexible and addressable functions, the DNA origami is always limited by the high cost, harsh synthesis and operating conditions. Consequently, developing other 2D materials as the tracks to improve the walking surface area and working condition are significantly important. Electrodes as prominent 2D plane have been applied to be tracks for DNA walkers. As shown in Fig. 4C, Yuan's team [65] developed a super-sensitive electrochemiluminescence (ECL) biosensor for miRNA-21 detection, relying on a DNA walking machine with triple-stranded DNA (tsDNA) nanostructures as tracks. Benefiting from the tsDNA nanostructures modified on the electrode interface averting probe entanglement and decreasing local overcrowding effects, this ECL biosensor could detect the miRNA-21 with a low detection limit of 6.67 amol/L. In addition, researchers developed some walkers based on toehold-mediated strand displacement amplification reaction. Xu's group [66] combined gold nanofilms as novel surface-enhanced Raman scattering (SERS)-electrochemistry active substrates to develop a DNA walker for dual-mode measurement of miRNA. Triggered by target miRNA and fueled by signal DNA strands labeled with rhodamine6G (R6G), this walking machine realized an enzyme-free amplification detection of miRNA with a low limit of detection. As shown in Fig. 4D, to improve the walking efficiency, Yuan's group [67] reported an intelligent Zn²⁺-driven DNA rolling machine constructed by numbers of leg DNA modified on Au nanoparticle, which matched with their tracks on the glassy carbon electrode. Through increasing the concentration of the leg DNA and track DNA, the walking efficiency of this walker could be improved, reducing the derailment of leg DNA. The highly speedy walking machine achieved signal amplification and provided good application in ultrasensitive strategy of miRNA detection. Therefore, the DNA walker based on 2D tracks opened a new avenue for sensitive detection of diverse biomolecules with signal amplication.

Up to now, the walkers that walked along 1D or 2D tracks showed excellent application in biosensor and biomedicine with signal amplification. Compared with 1D and 2D tracks, the 3D tracks constructed by the AuNPs, upconversion nanoparticles (UCNPs), microspheres, magnetic nanoparticles, and 3D origami possessing abundant specific surface area allowed the walker assembled more tracks on their surface and exhibited high walking efficiency.

Based on the origami technique, the DNA origami could be designed as 3D scale. Liu's group [68] demonstrated a plasmonic system, in which a gold nanorod could perform directional, progressive and reverse nanoscale walking on 2D or 3D DNA origami. When the walking machine carried out stepwise movements along the 3D double-layer DNA origami, the walking steps could be real-time monitored by optical spectroscopy. This walker provided another way to develop DNA walker that showed directional and progressive walking.

Besides 3D DNA origami, the AuNPs taking the advantage of unique optical property and versatile biofunctionality, have been increasingly used as good conjugated-nanomaterials for DNA walker. Tan's team [55] demonstrated a Na⁺-specific DNAzyme-based DNA motor with AuNPs tracks, which could differentiate cell subtypes by simultaneously sensing miRNA-21 and miRNA-205 in live cells (Fig. 4E). Benefiting from sufficient intracellular Na⁺, this miRNA-initiated Na⁺-fueled DNAzyme motor avoided the adding external metallic cofactors, showing the potential application in cancer clinical diagnosis and prognosis.

UCNPs, possessing several unique features and presenting emission in the visible region upon irradiation with NIR light through a multiphoton mechanism, have been widely applied in biosensing and bioimaging. Sun's group [69] reported an upconversion luminescence-initiated DNA walker, that was based on a 808 nm near-infrared light-excited low-heat upconversion nanoparticle (Fig. 4F). This light-controlled target could improve the sensitivity and specificity, due to the pre-triggering of the biosensor during the biological delivery. This DNA walker demonstrated an outstanding sensitivity with the detection limit of 0.51 pmol/L, showing the potential to distinguish single-base mismatching.

Streptavidin-coated microspheres having a spherical surface also could be regarded as nice 3D tracks. Through the streptavidin-biotin bind, the DNA strands could be modified on the surface of the streptavidin-coated microspheres. Xie's group [70] reported nicking endonuclease-powered 3D bipedal DNA walkers (BDW). The polystyrene microsphere was assembled with bipedal DNA walkers and fluorescence-labeled DNA strands. This BDW showed faster walking speed than single-legged DNA walker, and 70 min is enough for the reaction time. Meanwhile, this DNA walker have a wide linear range from 10 fmol/L to 5 nmol/L with a low detection limit of 5.2 fmol/L. Currently, Chu's group [71] also reported a DNAzyme walker, which was constructed by several DNAzyme walking strands and abundant FAM-labeled substrate strands on the surface of AuNP. This DNA walker could progressively walk along the tracks powered by DNAzyme-catalyzed substrate cleavage and realize intracellular miRNA image.

Magnetic nanoparticles were a series of nanoparticles controlled by magnetic fields, and be developed their application in medical diagnostics, imaging, drug and gene delivery. Particularly, the magnetic nanoparticles could be used in magnetic particle imaging (MPI) technology, which allowed excellent spatial and temporal resolution to enable targeted contrast enhancement and real-time assistance during medical interventions. Liu's group [72] reported a versatile single magnetic nanoparticle-confined, click chemistry actuated digital DNA walker for miRNA detection, which was fixed one DNA walking leg on a single magnetic nanoparticle. This DNA walker provided less than a 100-round walking efficiency of each swing arm, resulting in recovering sufficient fluorophores on the magnetic nanoparticle, achieving digital miRNA quantification.

Electrochemical biosensors were high-profile biosensors, because of their unique features, including high detection sensitivity, excellent specificity, fast response, easy miniaturization and low-cost instruments.

As is well known, the general electrode interface could be modified with single-stranded DNA or double-stranded DNA. Xu's group [73] developed a DNA walking machine based on ECL aptasensor to quantitatively sense tumor exosomes. The DNA walking machine was constructed by the two types of DNA. One was anchor DNA as a track including nicking enzyme recognition sequences (GCTGAGG) on the loop of a hairpin strand. And another one was the swing arm-CD63 aptamer double strand. In the presence of exosomes, CD63 aptamer in the swing arm-CD63 aptamer double strand could conjugate with the CD63 protein on the exosome surface, realizing the release of CD63 aptamer and the freedom of the swing arm. Then, anchor DNA could bind with the free swing arm, generating the nicking endonuclease cleavage spot. And the movement of the DNA walking machine exhibited high controllability, providing feasible detection of exosomes and outstanding identification of different cell lines. To improve the walking efficiency, as shown in Fig. 5A, Yuan's group [74] reported an unconfined DNA walker based on a DNA three-way junction with multiple recognition regions to detect miRNA-182–5p, possessing rapid walking speed and high sensitivity. The hairpins on this DNA walker would be successively opened by target miRNA to produce an annular DNA walker, which operated on the entire scope of the electrode surface without the confine for the length of DNA walker legs, improving the walking efficiency. Wang's group [75] reported a multipedal polydopamine nanoparticles–DNA (PDANs-DNA) walker with electrochemical-driven metal–organic frameworks (MOFs) conversion-enabled biosensing platform (Fig. 5B). This walking machine has high walking efficiency due to the fact that this DNA walker with multiple legs walked fast powered by exonuclease Ⅲ driving, forming DNA dendrimers through two hairpins assembly. This walker with multiple legs realized miRNA-21 detection ranging from 10 amol/L to 10 pmol/L. Besides, Lei's group [76] reported a multipedal DNA walker, which had multiple DNAzyme cores via rolling circle amplification. This walker was constructed by two components, the swing strand and the track. The swing strand was prepared by a padlock, an RCA primer and a block DNA as a recognition element in the DNA walker system. Meanwhile, the tracks fixed on a gold electrode, was labeled with ferrocene as signal output. Once the target DNA and an RCA kit were presented in system, RCA reaction could be activated and produce multiple DNAzyme strands for the cleavage of the substrates. Compared with a single and a bipedal DNA walking machine, the multipedal DNA walking machine showed a rapider cleavage speed and a lower ratio of the swing strand to the track. Despite sensitive and controllable, the above modification methods of single-stranded DNA or double-stranded DNA conjugating on the electrode suffered from local overcrowding effects and probe entanglement. To improve the steric hindrance effect, in Fig. 5C, Miao's group [77] took the tetrahedral DNA as track modified on the electrode, which could avoid spacer molecules for electrode surface modulation and enhance molecular recognition efficiency of the supported DNA track. Meanwhile, this team designed a bipedal DNA walker for electrochemical genosensing, with ultrahigh sensitivity and specificity. This proposed DNA walker provided a novel pathway to design the bipedal DNA walker with signal amplification.

Despite high detection sensitivity, excellent specificity, fast response, easy miniaturization and low-cost instruments, the electrochemical biosensors suffering from poor cell compatibility were not appropriate to be applied in vivo. In consequence, it is of great significance to explore other methods used in vivo. Fluorescence is another signal output mode, which was widely applied to design biosensor. Due to the excellent biocompatibility, good specificity, extremely high sensitivity, and amplification capabilities, DNA walker-based fluorescent biosensors could realize in situ real-time biomolecules image in cells. Currently, these biosensors have been widely applied for the measurement of enzymes, pH, nucleic acids and so on [78,79].

Enzymes with catalytic effects are produced by living cells. Nowadays, some studies have reported that DNA walker-based fluorescent biosensors could achieve enzymes image in living cells. As shown in Fig. 6A, Lei's group reported a telomerase-triggered 3D DNA walker, which employed the Cy5 as the signal output [80]. The walking strand of this walker was prepared by a superhairpin structure with a bulged loop in the stem. And the dye-labeled nicking endonuclease substrates as tracks. Once the telomerase in the system, the superhairpin structure could be open, and expose the binding site of the nicking endonuclease. Then, a swing arm with the open superhairpin structure could bind with the track, achieving the fluorescence recovery by Cy5-labeled fragments released from the surface of gold nanoparticles. This proposed walker provided a new platform for the telomerase detection and broadened the design of DNA walker.

Furthermore, the DNA walker-based fluorescence biosensor was also used to apply in the detection of nucleic acids. Chu's group [71] reported a DNAzyme-based walker, initiated by intracellular miRNA-21. This DNA walker assemble DNAzyme walking strands and FAM-labeled tracks on the surface of AuNPs. Once this DNA walker entered in living cells, it could autonomously walk along the tracks fueled by DNAzyme-catalyzed substrate cleavage, and company with the FAM-labeled DNA strand release by the cleavage of substrate strand. This DNA walking machine allowed real-time operation monitor, and held the potential in the miRNA associated biological research and disease diagnostics. Furthermore, Chu's group [81] developed a photocontrolled DNA walker with bipedal DNAzyme walkers for intracellular miRNA image (Fig. 6B). Compared with a single walking machine [71] with a long operating time (180 min), this bipedal DNA walking machine enhanced the walking machine and decreased the operating time (60 min), because of the increased number of walkers moving along the tracks. At the same time, this bipedal DNAzyme walker with a lower detection limit (3.51 pmol/L) exhibited high sensitivity and specificity. Beside the DNAzyme powered DNA walker, some researchers also developed DNA-fueled and catalytic molecule machinery. Wu's group [82] reported a self-protected DNAzyme walker capable of fully stepping on the substrate-modified AuNP for miRNAs image in live cells, exhibiting more than 8-fold enhanced catalytic activity and 5-fold enhanced serum resistance. The cleavage activity of the DNAzyme was pre-inhibited, because the catalytic core of DNAzyme walker was separated by a target binding domain. And the catalytic activity of DNAzyme walker was activated by target miRNA, resulting in the cleavage of substrates and enhancement of fluorescence signals.

Besides the above application, pH is also identified as a trigger to activate the DNA walker operate. Chao et al. [29] developed a pH-responsive DNA walker to perform multilayer DNA cascades, achieving calculation of intracellular biomolecules and precise sensing (Fig. 6C). Based on sequence tuning, this DNA walker could precisely respond to intracellular pH variations, because the Au nanoparticle was assembled by four-stranded DNAzyme walker precursor with a pH-responsive DNA switch, which was sensitive to pH variation of 5.0–7.0 by comformational change from linear to triplex. After the walker enter in cells, the DNA walker pass through intracellular lysosomes with acidic pH and become unlocked, enabling intracellular target miRNA image. This strategy applied an sensing-computing-output functional integration, promoting the application of programmable nanomachines in chemistry, biomedicine, and nanoengineering.

Furthermore, the DNA walker-based fluorescent biosensors were also used to detect bacteria, virus, and other micromolecule. Pei's group [61] reported stochastic DNA walkers, realizing ultrahigh-throughput bacterial detection. Through embedding intensity coded barcodes into a sequence of color-multiplexed barcodes, this multiple-step DNA walker provided a super-multiplex droplet-encoding strategy, and identified 20 distinct bacterial phenotype. By mixing encapsulated bacteria in droplets, the DNA walking machine could be triggered and the detection signals were amplified. This method provided digital and independent tuning of color and intensity, by combining with controlled ratios and amounts. And the coding capacity would grow exponentially with the increase of the number and intensity levels of the colors. This super-multiplexed and high-throughput biological assays provided enormous potential in multiple biological detection. Human immunodeficiency virus (HIV) is a virus that leads to AID by attacking the body's immune system. It is of great significance to sensitively detect HIV. Huang et al. [83] present an exonuclease Ⅲ-propelled stochastic DNA walker for signal amplification to detect HIV DNA, achieving the detection range of 0.05–1.2 nmol/L. The AuNPs were modified with fluorescent dye-labeled hairpin DNA. The target HIV DNA can initiate the DNA walker operating powered by Exo Ⅲ, generating "one-to-more" amplified fluorescence signals. This DNA walker presented good explanation of the intricate interactions among enzymes, DNA and nanoparticles, showing high promise in disease diagnosis. In addition, the DNA walker is also used to measure other micromolecules. Kanamycin as an important aminoglycoside antibiotics, has been extensively applied in the bacterial infections, pneumonia, and mastitis, because of a strong inhibiting capacity against Gram-negative bacteria. Yu's group [84] constructed an Endo Ⅳ (endonuclease Ⅳ)-powered DNA walker for ultrasensitive Kanamycin detection. The Kanamycin could bind with its aptamer, which was blocked by the complementary strand containing the Endo-Ⅳ recognition site. Through Endo Ⅳ-mediated hydrolysis of the anchorages, this DNA walker could move progressively and autonomously along 3D tracks. The cleavage fragment would be an initiator to induce the HCR reaction and Endo Ⅳ-assisted cyclic cleavage of fluorescence reporter probes. This DNA walker based on HCR amplification applied dual-amplified strategy, achieving ultrasensitive and accurate strategy to detect Kanamycin, providing effective platform for tracing amounts of analytes in molecular and bioanalysis diagnosis. Besides, the adenosine triphosphate (ATP) became an initiator to trigger the DNA walker. As shown in Fig. 6D, Xian et al. [85] reported a NIR-photoactivatable DNA walker powered by ATP for intracellular miRNA image with temporal and spatial resolution. In this system, UCNPs were employed to be the carriers of the DNA probe and could convert NIR to UV light. And the DNA walker could be initiated by the NIR light, fueled by intracellular ATP, achieving autonomous and progressive moving along the 3D-based track. This strategy realized intracellular miRNA image in various cell lines. Recently, tumor-derived exosomes have been attracted extensive attention, as the exosomes could be biomarkers for cancer diagnosis, due to multiple proteins on the surface of exosomes. Su's group [54] reported an exosome-responsive DNA walker, by consisting of aptamer-locked motor strands and fluorescein-labeled substrate strands. Through the recognition between the target protein on exosome and its aptamer modified on the gold nanoparticle, the DNA walker triggered by exosome could be propelled by restriction endonuclease, enabling the autonomous walking along the gold nanoparticle. Each walking step along with the cleavage of the fluorescein-labeled substrate displayed a low detection limit of 8.2 particles/µL in PBS. This proposed strategy showed great potential for developing a simple and cost-effective DNA walker for clinical diagnosis. Despite recognition of the proteins and small biomolecules, researchers also explored some DNA walkers to measure some metal ions. He's group [86] reported a programmable target-initiated DNAzyme walker with rationally designed DNAzyme that was triggered by adenosine, Ag⁺, and target DNA sequence and so on.

In addition to electrochemical and fluorescent method, some DNA walkers based on other signal readout such as colorimetric patterns [87], mass spectrometric [88] and SERS [89] were presented, providing new avenues to integrate intelligent and multifunctional DNA walkers.

The DNA walkers based on the colorimetric assay were developed to detect biomolecules. As shown in Fig. 7A, Liang's group [87] described colorimetric assay based on Au-Fe₃O₄ nanocomposite, which was modified with hundreds of DNA tracks and dozens of blocked walking strands. This DNA walker could be powered by DNAzyme cleavage, and activated by the miRNA-155, producing a great deal of single-stranded fragments (displaced ssDNA). After magnetic separation, displaced ssDNA in the solution could protect AuNPs from aggregation in high-salt solutions. The miRNA-155 could be determined by UV–vis spectroscopy, and the solution color would change from grayish black to purple. This facile, sensitive and specific colorimetric assay achieved the miRNA-155 detection in real human serum samples without complicated sample pretreatment.

Mass spectrometry as a superior accurate analytic technique for ratiometric measurements, showing a mass spectrum that plots the mass-to-charge (m/z) ratio of compounds in a mixture, has been extensively applied in metal stable isotope tagging. As shown in Fig. 7B, Liu et al. [88] developed a ratiometric DNA walker based on the elemental isotope ratio measurement for the detection of target DNA in human serum and cell lysates. Through a burnt-bridge mechanism, this Endo Ⅳ powered DNA walker could convert and amplify biological signals into mass spectrometric ratio signals (¹⁹⁷Au/¹¹⁵In). This target-triggered DNA walker could autonomously walk along the 3D-AuNPs. Then magnetic microparticle was used to separate the AuNPs after hybridization of DNA tracks and the target DNA. This strategy successfully converted the output signal to a mass spectrometric signal for ratio analysis. This proposed DNA walker provided a versatile platform for small biomolecules and proteins measure.

Additionally, SERS possesses the advantages of extremely narrow peak width, high sensitivity multivariate detection ability and wide dynamic range (Fig. 7C). Recently, Duan's group [89] developed a 3D DNA walker based on the SERS strategy for quantitative analysis of Salmonella typhimurium (S.ty). This DNA walker taking AuNPs modified magnetic nanoparticles as 3D walking surface, could be triggered by the complementary DNA of S.ty-recognizing aptamer replaced from the double-stranded DNA (dsDNA) of Apt@cApt reacting with S.ty. Under the driving force of nicking endonuclease, this system could induce the SERS signal. This DNA walker showed highly selective and sensitive method for the S.ty detection, achieving the signal amplification, presenting new methods for SERS assay of biomaterials.