Thrombin Aptamer Research Articles

Platform- and label-free detection of lead ions in environmental and laboratory samples using G-quadraplex probes by circular dichroism spectroscopy

Guanine-rich quadruplex (G-QD) are formed by conversion of nucleotides with specific sequences by stabilization of positively charged K+ or Na+. These G-QD structures differentially absorb two-directional (right- and left-handed) circularly polarized light, which can discriminate the parallel or anti-parallel structures of G-QDs. In this study, G-QDs stabilized by Pb2+ were analyzed by a circular dichroism (CD) spectroscopy to determine Pb2+ concentration in water samples. Thrombin aptamer (TBA), PS2.M, human telomeric DNA (HTG), AGRO 100, and telomeric related sequence (T2) were studied to verify their applicability as probes for platform- and label-free detection of Pb2+ in environmental as well as laboratory samples. Among these nucleotides, TBA and PS2.M exhibited higher binding constants for Pb2+, 1.20–2.04 × 106/M at and 4.58 × 104–1.09 × 105/M at 100 micromolar and 100 mM K+ concentration, respectively. They also exhibited excellent selectivity for Pb2+ than for Al3+, Cu2+, Ni2+, Fe3+, Co2+, and Cr2+. When Pb2+ was spiked into an effluent sample from a wastewater treatment plant (WWTP), its existence was detected by CD spectroscopy following a simple addition of TBA or PS2.M. By the addition of TBA and PS2.M, the Pb2+ signals were observed in effluent samples over 0.5 micromolar (100 ppb) concentration. Furthermore, PS2.M caused a Pb2+-specific absorption band in the effluent sample without spiking of Pb2+, and could be induced to G-QD structure by the background Pb2+ concentration in the effluent, 0.159 micromolar concentration (3.30 ppb). Taken together, we propose that TBA and PS2.M are applicable as platform- and label-free detection probes for monitoring Pb2+ in environmental samples such as discharged effluent from local WWTPs, using CD spectroscopy.

A Heparin based dual ratiometric sensor for Thrombin

Thrombin is an important enzyme that plays a pivotal role in the blood clotting pathways. An imbalance in the activity of this enzyme is clinically known to be associated with various diseases, such as thrombosis, inflammation, atherosclerosis, and haemophilia, suggesting the need to devise sensors for Thrombin detection. However, the majority of the fluorescence-based Thrombin assays rely on fluorescence labelling assays or Thrombin specific recognition biomolecules, such as, aptamers or antibody which requires sophisticated techniques and makes it very expensive. Herein, we report a simple, selective, sensitive and label-free fluorescence detection scheme for Thrombin which is based on the interaction between Thrombin and a fluorescent complex of Heparin with a molecular rotor dye, Thioflavin-T. The detection scheme exploits selective interaction between cationic Thrombin and anionic Heparin to modulate the monomer-aggregate equilibrium of the Thioflavin-T–Heparin system. Importantly, the present system offers a ratiometric response that has the ability for robust quantification of Thrombin concentration even in complex medium. The involvement of all commercially available components is a crucial advantage of this detection scheme. Further, the detection scheme also shows reasonable response in diluted serum matrix.

Electrochemical sandwich-type thrombin aptasensor based on silver nanowires& particles decorated electrode and the signal amplifier of Pt loaded hollow zinc ferrite

An ultrasensitive enzyme-free electrochemical sandwich-type thrombin aptasensor is proposed in this work, with the amplifier of Pt loaded hollow zinc ferrite nanospheres. Silver nanowires& particles (AgNWs&PCs) are used as the substrate materials to decorate the working electrode. Silver particles (AgPCs) are introduced for welding of AgNWs to further improve the electrical performance of the working electrode. The conductive electrode decorated with AgNWs&PCs can provide a substrate for the immobilization of thrombin aptamer 1 (Apt1). Moreover, AgNWs&PCs possess the catalytic activity for H2O2 reduction, contributing to the signal amplification. The prepared Pt loaded hollow zinc ferrite nanospheres with an excellent electrocatalytic activity for H2O2 reduction can also be used to immobilize aptamer 2 (Apt2). Under the optimized condition, the performance of the biosensor is evaluated by amperometry. The proposed thrombin aptasensor has a wide linear detection range from 0.05 pM to 35 nM and a low detection limit of 0.016 pM (S/N = 3), due to the synergistic effect of AgNWs&PCs electrode and the Pt loaded hollow zinc ferrite nanospheres amplifier. This aptasensor displays high sensitivity, good reproducibility and stability, providing a promising platform for the analysis of thrombin.

Self-assembled perylenetetracarboxylic acid-reduced graphene oxide film for high-sensitive impedimetric determination of thrombin

Graphene-based thin film has been widely used as signal conductor and platform material in electrochemical sensors. For commonly used reduced graphene oxide (rGO), oxygen-containing functional groups can provide coupling sites to conjugate with bio-cognitive elements, but high degree of oxidation can harm its structural integrity and signal conductivity. In this work, a new strategy is developed to balance the conductivity and functionality of rGO. The PTCA-rGO composite film was prepared by self-assembly and used to fabricate electrochemical biosensor. To form reusable thrombin aptasensor, an in situ layer-by-layer self-assembling method was adopted to stepwise deposit reduced graphene oxide (rGO), 3, 4, 9, 10-perylenetetracarboxylic acid (PTCA), and thrombin aptamer (TBA) onto 3-aminopropyltriethoxy silane (APTES)-modified glassy carbon electrode (GCE). The as-prepared aptasensor was used to determine thrombin in response to impedimetric signal by electrochemical impedance spectroscopy (EIS). The as-prepared GCE/rGO/PTCA/TBA sensor provided outstanding performance for determination of thrombin concentration with wide linear range (1 pM-100 nM), low detection limit (0.2 pM), reusability, and good specificity to thrombin. The good performance of GCE/rGO/PTCA/TBA aptasensor can be attributed to the electrochemical property of rGO-PTCA composite film and the in situ layer-by-layer fabricated strategy. This PTCA-modification method is promising to be widely used in the fabrication of graphene-based electrochemical devices.

A Stable Biotin-Streptavidin Surface Enables Multiplex, Label-Free Protein Detection by Aptamer and Aptamer-Protein Arrays Using Arrayed Imaging Reflectometry.

While label-free multiplex sensor technology enables “mixing and matching” of different capture molecules in principle, in practice this has been rarely (if ever) demonstrated. To fill this gap, we developed protocols for the preparation of mixed aptamer-protein arrays on the arrayed imaging reflectometry (AIR) sensing platform using streptavidin as a common attachment point for both biotinylated proteins and aptamers. Doing so required overcoming the noted instability of dried streptavidin monolayers on surfaces. After characterizing this degradation, stable surfaces were obtained using a commercial microarray product. Microarraying through the layer of stabilizer then provided mixed aptamer-antibody arrays. We demonstrate that sensor arrays prepared in this manner are suitable for several probes (thrombin and TGF-β1 aptamers; avi-tagged protein) and targets.

A simple label-free photoelectrochemical aptasensor for ultrasensitive detection of thrombin

This work demonstrates a label-free photoelectrochemical (PEC) aptamer sensor based on poly(5-formylindole)/Au (P5FIn/Au) nanocomposites for the ultrasensitive determination of thrombin (TB). As a photosensitive material, P5FIn exhibits excellent photocurrent response under visible light irradiation, and can capture more thrombin aptamers (TBA) due to large specific surface area and surface-rich aldehyde group. In addition, the aldehyde groups of P5FIn are conducive to the stable and efficient immobilization of aptamers or bioactive molecules, enhancing their biological activity. Au increases the absorption of visible light and effectively inhibits the recombination of photogenerated electron-hole pairs, thereby improving the photocurrent conversion efficiency of P5FIn. Therefore, after TB is specifically identified, the photocurrent decreases linearly. Under optimal conditions, this PEC aptasensor has a linear response ranging from 0.001 to 10 nM with detection limits as low as 0.2 pM. This sensor also has good selectivity, repeatability, stability and simple construction process. The determination results of TB in real human serum samples are also satisfactory, which will provide a practical PEC platform for the rapid detection of proteins in the future.

Visible light driven photoelectrochemical thrombin aptasensor based on uniform TiO2 nanotube arrays modified with CuOx and perylene-3,4,9,10-tetracarboxylic acid

In this work, a sensitive signal-off photoelectrochemical (PEC) thrombin aptasensor with satisfying specificity is proposed, in which the evident initial photocurrent response is produced by the photoanode and the signal amplification strategy toward the target is based on the specific bio-affinity properties between thrombin and their aptamers. The photoanode was fabricated with uniform TiO2 nanotube arrays decorated with CuOx and perylene-3,4,9,10-tetracarboxylic acid (PTCA). After the decoration of CuOx and PTCA, both possessing a strong absorption in visible region, the photocurrent response is improved greatly. Pt nanoparticles were modified on the photoanode electrode to immobilize thrombin aptamers, which could obviously improve the conductivity of the photoanode electrode owing to the excellent conductivity of Pt nanoparticles. The non-conductive complex leads to dramatical decrease in the photocurrent value due to the steric hinder effect. Based on the joint action of evident photocurrent response of photoanode and significant signal amplification strategy, the aptasensor in this project offers a wide linear range from 0.0003 nM to 10 nM, with a detection limit (3S/N) of thrombin down to 55 fM. Moreover, the as-fabricated aptasensor exhibits acceptable selectivity and stability, providing a meaningful attempt for the detection of thrombin.

Flexible flower-like MOF of Cu2(trans-1,4-cyclohexanedicarboxylic acid)2 as the electroactive matrix material for label-free and highly sensitive sensing of thrombin

A flexible metal-organic framework (MOF) of Cu2(trans-1,4-cyclohexanedicarboxylic acid)2 (Cu2(CHDC)2) has been synthesized by a facile hydrothermal method. The physical characterization experiments show that the Cu2(CHDC)2 presents unique flower-like shape, large surface area and high electroactivity. Then gold nanoparticles (AuNPs)/Cu2(CHDC)2 modified electrode was prepared and utilized as an electroactive matrix for thrombin aptamer immobilization. The electrochemical assay shows that the AuNPs can effectively enhance the electrochemical signal of the Cu2(CHDC)2. Upon the binding of the electrode-confined aptamer with the thrombin, the electrochemical signal of the Cu2(CHDC)2 decreases obviously due to the change of the microenvironment of the sensing interface. Thus a label-free electrochemical sensing strategy for thrombin was realized. The aptasensor displays excellent performance with wide linear range, ultralow detection limit of 0.01 fM (S/N = 3), and favorable specificity. Also, the aptasensor was applied for determination of thrombin in the human serum samples, and the satisfactory results are obtained. The results imply that the proposed sensor can be potentially used for the early diagnosis of disease and clinical monitoring.

Surface plasmon resonance-based aptasensor for direct monitoring of thrombin in a minimally processed human blood

Optical affinity biosensors are pursued for timely monitoring of thrombin in human blood, which is of urgent need in tailored anticoagulation therapies. However, the unspecific deposition of molecules, cells, and aggregates from the blood at their surface (also termed fouling) severely hinders their development and impedes the deploying of this technology to everyday clinical practice. We addressed this challenge by designing surface plasmon resonance (SPR) sensor chip with an antifouling polymer brush architecture and incorporated thrombin aptamer bioreceptors. Poly[(N-(2-hydroxypropyl)-methacrylamide)-co-(carboxybetaine methacrylamide)] brushes were synthesized on gold sensor chip surface via photoinduced single-electron transfer living radical polymerization and postmodified with three thrombin aptamers (HD1 short, HD1 and HD22). The affinity interaction of the aptamer bioreceptors with thrombin (as well as with other molecules present in the blood) was investigated and changes in their performance when incorporated into the polymer brushes were characterized. The combination of brushes and aptamer bioreceptors allowed for the analysis of medically relevant concentrations of thrombin in the 10 % blood by direct SPR detection format. This is the first time that the optical affinity biosensor is demonstrated for label-free analysis of biomarkers in a minimally processed human blood without a need for pre-separation steps. We believe that this system constitutes a basis for the future affinity biosensor applications that are suitable for the clinical settings and can be readily adapted to detect a range of important biological markers.

Measuring Aptamer Folding Energy Using a Molecular Clamp.

Folding energy (ΔGfold) offers a useful metric for characterizing the stability and function of aptamers. However, experimentally measuring the folding energy is challenging, and there is currently no general technique to measure this parameter directly. In this work, we present a simple approach for measuring aptamer folding energy. First, the aptamer is stretched under equilibrium conditions with a double-stranded DNA "molecular clamp" that is coupled to the aptamer ends. We then measure the total internal energy of stressed DNA molecules using time-lapse gel electrophoresis and compare the folding and unfolding behavior of molecular clamp-stressed molecules that incorporate either the aptamer or unstructured random single-stranded DNA in order to derive the aptamer folding energy. Using this approach, we measured a folding energy of 10.40 kJ/mol for the HD22 thrombin aptamer, which is consistent with other predictions and estimates. We also analyzed a simple hairpin structure, generating a folding energy result of 9.05 kJ/mol, consistent with the value predicted by computational models (9.24 kJ/mol). We believe our strategy offers an accessible and generalizable approach for obtaining such measurements with virtually any aptamer.

Functional DNA Structures and Their Biomedical Applications

Since the discovery of the double-helix structure in 1953, nucleic acids have been developed from natural genetic codes into functional building blocks in a wide range of biotechnology and material...

Laser-induced graphene interdigitated electrodes for label-free or nanolabel-enhanced highly sensitive capacitive aptamer-based biosensors

Highly porous laser-induced graphene (LIG) is easily generated in complex electrode configurations such as interdigitated electrodes (IDEs). Here, we demonstrate that their superior capacitive response at low frequencies can be exploited in affinity biosensors using thrombin aptamers as model biorecognition elements. Of specific interest was the effect of electrode surface area on capacitance detection, and the comparison between a label-free format and enhancement strategies afforded by carboxy group bearing polymeric nanoparticles or liposomes. Electrochemical impedance spectroscopy (EIS) was used to investigate the LIG performance and optimize the biosensor design. Interestingly, the label-free strategy performed extremely well and additional labels decreased the limit of detection or increased the sensitivity only minimally. It is assumed that the highly porous nature of the LIG structures dominates the capacitive response so that labels removed from the surface have only limited influence Also, while slight performance changes can be observed for smaller vs. larger electrode structures, the performance of a LIG IDE is reasonably independent of its size. In the end, a dynamic range of 5 orders of magnitude was obtained (0.01 nM–1000 nM) with a limit of detection as low as 0.12 pM. When measured in serum, this increased to 1.3 pM. The good reproducibility (relative standard deviation (RSD), 4.90%) and repeatability (RSD, 2.59%) and good long-term stability (>7 weeks at 4 °C) prove that a LIG-based capacitance sensor is an excellent choice for affinity-based biosensor. The ease-of-production, the simplicity of modification and the superior performance even in a label-free format indicate that LIG-based biosensors should be considered in point-of-care diagnostics in the future.

Highly sensitive host-guest mode homogenous electrochemical thrombin signal amplification aptasensor based on tetraferrocene label

The development of sensitive and convenient detection methods to monitor thrombin without the use of enzymes or complex nanomaterials is highly desirable for the diagnosis of cardiovascular diseases. In this article, tetraferrocene was first synthesized and then a sensitive and homogeneous electrochemical aptasensor was developed for thrombin detection based on host-guest recognition between tetraferrocene and β-cyclodextrin (β-CD). In the absence of thrombin, the double stem-loop of thrombin aptamer (TBA) prevented tetraferrocenes labeled at both ends from entering the cavity of β-CD deposited on gold electrode surface. After binding with thrombin, the stem-loop structure of TBA opened and transformed into special G-quarter structure, forcing tetraferrocene into the cavity of β-CD. As a result, thrombin allowed eight ferrocene molecules to reach the gold electrode surface, greatly amplifying the response signal. The obtained aptasensors showed dynamic detection range from 4 pM to 12.5 nM with detection limit around 1.2 pM. Overall, the results indicate that the proposed aptasensors are promising for future rapid clinical detection of thrombin and development of signal amplification strategies for detection of various proteins.

Highly Enhanced Electrochemiluminescence Luminophore Generated by Zeolitic Imidazole Framework-8-Linked Porphyrin and Its Application for Thrombin Detection.

Novel and distinct enhancement in electrochemiluminescence (ECL) signals of advanced organic luminophores are of importance for expanding their applications in early diagnosis. This work reported the construction of an ultrasensitive label-free ECL aptasensor for thrombin (TB) detection by grafting zinc proto-porphyrin IX (ZnP) onto an aminated zeolitic imidazole framework-8 (defined as ZnP-NH-ZIF-8 for clarity) as the luminophore. The structure and optical properties of the resulting ZnP-NH-ZIF-8 were carefully characterized. For that, there appeared to be weak ECL radiation for ZnP in dichloromethane (DCM) containing tetra-n-butylammonium perchlorate (TBAP) because of the as-formed singlet-state oxygen via the "reduction-oxidation" route. More notably, the ECL signals display 153-times enhancement for ZnP-NH-ZIF-8, thanks to the excellent catalytic kinetics for the oxygen reduction reaction (ORR). By virtue of the specific interactions of the TB aptamer (TBA) with the TB protein and the highly efficient catalysis of the ZnP-NH-ZIF-8 for ORR, the as-prepared aptasensor showed a wider linear range (0.1 fM∼1 pM) and a lower detection limit (ca. 58.6 aM). This work provides some useful guidelines for synthesis of an advanced organic luminophore with largely boosted ECL signals in ultrasensitive analysis and clinical diagnosis.

An Ultrasensitive Electrochemical Aptasensor for Thrombin Detection Using MoS2 Nanoparticles Loaded Iron-Porphyrinic Metal-Organic Framework as Signal Amplifier

An ultrasensitive electrochemical aptasensor for thrombin (TB) was designed using MoS2 nanoparticles loaded iron-porphyrinic metal-organic framework (PCN-223-Fe) as the electrochemical tracer. Spindle-shaped PCN-223-Fe was synthesized with zirconium ion as node and iron-porphyrin as linker, and in situ loaded with MoS2 nanoparticles to obtain MoS2/PCN-223-Fe composite. MoS2/PCN-223-Fe was characterized by electron microscopies, X-ray diffraction and X-ray photoelectron spectroscopy. Due to the synergistic effect of MoS2 and PCN-223-Fe, MoS2/PCN-223-Fe modified electrode exhibited enhanced electrocatalytic activity to oxygen reduction. Sequentially, MoS2/PCN-223-Fe was combined with TB aptamer 2 to prepare signal probe. A sandwich-type electrochemical aptasensor was constructed for TB detection. TB aptamer 1 was fixed on electrode surface to capture TB molecules, and then TB aptamer 2 functionalized MoS2/PCN-223-Fe tracer was introduced on electrode surface. By detecting the electrochemical signal of oxygen reduction catalyzed by MoS2/PCN-223-Fe, the designed TB sensor was monitored. The proposed TB aptasensor showed a wide linear range from 0.1 pM to 100 nM and a low detection limit of 0.03 pM (S/N = 3). Meanwhile, the aptasensor showed good reproducibility, stability and selectivity, which was successfully applied for TB detection in human serum sample. This strategy broadens application of metal-organic frameworks, and could be used for other biomolecules detection.

A single-molecule study reveals novel rod-like structures formed by a thrombin aptamer repeat sequence.

Thrombin aptamers (TBAs) have attracted much attention due to their various applications. The structures and properties of long ssDNA chains with multiple TBA repeat sequences are interesting and distinct from those of their monomers. Due to the complexity of the sample system, it is quite difficult to reveal the structure of such a long-chain ssDNA using traditional methods. In this work, we investigated the repeated ssDNA by using single-molecule magnetic tweezers and AFM imaging. To do that we developed the polymerase change-rolling circle amplification (PC-RCA) synthetic method and prepared two-end modified repeated ssDNA. The rod-like G4 structures formed by intramolecular stacking of the repeat sequence were for the first time identified. This novel structure is different from those higher-order quadruplex structures formed by G-tetrads or loop-mediated interactions. It is also quite interesting to find that the increase of the TBA copy number can unitize the diversity of TBA conformation to the best-fit binding structure for thrombin. The methodology developed in this work can be used for studying other repeat sequences in the genome, such as telomeric DNA as well as interactions of ssDNA with the binding molecule.

Improving Spinach2-and Broccoli-based biosensors for single and double analytes

The use of “Spinach”-based RNA sensors for the detection of small metabolites and proteins has received growing interest in the recent years. While this approach can be used in vivo for cell sensing and in vitro for microfluidic assays, their overall utility is limited as a result of a high magnesium ion dependence. Here, we alleviate this limitation through incorporating a human tRNALys3 or an engineered viral F30 (a three-way junction RNA motif) scaffold to facilitate aptamer folding in vitro and improve the performance of selected streptavidin, tyrosine, and thrombin aptamers as exemplary cases. Furthermore, we demonstrate the use of a Broccoli aptamer scaffold in conjunction with the viral F30 to enable simultaneous sensing of two molecules in a logic gate type fashion. Our proof-of-concept results demonstrate the ability to redesign these aptamer sensors for improved brightness as well as signal stability without the need for high magnesium—both traits that can further enhance downstream screening applications.

Application of Piezo-Based Measuring System for Evaluation of Nucleic Acid-Based Drugs Influencing the Coagulation.

During open-heart surgery, the status of hemostasis has to be constantly monitored to quickly and reliably detect bleeding or coagulation disorders. In this study, a novel optimized piezo-based measuring system (PIEZ) for rheological monitoring of hemostasis was established. The applicability of the PIEZ for the evaluation of nucleic acid-based drugs influencing coagulation was analyzed. Thrombin aptamers such as NU172 might be used during extracorporeal circulation (ECC) in combination with a reduced heparin concentration or for patients with heparin-induced thrombocytopenia (HIT). Therefore, the effect of the coagulation inhibiting thrombin aptamer NU172 and the abrogation by its complementary antidote sequence (AD) were investigated by this rheological PIEZ system. After the addition of different NU172 concentrations, the coagulation of fresh human blood was analyzed under static conditions and using an in vitro rotation model under dynamic conditions (simulating ECC). The clotting times (CTs) detected by PIEZ were compared to those obtained with a medical reference device, a ball coagulometer. Additionally, after the circulation of blood samples for 30 min at 37 °C, blood cell numbers, thrombin markers (thrombin-antithrombin III (TAT) and fibrinopeptide A (FPA)) and a platelet activation marker (β-thromboglobulin (β-TG)) were analyzed by enzyme-linked immunosorbent assays (ELISAs). The increase of NU172 concentration resulted in prolonged CTs, which were comparable between the reference ball coagulometer and the PIEZ, demonstrating the reliability of the new measuring system. Moreover, by looking at the slope of the linear regression of the viscous and elastic components, PIEZ also could provide information on the kinetics of the coagulation reaction. The shear viscosity at the end of the measurements (after 300 s) was indicative of clot firmness. Furthermore, the PIEZ was able to detect the abrogation of coagulation inhibition after the equimolar addition of NU172 aptamer´s AD. The obtained results showed that the established PIEZ is capable to dynamically measure the hemostasis status in whole blood and can be applied to analyze nucleic acid-based drugs influencing the coagulation.

Thrombin Aptamer-Modified Metal-Organic Framework Nanoparticles: Functional Nanostructures for Sensing Thrombin and the Triggered Controlled Release of Anti-Blood Clotting Drugs.

This paper features the synthesis of thrombin-responsive, nucleic acid-gated, UiO-68 metal–organic framework nanoparticles (NMOFs) loaded with the drug Apixaban or rhodamine 6G as a drug model. Apixaban acts as an inhibitor of blood clots formation. The loads in the NMOFs are locked by duplex nucleic acids that are composed of anchor nucleic acids linked to the NMOFs that are hybridized with the anti-thrombin aptamer. In the presence of thrombin, the duplex gating units are separated through the formation of thrombin–aptamer complexes. The unlocking of the NMOFs releases the drug (or the drug model). The release of the drug is controlled by the concentration of thrombin. The Apixaban-loaded NMOFs revealed improved inhibition, as compared to free Apixaban, toward blood clot formation. This is reflected by their longer time intervals for inducing clot formation and the decreased doses of the drug required to affect clots formation. The beneficial effects of the Apixaban-loaded NMOFs are attributed to the slow-release mechanism induced by the NMOFs carriers, where the inhibition of factor Xa in the blood clotting cycle retards the formation of thrombin, which slows down the release of the drug.

Binding-Induced 3D-Bipedal DNA Walker for Cascade Signal Amplification Detection of Thrombin Combined with Catalytic Hairpin Assembly Strategy.

As an important biomarker, thrombin (TB) is a major player in thrombosis and hemostasis and has attracted increasing attention involving its determination. Herein a universal and ultrasensitive fluorescence biosensor based on a binding-induced 3D-bipedal DNA walker and catalytic hairpin assembly (CHA) strategy has been proposed for cascade signal amplification detection of thrombin. In this study, we designed two proximity probes (foot 1 and foot 2) which include a specific affinity ligand for TB binding and a Pb2+-dependent DNAzyme tail sequence. In the presence of TB, the simultaneous binding of TB to foot 1 (F1) and foot 2 (F2) via TB aptamer (TBA) brings the tail sequences into close proximity and the melting temperature for tail sequences and track DNA is increased, allowing the Pb2+-dependent DNAzyme to cleave the track DNA into two short fragments which have lower affinities for the DNAzyme and, finally, leading to the release of trigger DNA (T-DNA) for subsequent CHA reaction. In the meantime, the dissociated DNA walkers (F1 and F2) explore adjacent unwound track DNA, and the walking procedure is conducted. Unlike the conventional unipedal DNA walkers that anchor foot DNA and track DNA on the same sensing surface, the proposed 3D-bipedal DNA walking machine can not only increase the local concentration of track DNA but can also improve the walking efficiency and expand the range of the walkers to some extent due to the two free feet. Moreover, with the advantages of superior sensitivity and excellent specificity, this biosensing platform exhibits a huge potential in practical application in biomedical research and clinical diagnosis.