Biosensor Based on a Dynamic DNA Nanostructure Driven by Strand Displacement Reaction

Abstract

The present invention belongs to the field of biological detection technology, and relates to a biosensor based on a dynamic DNA nanosystem driven by strand displacement reaction, which is composed of a target recycling unit and a multi-legged DNA walker unit; in the target recycling unit, the closed multi-legged DNA walker can release the multi-legged DNA walker under the combined action of the fuel strand F and the target nucleic acid. In the multi-legged DNA walker unit, the closed gold electrode can release the closed strand B2 under the action of the labeled DNA and the released multi-legged DNA walker, so as to realize the ultrasensitive quantitative detection of the target nucleic acid through the detection of the electrochemical signal ratio between the first electrochemical signal material and the second electrochemical signal material. The biosensor based on the dynamic DNA nanosystem driven by strand displacement reaction provided by the present invention can ultrasensitively detect nucleic acids.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and relates to a biosensor based on a dynamic DNA nanosystem driven by strand displacement reaction. Background Art

[0002] Disclosing the information of this background art section is only intended to increase the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] DNA walkers show great potential in biosensing applications by autonomously moving on a preset track to amplify and convert signals. So far, the number of walking strands of DNA walkers has gradually developed from single-foot to double-foot and even multi-foot. In contrast, multi-foot DNA walkers have better walking continuity and higher reaction efficiency due to the high local concentration of walking strands. During the walking process, the Gibbs free energy gradient caused by biochemical reactions (such as strand displacement, nuclease-assisted strand breakage, and hydrolysis) drives the DNA walker to move spontaneously towards the lower energy level. However, DNA walkers need to provide nucleases as the driving force, and the enzyme activity has a great influence on the detection effect of DNA walkers. Therefore, a new type of enzyme-free isothermal amplification strategy is needed for point-of-care testing (POCT) devices. Summary of the Invention

[0004] Compared with the driving force provided by nucleases, toehold-mediated strand displacement reaction (TMSDR) can be carried out under enzyme-free and isothermal conditions, leaving great room for the development of low-cost and robust POCT devices. As a DNA nanotechnology with powerful computing capabilities, TMSDR can promote the key processes of dynamic DNA nanotechnology, and sensors based on TMSDR are naturally most suitable for detecting nucleic acid analytes. Importantly, it is how to combine TMSDR-driven dynamic DNA nanotechnology with effective target recognition and appropriate detection methods to output detection signals.

[0005] To solve the deficiencies of the prior art, the purpose of the present invention is to provide a biosensor based on a dynamic DNA nanosystem driven by strand displacement reaction, which is integrated into a ratio-type electrochemical biosensor and can detect nucleic acids with ultra-high sensitivity.

[0006] To achieve the above purpose, the technical solution of the present invention is as follows:

[0007] On the one hand, a biosensor based on a dynamic DNA nanosystem driven by strand displacement reaction is composed of a target recycling unit and a multi-foot DNA walker unit;

[0008] The target substance circulation unit includes a multi-legged DNA walker, auxiliary strand A1, blocking strand B1, and fuel strand F. The multi-legged DNA walker is formed by connecting at least two walking strands W to the surface of a gold nanoparticle, with one end of the walking strand W connected to the gold nanoparticle. The blocking strand B1 is provided with a W region, an A1 region, and a first toehold region. The W region can be complementary to the DNA fragment at the other end of the walking strand W. The A1 region can be complementary to the auxiliary strand A1. The first toehold region can hybridize with the target nucleic acid, and through strand displacement reaction, the target nucleic acid displaces the auxiliary strand A1, and at the same time, a second toehold region is exposed in the middle part of the blocking strand B1. The fuel strand F can hybridize with the second toehold region and replace the walking strand W and the target nucleic acid through strand displacement reaction.

[0009] The multi-legged DNA walker unit includes a gold electrode, auxiliary strand A2, blocking strand B2, and labeled DNA. A number of substrate strands S are connected to the surface of the gold electrode. The substrate strand S is provided with a B2 region, an A2 region, and a third toehold region. The B2 region can be complementary to the blocking strand B2. The A2 region can be complementary to the auxiliary strand A2. The first toehold region can hybridize with the walking strand W, and through strand displacement reaction, the DNA fragment at the other end of the walking strand W displaces the auxiliary strand A2, and at the same time, a fourth toehold region is exposed in the middle part of the substrate strand S. The labeled DNA can hybridize with the fourth toehold region and replace the blocking strand B2 and the DNA fragment at the other end of the walking strand W through strand displacement reaction. One end of the blocking strand B2 is connected to a first electrochemistry signal material. After the blocking strand B2 hybridizes with the substrate strand S, the first electrochemistry signal material is close to the gold electrode. One end of the labeled DNA is labeled with a second electrochemistry signal material. After the labeled DNA hybridizes with the substrate strand S, the second electrochemistry signal material is close to the gold electrode.

[0010] During use, in the target substance circulation unit, the multi-legged DNA walker, auxiliary strand A1, and blocking strand B1 form a closed multi-legged DNA walker through complementary hybridization. Each walking strand W in the closed multi-legged DNA walker is complementary hybridized with the auxiliary strand A1 and the blocking strand B1. The closed multi-legged DNA walker can release the multi-legged DNA walker under the combined action of the fuel strand F and the target nucleic acid.

[0011] During use, in the multi-legged DNA walker unit, the gold electrode, auxiliary strand A2, and blocking strand B2 form a closed gold electrode through complementary hybridization. Each substrate strand S in the closed gold electrode is complementary hybridized with the auxiliary strand A2 and the blocking strand B2. The closed gold electrode can release the blocking strand B2 under the action of the labeled DNA and the released multi-legged DNA walker, so as to realize ultrasensitive quantitative detection of the target nucleic acid through the detection of the electrochemical signal ratio between the first electrochemistry signal material and the second electrochemistry signal material.

[0012] The target recycling unit can not only recognize the target nucleic acid, but also dynamically activate the multi-legged DNA walker unit. Both the target recycling unit and the multi-legged DNA walker unit are driven by TMSDR, realizing enzyme-free and isothermal amplification. As the activated multi-legged DNA walker rolls on the sensor surface, it ingeniously amplifies the bidirectional signal and outputs a ratio-type signal, thus realizing the detection of target nucleic acid.

[0013] On the other hand, an application of the above-mentioned biosensor based on the strand displacement reaction-driven dynamic DNA nanosystem in nucleic acid detection.

[0014] In the third aspect, a method for detecting nucleic acid provides the above-mentioned biosensor based on the strand displacement reaction-driven dynamic DNA nanosystem, including the following steps:

[0015] Incubate the multi-legged DNA walker, auxiliary strand A1, and blocking strand B1 to form a blocked multi-legged DNA walker;

[0016] Incubate the gold electrode, auxiliary strand A2, and blocking strand B2 to form a blocked gold electrode;

[0017] Mix and react the blocked multi-legged DNA walker, fuel strand F, and the target nucleic acid to be detected. After mixing the reaction solution with the labeled DNA, drop it on the surface of the blocked gold electrode and incubate, and then detect the electrochemical signal after incubation.

[0018] In the fourth aspect, a nucleic acid detection kit includes the above-mentioned biosensor based on the strand displacement reaction-driven dynamic DNA nanosystem and a buffer solution.

[0019] The beneficial effects of the present invention are as follows:

[0020] The present invention constructs a TMSDR-driven dynamic DNA nanosystem. The synergistic effect of the target recycling unit and the multi-legged DNA walker unit in the dynamic DNA nanosystem, as well as the ratio-type readout mode, realize cascaded signal amplification and ultrasensitive detection of target nucleic acid in complex biological samples. As the driving force of the dynamic DNA nanosystem, TMSDR not only realizes an enzyme-free isothermal amplification strategy, but also ensures high specificity of detection. By appropriately adjusting the target recycling unit, the proposed sensor can be extended to detect a wide range of nucleic acid targets, providing a general strategy for various clinical diagnostic applications. The successful preparation of the multi-legged DNA walker and the feasibility of the scheme are fully confirmed by means such as PAGE, TEM, UV-vis, and Zeta potential. The analytical performance of the sensor is comprehensively evaluated by electrochemical tests, with a linear range of 100 aM - 10 pM and a detection limit as low as 36.71 aM.

[0021] In summary, the dynamic DNA nanosystem driven by TMSDR provided by the present invention can be used as a novel ratio-type electrochemical biosensor for ultrasensitive detection of nucleic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0023] Figure 1 Schematic diagram for enzyme-free detection of nucleic acids by the ratio-type electrochemical biosensor based on multi-legged DNA walker according to the embodiment of the present invention; (A) Target recognition stage, (B) Signal output stage.

[0024] Figure 2 Polyacrylamide gel electrophoresis diagram according to the embodiment of the present invention; (A) Target recognition stage. 1, HIV-DNA; 2, A1; 3, B1; 4, F; 5, W; 6, W / B1; 7, W / B1 / A1; 8, W / B1 / A1 + HIV-DNA; 9, W / B1 / A1 + HIV-DNA; 10, W / B1 / A1 + HIV-DNA + F; (The concentration of F is 1 μM, and the concentration of other DNAs is 200 nM); (B) Signal output stage. 1, A2; 2, B2; 3, LD; 4, W; 5, S; 6, S / A2; 7, S / B2; 8, S / (A2 + B2); 9, S / (A2 + B2) + W; 10, S / (A2 + B2) + LD, 11, S / (A2 + B2) + W + LD; (The concentration of W and LD is 1 μM, and the concentration of other DNAs is 200 nM); M, DNA marker (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, 500 bp).

[0025] Figure 3 Polyacrylamide gel electrophoresis diagram of the signal output stage according to the embodiment of the present invention; 1, A2'; 2, B2; 3, LD; 4, W; 5, S; 6, S + A2'; 7, S + B2; 8, S / A2' / B2; 9, S / A2' / B2 + W; 10, S / A2' / B2 + LD, 11, S / A2' / B2 + W + LD; (The concentration of W and LD is 1 μM, and the concentration of other DNAs is 200 nM); M, DNA marker (20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, 400, 500 bp).

[0026] Figure 4TEM images of the multi-legged DNA walker of the embodiments of the present invention; (A) AuNPs, (B) AuNP-W, (C) AuNP-W / B1 / A1.

[0027] Figure 5 In the embodiments of the present invention, (A) UV-visible absorption spectra of the walking strand W(a), gold nanoparticles (b), and multi-legged DNA walker (c); (B) Electrochemical impedance spectroscopy Nyquist plots of bare gold electrode (a), gold electrode / S (b), gold electrode / S / MCH (c), gold electrode / S / MCH / (A2 + B2) (d), gold electrode / S / MCH / (A2 + B2) / W (e), and gold electrode / S / MCH / (A2 + B2) / (W + LD) (f) in 0.1 M KCl containing 5.0 mM [Fe(CN)6] 3- / 4- ; (C) ACV signal responses of the sensor based on the multi-legged DNA walker to 0 pM HIV-DNA (a) and 1 pM HIV-DNA (b), and the sensor based on the single-legged DNA walker to 1 pM HIV-DNA (c).

[0028] Figure 6 ACV response curves of the ratiometric electrochemical biosensor of the embodiments of the present invention in the absence of W (a), 1 μM single-stranded W (b), and 1 nM multi-legged DNA walker (c).

[0029] Figure 7 Results of condition optimization of the embodiments of the present invention; (A) NaCl concentration in the buffer, (B) concentration of AuNP-W / B1 / A1, (C) target recognition time, (D) rolling time of the multi-legged DNA walker.

[0030] Figure 8 In the embodiments of the present invention, ACV signal responses of the ratiometric electrochemical biosensor based on the multi-legged DNA walker to different concentrations of HIV-DNA: 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, and 10 pM (from a to g); (B) I MB / I Fc and log c HIV-DNA linear relationship of the logarithm; (C) ACV signal responses of the sensor when detecting HIV-DNA, Sm-T, Tm-T, and N-T.

[0031] Figure 9 ACV signal responses of the sensor of the embodiments of the present invention to HIV-DNA after being stored at 4 °C for 0, 1, 3, 5, 7, and 14 days respectively. Detailed implementation manners

[0032] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention belongs.

[0033] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0034] In order to integrate a multi-legged DNA walker into a ratio-type electrochemical biosensor for ultrasensitive nucleic acid detection, the present invention proposes a biosensor based on a strand displacement reaction-driven dynamic DNA nanosystem.

[0035] A typical embodiment of the present invention provides a biosensor based on a strand displacement reaction-driven dynamic DNA nanosystem, which consists of a target recycling unit and a multi-legged DNA walker unit;

[0036] The target recycling unit includes a multi-legged DNA walker, an auxiliary strand A1, a blocking strand B1, and a fuel strand F. The multi-legged DNA walker is formed by connecting at least two walking strands W to the surface of a gold nanoparticle, and one end of the walking strand W is connected to the gold nanoparticle. The blocking strand B1 is provided with a W region, an A1 region, and a first toehold region. The W region can be complementary to the DNA fragment at the other end of the walking strand W. The A1 region can be complementary to the auxiliary strand A1. The first toehold region can hybridize with the target nucleic acid. Through strand displacement reaction, the target nucleic acid displaces the auxiliary strand A1, and at the same time, a second toehold region is exposed in the middle part of the blocking strand B1. The fuel strand F can hybridize with the second toehold region and replace the walking strand W and the target nucleic acid through strand displacement reaction;

[0037] The multi-legged DNA walker unit includes a gold electrode, auxiliary strand A2, blocking strand B2, and labeled DNA. A number of substrate strands S are connected to the surface of the gold electrode. The substrate strand S is provided with a B2 region, an A2 region, and a third toehold region. The B2 region can be complementary to the blocking strand B2. The A2 region can be complementary to the auxiliary strand A2. The first toehold region can hybridize with the walking strand W. Through strand displacement reaction, the DNA fragment at the other end of the walking strand W displaces the auxiliary strand A2, and at the same time, a fourth toehold region is exposed in the middle part of the substrate strand S. The labeled DNA can hybridize with the fourth toehold region and replace the blocking strand B2 and the DNA fragment at the other end of the walking strand W through strand displacement reaction. One end of the blocking strand B2 is connected to a first electrochemical signal material. After the blocking strand B2 hybridizes with the substrate strand S, the first electrochemical signal material is close to the gold electrode. One end of the labeled DNA is labeled with a second electrochemical signal material. After the labeled DNA hybridizes with the substrate strand S, the second electrochemical signal material is close to the gold electrode.

[0038] The walking strand W, auxiliary strand A1, blocking strand B1, fuel strand F, substrate strand S, auxiliary strand A2, blocking strand B2, and labeled DNA described in the present invention are all single-stranded DNAs.

[0039] In some embodiments, the gold nanoparticles are connected to the walking strand W through gold-sulfur bonds.

[0040] In some embodiments, the gold electrode is blocked with mercaptohexanol (MCH).

[0041] In some embodiments, the first electrochemical signal material is ferrocene, and the second electrochemical signal material is methylene blue.

[0042] The present invention selects the human immunodeficiency virus-specific related DNA fragment (HIV-DNA) as the target for verification, indicating better detection effects. Specifically, the sequence of the auxiliary strand A1 is as shown in SEQ ID NO.2, the sequence of the blocking strand B1 is as shown in SEQ ID NO.3, the sequence of the fuel strand F is as shown in SEQ ID NO.4, the sequence of the walking strand W is as shown in SEQ ID NO.5, the sequence of the substrate strand S is as shown in SEQ ID NO.6, the sequence of the auxiliary strand A2 is as shown in SEQ ID NO.7, the sequence of the blocking strand B2 is as shown in SEQ ID NO.9, and the sequence of the labeled DNA is as shown in SEQ ID NO.10.

[0043] Another embodiment of the present invention provides an application of the biosensor of the above-described dynamic DNA nanosystem driven by strand displacement reaction in nucleic acid detection.

[0044] Specifically, the nucleic acid is the nucleic acid of human immunodeficiency virus. More specifically, the nucleic acid is a specific related DNA fragment of human immunodeficiency virus, and its sequence is shown as SEQ ID NO.1.

[0045] The third embodiment of the present invention provides a method for detecting nucleic acid. A biosensor based on the strand displacement reaction-driven dynamic DNA nanosystem as described above is provided, including the following steps:

[0046] Incubate the multi-legged DNA walker, auxiliary strand A1, and blocking strand B1 to form a blocked multi-legged DNA walker;

[0047] Incubate the gold electrode with several substrate strands S connected to its surface, auxiliary strand A2, and blocking strand B2 to form a blocked gold electrode;

[0048] Mix and react the blocked multi-legged DNA walker, fuel strand F, and the nucleic acid to be detected. After mixing the reaction solution with the labeled DNA, drop it on the surface of the blocked gold electrode and incubate, and then detect the electrochemical signal after incubation.

[0049] Each step of the detection method of the present invention is carried out under room temperature conditions. The room temperature mentioned in the present invention refers to the temperature of the indoor environment, generally 15 - 30 °C.

[0050] In some embodiments, the incubation time of the multi-legged DNA walker, auxiliary strand A1, and blocking strand B1 is 70 - 90 min.

[0051] In some embodiments, the incubation time of the gold electrode with several substrate strands S connected to its surface, auxiliary strand A2, and blocking strand B2 is 70 - 90 min.

[0052] In some embodiments, the mixing and reaction time of the blocked multi-legged DNA walker, fuel strand F, and the nucleic acid to be detected is 70 - 90 min.

[0053] In some embodiments, the incubation time after mixing the reaction solution with the labeled DNA and dropping it on the surface of the blocked gold electrode is 50 - 70 min.

[0054] The fourth embodiment of the present invention provides a nucleic acid detection kit, including the biosensor based on the strand displacement reaction-driven dynamic DNA nanosystem as described above and a buffer solution.

[0055] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0056] Examples

[0057] Reagents and materials:

[0058] Chloroauric acid (HAuCl4) and trisodium citrate dihydrate (C6H5Na3O7·2H2O) were provided by Sinopharm Chemical Reagent Co., Ltd. Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) was provided by BBI Life Sciences Co., Ltd. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was provided by Shanghai Aladdin Biochemical Technology Co., Ltd. 6× glycerol gel loading buffer, N,N,N,N-Tetramethylethylenediamine (TEMED), and ammonium persulfate were purchased from Shanghai Sangon Biotech Co., Ltd. Potassium ferricyanide (K3[Fe(CN)6]) and potassium ferrocyanide (K4[Fe(CN)6]) were provided by Sinopharm Chemical Reagent Co., Ltd. Normal human serum was provided by Beijing Solarbio Science & Technology Co., Ltd. Tris-HCl buffer (10 mM Tris-HCl, 400 mM NaCl, 10 mM MgCl2, pH 8.0) was used to dilute all DNA oligonucleotides. Ultrapure water obtained from the Youpu purification water system was used to prepare aqueous solutions used throughout the process (resistivity > 18.25 MΩ·cm). All reagents were of analytical grade and did not require further purification unless otherwise specified. All DNA oligonucleotides were provided by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China), and their detailed sequences are shown in Table 1:

[0059] Table 1 DNA and sequences used in this example

[0060]

[0061]

[0062] Experimental instruments:

[0063] The modification of gold nanoparticles (AuNPs) was characterized using a transmission electron microscope (TEM, HT-7700, Hitachi, Japan), a TU-1901 double-beam ultraviolet-visible spectrophotometer (UV-vis, Purkinje General Instrument Co., Ltd., Beijing, China), and a Zetasizer nano instrument (Malvern Instruments Ltd., UK). All electrochemical measurements were carried out on a CHI 660E electrochemical workstation (Chenhua, Shanghai, China) using a traditional three-electrode system consisting of a modified gold electrode (Au E, Φ = 3 mm) as the working electrode, Ag / AgCl (in saturated KCl solution) as the reference electrode, and a platinum wire as the counter electrode. ZWY-103B constant temperature incubator shaker (Zhicheng, Shanghai, China), K30 series dry bath incubator (Aosheng, Hangzhou, China), UPR-II-10T ultrapure water system (Youpu, Sichuan, China), DYY-6D electrophoresis apparatus (Liuyi, Beijing, China), TGL-16 high-speed refrigerated centrifuge (Xiangyi, Hunan, China). DSN-280B portable pressure steam sterilizer, KQ2200E ultrasonic cleaner. ME104 electronic balance, laboratory pH meter FE20.

[0064] Synthesis of gold nanoparticles:

[0065] Before synthesizing gold nanoparticles, all glassware and magnetic stirrers were soaked in aqua regia for at least 4 hours, thoroughly washed with ultrapure water, and then dried. While vigorously stirring, a sodium citrate solution (5 mL, 38.8 mM) was rapidly added to the boiling chloroauric acid aqueous solution (50 mL, 1 mM). After the solution turned wine red, it was kept boiling for 10 minutes while vigorously stirring, and then the heating was stopped and the mixed solution was continuously stirred for 15 minutes. Under continuous slow stirring, the temperature of the solution was gradually reduced to room temperature. The prepared gold nanoparticle solution was filtered using a filter membrane with a pore size of 0.2 μm. After filtration, the synthesized gold nanoparticle solution was stored at 4 °C in the dark for future use.

[0066] Preparation of AuNP-W / B1 / A1 bioconjugate:

[0067] The conjugation of W on the surface of gold nanoparticles is achieved through Au-S bond chemistry. Briefly, W was reduced with TCEP for one hour in the dark to activate the thiol groups. The activated W (final concentration of 4 μM) was incubated with 500 μL of gold nanoparticles for 16 hours. Subsequently, 2 M NaCl solution was added dropwise to the mixed solution as slowly as possible six times at 30-minute intervals to make the final salt concentration 0.1 M. After at least 40 h of "salt aging", the solution was centrifuged and washed three times (12,000 rpm, 30 min), and finally redissolved in 500 μL of 10 mM Tris-HCl buffer solution containing 0.4 M NaCl, completing the preparation of the multi-legged DNA walker (AuNP-W). The prepared AuNP-W was mixed and incubated with excess A1 and B1 for 80 minutes to block the hybridization sites on W. After centrifuging and washing three times to remove unbound A1 and B1 in the solution, the preparation of the AuNP-W / B1 / A1 bioconjugate was finally completed.

[0068] Construction of the AuE / S / (A2 + B2) sensing platform:

[0069] Before the surface modification of the electrode, the gold electrode was pretreated according to the method reported previously. First, the gold electrode was polished with 50-nm alumina slurry until a mirror surface was obtained, and then the electrode was rinsed twice with absolute ethanol and ultrapure water, respectively. Finally, the gold electrode was electrochemically activated by cyclic voltammetry (CV) in 0.5 M H2SO4 solution, scanned at a sweep rate of 0.1 V / s in the potential range from -0.2 V to 1.6 V until a stable cyclic voltammogram was obtained. After thorough rinsing with ultrapure water, the activated gold electrode was obtained.

[0070] S at 1 μM was reduced with TCEP for 1 hour in the dark at room temperature to activate the thiol groups. Subsequently, 5 μL of the above solution was dropped onto the surface of the pretreated gold electrode respectively, and incubated at 4 °C for 12 h, so that S was anchored on the surface of the gold electrode through Au-S interaction. After washing, the electrode was immersed in 1 mM MCH for 1 h to block the excess active sites on the electrode surface. Finally, 2 μM of the mixed solution containing A2 and B2 was dropped onto the surface of the washed electrode and incubated at room temperature for 80 minutes. After washing, the construction of the AuE / S / (A2 + B2) sensing platform was completed.

[0071] Quantitative analysis of HIV-DNA:

[0072] Mix the HIV-DNA and an excess of F with the prepared AuNP-W / B1 / A1 bioconjugate and react for 80 minutes. After the reaction is completed, mix the final solution with LD and drop it onto the surface of the AuE / S / (A2+B2) sensing platform and incubate at room temperature for 1 h. Finally, test the obtained sensor by alternating current voltammetry (ACV) in a 10 mM Tris-HCl buffer solution, with a scanning potential range of -0.6 V to 0.6 V, a step potential of 4 mV, a frequency of 25 Hz, and an amplitude of 25 mV.

[0073] 12% polyacrylamide gel electrophoresis:

[0074] Gel electrophoresis experiments were used to verify the reaction between DNAs during the detection process. First, wipe the glass plates unidirectionally with ultrapure water and absolute ethanol in sequence, dry them and assemble them, and then start preparing the gel (12% polyacrylamide gel). The specific process is as follows: sequentially add 25 mL of ultrapure water, 5 mL of 10×TBE, 20 mL of polyacrylamide (30%), 250 μL of 10% ammonium persulfate, and 25 μL of TEMED into a beaker, stir magnetically for about 5 minutes, pour the mixture into the pre-treated gel plate as slowly as possible (to prevent the appearance of bubbles), slowly insert the comb teeth to generate sample loading ports, then let the gel plate stand at an inclination angle of about 10° for 6 hours, and finally slowly pull out the comb teeth to complete the gel preparation. Take out the glass gel chamber in the gel plate and fix it in the electrophoresis tank, and add 1×TBE to submerge the gel. Then comes the sample loading process. The loading buffer and the sample solution are mixed evenly at a ratio of 1:5, clean the bubbles on the sample loading ports, keep the tip of the pipette perpendicular to the sample loading ports, and add 12 μL of the above mixed solution to the bottom of the sample loading ports. Subsequently, turn on the electrophoresis instrument and perform the electrophoresis process under a constant temperature condition of 10 °C for 1.5 hours. Finally, for the imaging of the gel electrophoresis, stain the gel after electrophoresis with 200 mL of 1×gel Red for 30 minutes, and place it in the sample chamber of the Bio-Rad Gel Doc XR+ instrument and set the specific parameters for imaging.

[0075] Results and discussion:

[0076] Detection principle:

[0077] As Figure 1 shown, consisting of the target recognition stage ( Figure 1 A) and the signal output stage ( Figure 1B). In the target recognition stage, first, the thiolated walking strand W is immobilized on the surface of gold nanoparticles through gold-sulfur bonds to prepare a multi-legged DNA walker. Then, it interacts with an excess of blocking strand B1 and auxiliary strand A1 to form an AuNP-W / B1 / A1 bioconjugate, leaving a toehold region at the outer end of B1 to respond to HIV-DNA. In the presence of HIV-DNA, it can attach to the end of B1 through hybridization with the toehold. Compared with the double-stranded B1 / A1, a stronger binding force between HIV-DNA and B1 is preset by designing more complementary base pairs. Therefore, A1 can be gradually replaced by HIV-DNA through branch migration, and at the same time, a new toehold region is exposed in the middle part of B1. Accordingly, the fuel strand F can gradually replace W and HIV-DNA through a similar TMSDR. In this way, the blocked W is activated, and the released HIV-DNA continues to react with other W / B1 / A1 complexes on the AuNP, further leading to the repetition of the above cycle. Through this target cycling process, even a very small amount of HIV-DNA can greatly activate the multi-legged DNA walker AuNP-W for subsequent signal output.

[0078] In the signal output stage, the AuE is modified with the substrate strand S as the walking track, and then the electrode is blocked with MCH. Subsequently, S is partially blocked by the auxiliary strand A2 and the blocking strand B2 (modified with ferrocene (Fc)), leaving a toehold region at the top end of S. After introducing the activated multi-legged DNA walker AuNP-W and the labeled DNA (LD, modified with methylene blue (MB)), W first binds to the toehold region of S, and then gradually replaces A2 through branch migration, exposing a new toehold region in the middle of S for the binding of LD. Driven by TMSDR, LD gradually displaces W and B2, causing the AuNP-W to roll and further triggering a new strand displacement process. Finally, the Fc label on the electrode surface decreases, while the MB label increases, and the final electrochemical signal ratio (I MB / I Fc ) can be used to reflect the content of HIV-DNA. On the contrary, in the absence of HIV-DNA, the blocked multi-legged DNA walker cannot be activated at all and cannot roll on the sensing platform, so the electrochemical signals of MB and Fc do not change.

[0079] Gel electrophoresis verification:

[0080] The proposed strategy was verified by 12% polyacrylamide gel electrophoresis (PAGE) experiments. The results corresponding to the target recognition stage are as Figure 2As shown in A, the bands in lanes 1 to 5 are HIV-DNA, A1, B1, F, and W respectively. After W is mixed with A1 and B1, the relevant bands become slower and slower (lanes 6 and 7), indicating the formation of the W / B1 / A1 complex; after the W / B1 / A1 complex is incubated with F alone, two bright bands appear in lane 8, representing the W / B1 / A1 complex and F respectively, and a faint band representing F / B1 is also seen. This may be because the excess F directly undergoes a strand displacement reaction with the W / B1 / A1 complex without the toehold being exposed. This leakage reaction is also a common problem in TMSDR, but through reasonable sequence design, the leakage has been minimized as much as possible, so the band of F / B1 is very faint. All these indicate that in the absence of the target, the excess F hardly reacts with the W / B1 / A1 complex; lane 9 reflects the interaction between the W / B1 / A1 complex and HIV-DNA alone. It is observed that the band representing T disappears, the band representing A1 reappears, and at the same time, a band of the W / B1 / HIV-DNA complex slightly slower than the band representing the W / B1 / A1 complex can be seen, indicating that T successfully displaces A1; after the W / B1 / A1 complex is incubated with T and F simultaneously (lane 10), it is observed that the bands representing W, A1, and HIV-DNA reappear, a brighter band of F / B1 appears, and the band of the triple-stranded complex almost disappears, indicating that under the condition of the simultaneous presence of T and F, it can undergo a strand displacement reaction with the W / B1 / A1 complex and cycle continuously, resulting in the full exposure of W in the closed state.

[0081] The signal output stage, that is, the walking process of the DNA walker, was also verified by PAGE. As Figure 2 shown in B, the bands in lanes 1 to 5 are A2, B2, LD, W, and S respectively, and Figure 3Compared with the early experimental results, the sequence design of the current auxiliary strand A2 has a significant impact on the stability of the S / A2 duplex and subsequent reactions, even with a difference of only one base. In the current design, adding a guanine base to A2 effectively enhances the stability of the relevant S / A2 duplex (lane 6) and S / (A2+B2) complex (lane 8), thus making the proposed strategy accurate. After incubating the S / (A2+B2) complex with W, the band representing the S / (A2+B2) complex became darker and a lighter band representing A2 was observed. At the same time, a significantly slower new band appeared in lane 9, indicating that W successfully replaced A2 to form the S / (W+B2) complex; after incubating the S / (A2+B2) complex with LD alone, only two bands representing the S / (A2+B2) complex and LD were observed (lane 10), indicating that the S / (A2+B2) complex has excellent thermodynamic stability and LD cannot react with it in the absence of W; lane 11 shows the bands after the reaction of the S / (A2+B2) complex, W, and LD together. The appearance of the band corresponding to A2 implies the TMSDR-driven transformation from the S / (A2+B2) complex to the S / (W+B2) complex. In addition, the presence of the band representing B2 and the absence of the band related to the S / (W+B2) complex indicate the dissociation of the S / (W+B2) complex because the TMSDR driven by LD forms a stable S / LD duplex. As seen in lane 11, the band position of the newly formed S / LD duplex is basically the same as that of the S / (A2+B2) complex because the number of bases in LD is the same as the sum of A2 and B2. The results of the PAGE experiment confirm the feasibility of the strategy proposed in this example.

[0082] Characterization of the multi-legged DNA walker:

[0083] First, the preparation process of the multi-legged DNA walker was characterized by transmission electron microscopy (TEM). As Figure 4 shown in A, the synthesized AuNPs are spherical in the TEM image, with an average particle size of about 13.4 nm. Due to the electrostatic repulsion generated by the citrate ions wrapped on the surface, they show sufficient dispersibility. After modifying the gold nanoparticles with W ( Figure 4 B) and further blocking with B1 and A1 ( Figure 4 C), the particle sizes increased to 15.0 nm and 15.81 nm respectively, which is attributed to the residual salts attached to the DNA strands. In addition, compared with image A, the dispersibility of the particles in images B and C is significantly improved, mainly because the negative charges of the high-density DNA strands significantly increase the electrostatic repulsion between the particles. These TEM images indicate the successful preparation of AuNP-W and AuNP-W / B1 / A1.

[0084] Zeta potential measurements were performed for each step in the preparation process of the AuNP-W / B1 / A1 bioconjugate. The Zeta potential test of AuNPs showed -26.7 mV, mainly due to the presence of sodium citrate carboxylate during the synthesis process, which coated the entire particle. After modifying the surface of AuNPs with W, the Zeta potential shifted positively to -18.9 mV because the thiol group of W replaced sodium citrate. Although the attached DNA strand contains a large amount of phosphate, the negatively charged groups near the AuNP surface still decreased because the multi-stage ionization constant of phosphoric acid is much smaller than that of citric acid, so the Zeta potential of AuNP-W shifted positively. After incubation of AuNP-W with A1 and B1, the Zeta potential shifted negatively to -24.7 mV because the increased DNA molecules brought more negative charges.

[0085] The preparation of AuNP-W was characterized by ultraviolet-visible absorption spectroscopy (UV-vis), as Figure 5 shown in A. The maximum absorption peak of W appears at 260 nm (curve a), which is consistent with the characteristic peak position of DNA. The synthesized gold nanoparticles have an absorption peak near 520 nm (curve b), which is consistent with the size of the gold nanoparticles observed in the TEM image. After modifying W on the surface of AuNPs, the DNA characteristic peak and the AuNP characteristic peak were observed (curve c). In addition, the latter showed an obvious red shift at 525 nm. After further blocking AuNP-W with B1 and A1, the DNA characteristic peak and the AuNP characteristic peak red-shifted to 527 nm were also observed, indicating the successful preparation of AuNP-W and AuNP-W / B1 / A1 bioconjugates.

[0086] Electrochemical characterization:

[0087] Electrochemical impedance spectroscopy (EIS) was used to analyze the stepwise modification and reaction process of the sensor in 0.1 M KCl containing 5.0 mM [Fe(CN)6] 3- / 4- . As Figure 5 shown in B, the inset is the equivalent fitting circuit, and the symbols in the figure are respectively represented as: R ct is the surface charge transfer resistance, Z W is the Warburg impedance generated by the diffusion of the redox probe, R Ω is the electrolyte solution resistance, C d is the double-layer capacitance. Curve a is almost a straight line, which is the EIS spectrum of the bare gold electrode, indicating that the electron transfer rate is very fast and the R ct value is only 3.850 Ω. Subsequently, after anchoring S and blocking the electrode with MCH, due to the negatively charged phosphate backbone of DNA and [Fe(CN)6] 3- / 4-The repulsion between anions causes a semicircular area to appear on curve b. ct The value increases to 161.4Ω. After S is combined with A2 and B2, a larger semicircle appears, and its R ct The value continued to increase to 244.6Ω (curve c). This is the gradual modification and construction process of the sensor. In the subsequent reaction process, after the AuE / S / A2+B2 sensor was incubated with AuNP-W, the semicircle radius was significantly enlarged, and R ct The value increased by 338.2Ω (curve d), indicating that AuNP-W connected the orbital by replacing the A2. In the presence of both AuNP-W and LD, R ct The value dropped sharply to 255.6Ω (curve e), indicating that the LD-driven AuNP-W rolled continuously, leaving a large number of S / LD duplexes on the electrode surface. The above results fully confirmed that each step of the modification and reaction of the sensor occurred successfully as expected.

[0088] To further confirm the feasibility of the proposed sensing strategy, electrochemical tests were performed using alternating current voltammetry (ACV) in 10 mM Tris-HCl buffer. Figure 5 As shown in Figure C, when HIV-DNA is not present, a very small current peak is observed near -0.23V, and a large current peak is observed near +0.47V, which are the electrochemical signals belonging to MB and Fc, respectively. Without HIV-DNA, the closed AuNP-W / B1 / A1 bioconjugate cannot be activated, so it cannot be introduced into the walking track. In this case, the fuel chain LD also cannot function. Therefore, the attachments on the sensor surface remain almost in their original state. The reason for the existence of this small background signal is the leakage reaction that is difficult to eliminate by TMSDR itself. In TMSDR, in order to ensure the reaction rate, the fuel chain as the driving force is usually excessive. Although the three-chain complex in the absence of the target does not expose the toehold that interacts with the fuel chain, the leakage is considered to be caused by the "no toehold" chain displacement reaction between them, and the high concentration of the fuel chain will aggravate the leakage problem, which is also mutually confirmed by the results of the PAGE part. Therefore, this embodiment reduces the background signal as much as possible through clever chain design. In the presence of 1pM HIV-DNA, the oxidation peak current (I Fc ) was significantly reduced, while the oxidation peak current (I MB ) increased significantly (curve b). This indicates that the conversion from AuNP-W / B1 / A1 bioconjugate to AuNP-W is triggered by HIV-DNA, thus enabling the subsequent rolling of the multi-legged DNA walker and the replacement of Fc-labeled B2 with MB-labeled LD by TMSDR. The signal ratio of MB to Fc (I MB / IFc ) is used to evaluate the content of HIV-DNA. To further study the signal amplification effect of the multi-legged DNA walker, a single-legged DNA walker (W / B1 / A1) was used for a control experiment to detect the same concentration of HIV-DNA. The obtained curve c shows that W can also be released through target recycling and walk on the track through TMSDR, resulting in I Fc and I MB reverse movement. However, the corresponding current change amplitude is significantly smaller than that of using the multi-legged DNA walker AuNP-W. Compared with the control, the I MB / I Fc value of this strategy is increased by about 2.2 times, demonstrating the high reaction efficiency of the multi-legged DNA walker. The same conclusion was also obtained from Figure 6 . In the absence of W (curve a), a very small I MB and a large I Fc were observed, indicating that LD cannot react with S / (A2+B2) alone. The ACV test was carried out under the condition of adding 1 nM AuNP-W (curve b), and it can be observed that I MB and I Fc increased and decreased significantly respectively, indicating that AuNP-W can roll on the electrode surface driven by LD. Finally, a control experiment was carried out with 1 μM single-stranded W (curve c), and the I MB / I Fc is significantly smaller than that of AuNP-W with a concentration of only 1 nM, which may be due to the fact that the locally higher concentration of W on the surface of AuNPs can effectively prevent AuNP-W from derailing during the reaction, thereby improving the continuity of TMSDR and the reaction efficiency. The above results fully verify the feasibility of the strategy proposed in this embodiment.

[0089] Optimization of experimental conditions:

[0090] As the key unit for signal amplification, the relevant parameters of the AuNP-W / B1 / A1 bioconjugate will greatly affect the performance of the sensor, including the NaCl concentration in the buffer solution, the concentration of the AuNP-W / B1 / A1 bioconjugate, the target recognition time, and the rolling time of the multi-legged DNA walker. In this embodiment, they were optimized under the condition of the presence of 1 pM HIV-DNA. First, the effect of the NaCl concentration in all buffer solutions used for TMSDR was studied, ranging from 200 to 600 mM ( Figure 7 A). Since NaCl can promote the reaction between DNA molecules by shielding electrostatic repulsion, the signal-to-noise ratio (S / N) initially increases with the increase of c NaCl . However, with the increase of c NaClAbove 400 mM, the S / N value began to decline, indicating that excessive NaCl made the DNA double-strand too stable to be displaced. Therefore, 400 mM of NaCl is the most suitable salt concentration for this TMSDR system. Then, the concentration of AuNP-W / B1 / A1 was optimized in the range of 1 nM - 20 nM ( Figure 7 B). In the low concentration range, the I MB / I Fc value increased with the increase in the concentration of AuNP-W / B1 / A1 because more and more related DNA dynamic systems were activated. When the concentration of AuNP-W / B1 / A1 was higher than 8 nM, the response of the sensor began to decline. This may be attributed to the competitive reaction between Ws attached to adjacent AuNPs and T-DNA, which reduced the percentage of W strands released on each AuNP and further affected the rolling efficiency of the resulting multi-legged DNA walker. Therefore, 8 nM was selected as the working concentration of AuNP-W / B1 / A1. The length of the target recognition time directly affects the activation degree of the multi-legged DNA walker, which is very important for the subsequent rolling process. Figure 7 C shows the effect of the target recognition time on the sensor response. It was observed that as the target recognition time extended, the I MB / I Fc value gradually increased. Then a stable plateau appeared after 80 minutes, indicating the depletion of the AuNP-W / B1 / A1 substrate. Therefore, 80 minutes was sufficient for the target recognition reaction. Finally, the rolling time of the multi-legged DNA walker on the sensor surface was optimized. As Figure 7 shown in D, the sensor response continuously increased with the extension of t rolling and reached a plateau at 60 minutes, and then there was no significant change, indicating that the reaction process on the sensor surface had been fully carried out. Therefore, 60 minutes was selected as the rolling time of the multi-legged DNA walker.

[0091] Establishment of the standard curve and calculation of the detection limit:

[0092] Under the optimized experimental conditions, a series of ACV tests were performed on different concentrations of HIV-DNA (10 aM to 0.1 nM) using the prepared sensor. As Figure 8 shown in A, as the concentration of HIV-DNA increased, it could be observed that the I MB gradually increased and the I Fc gradually decreased. According to the mechanism of the ratio-type sensor, the signal ratio I MB / I Fc was used to evaluate the content of HIV-DNA.

[0093] Finally, the peak value of the obtained ACV curve was measured, and I MB / I FcAs the final output signal, a linear relationship is established with the logarithm of the HIV-DNA concentration, as Figure 8 shown in B. The obtained linear relationship is calculated to get an equation I MB / I Fc = 1.35507 + 0.06282 log c HIV-DNA . The correlation coefficient is 0.99229, and the linear range is from 100 aM to 10 pM. According to the 3σ rule, the lowest detection limit (LOD) of the sensor for HIV-DNA is calculated to be 36.71 aM, indicating that the sensor designed and developed in this example has good detection performance.

[0094] Selectivity, reproducibility and stability:

[0095] Selectivity, reproducibility and stability are important indicators for evaluating the practical application performance of biosensors. The evaluation of the selectivity of the sensor is achieved by detecting four different DNA fragments (all at a concentration of 1 fM), namely HIV-DNA, single-base mismatched DNA (Sm-T), triple-base mismatched DNA (Tm-T) and non-complementary DNA (N-T). As Figure 8 shown in C, it can be observed that the sensor has a strong signal response to HIV-DNA, while the signal responses to Sm-T, Tm-T and N-T are significantly reduced, indicating that the sensor designed in this example has good selectivity.

[0096] To test the reproducibility of the sensor, a group of five sensors were fabricated under the same conditions for detecting 1 fM HIV-DNA. Finally, the measured ACV signal responses were compared, and the relative standard deviation (RSD) between the responses of different sensors was 4.23%, indicating that the sensor has excellent reproducibility.

[0097] To study the stability of the proposed sensor, a batch of fabricated sensors were stored at 4°C. After 0, 1, 3, 5, 7, 14 days, the same concentration of HIV-DNA (1 fM) was detected respectively. As Figure 9 shown, the proposed sensor shows a relatively stable response. After storing for 7 days and 14 days, it still maintains 94.22% and 86.27% of the initial response respectively, indicating that the proposed sensor has acceptable stability.

[0098] Complex biological sample analysis:

[0099] To study the analytical performance of the designed sensor in complex biological samples, a spiking / recovery experiment was conducted in human serum. First, normal human serum was diluted 10-fold with 10 mM Tris-HCl buffer solution. Then, solutions containing 1.000, 10.00, 100.0, and 1000 fM HIV-DNA were prepared with the 10-fold diluted normal human serum and detected by the proposed sensor. As shown in Table 2, the recovery rates were 99.32%, 98.94%, 99.59%, and 97.81%, respectively, and the RSD was less than 6.2%. The above results indicate that the proposed sensor can be used as a potential tool for analyzing complex biological samples.

[0100] Table 2 Detection of HIV-DNA in serum by a ratio-type electrochemical biosensor based on a multi-legged DNA walker

[0101]

[0102] Conclusion:

[0103] In this example, a novel ratio-type electrochemical biosensor based on a TMSDR-driven dynamic DNA nanosystem was constructed for ultrasensitive detection of nucleic acids. The synergistic effect of the target recycling unit and the multi-legged DNA walker unit within the dynamic DNA nanosystem, as well as the ratio-type readout mode, achieved cascade signal amplification and ultrasensitive detection of HIV-DNA in complex biological samples. As the driving force of the dynamic DNA nanosystem, TMSDR not only realized an enzyme-free isothermal amplification strategy but also ensured the high specificity of this detection strategy. By appropriately adjusting the target recognition unit, the proposed sensor can be extended to detect a wide range of nucleic acid targets, providing a general strategy for various clinical diagnostic applications. The successful preparation of the multi-legged DNA walker and the feasibility of the scheme were fully verified by means such as PAGE, TEM, UV-vis, and Zeta potential. The analytical performance of the sensor was comprehensively evaluated by electrochemical tests. The linear range was 100 aM - 10 pM, and the detection limit was as low as 36.71 aM. The results showed that the sensing strategy in this example had high sensitivity. In addition, it also exhibited good specificity, reproducibility, and stability, and ideal recovery rates were also measured when analyzing the targets in serum. These results all indicate that the sensor in this example shows great application potential in clinical analysis and disease diagnosis.

[0104] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A biosensor based on a dynamic DNA nanosystem driven by strand displacement reaction, which is composed of a target recycling unit and a multi-legged DNA walker unit; The target recycling unit includes a multi-legged DNA walker, an auxiliary strand A1, a blocking strand B1, and a fuel strand F; the multi-legged DNA walker is formed by connecting at least 2 walking strands W to the surface of a gold nanoparticle, and one end of the walking strand W is connected to the gold nanoparticle; the blocking strand B1 is provided with a W region, an A1 region, and a first toehold region; the W region can be complementary to the DNA fragment at the other end of the walking strand W; the A1 region can be complementary to the auxiliary strand A1; the first toehold region can hybridize with the target nucleic acid, and through strand displacement reaction, the target nucleic acid displaces the auxiliary strand A1, and at the same time, a second toehold region is exposed in the middle part of the blocking strand B1; the fuel strand F can hybridize with the second toehold region and replace the walking strand W and the target nucleic acid through strand displacement reaction; The multi-legged DNA walker unit includes a gold electrode, an auxiliary strand A2, a blocking strand B2, and a labeled DNA; a number of substrate strands S are connected to the surface of the gold electrode; the substrate strand S is provided with a B2 region, an A2 region, and a third toehold region; the B2 region can be complementary to the blocking strand B2; the A2 region can be complementary to the auxiliary strand A2; the first toehold region can hybridize with the walking strand W, and through strand displacement reaction, the DNA fragment at the other end of the walking strand W displaces the auxiliary strand A2, and at the same time, a fourth toehold region is exposed in the middle part of the substrate strand S; the labeled DNA can hybridize with the fourth toehold region and replace the blocking strand B2 and the DNA fragment at the other end of the walking strand W through strand displacement reaction; one end of the blocking strand B2 is connected to a first electrochemistry signal material, and after the blocking strand B2 hybridizes with the substrate strand S, the first electrochemistry signal material is close to the gold electrode; one end of the labeled DNA is labeled with a second electrochemistry signal material; after the labeled DNA hybridizes with the substrate strand S, the second electrochemistry signal material is close to the gold electrode.

2. The biosensor of the dynamic DNA nanosystem driven by strand displacement reaction according to claim 1, characterized in that, The gold nanoparticle and the walking strand W are connected by a gold-sulfur bond.

3. The biosensor of the dynamic DNA nanosystem driven by strand displacement reaction according to claim 1, characterized in that, The gold electrode is blocked with mercaptohexanol.

4. The biosensor of the dynamic DNA nanosystem driven by strand displacement reaction according to claim 1, characterized in that, The first electrochemistry signal material is ferrocene, and the second electrochemistry signal material is methylene blue.

5. The biosensor of the dynamic DNA nanosystem driven by strand displacement reaction as claimed in claim 1, wherein, The sequence of the auxiliary strand A1 is shown in SEQ ID NO.2, the sequence of the blocking strand B1 is shown in SEQ ID NO.3, the sequence of the fuel strand F is shown in SEQ ID NO.4, the sequence of the walking strand W is shown in SEQ ID NO.5, the sequence of the substrate strand S is shown in SEQ ID NO.6, the sequence of the auxiliary strand A2 is shown in SEQ ID NO.7, the sequence of the blocking strand B2 is shown in SEQ ID NO.9, and the sequence of the labeled DNA is shown in SEQ ID NO.

10.

6. Application of the biosensor based on the dynamic DNA nanosystem driven by strand displacement reaction according to any one of claims 1 to 5 in nucleic acid detection.

7. A method for detecting a nucleic acid, characterized in that, Providing the biosensor based on the dynamic DNA nanosystem driven by strand displacement reaction according to any one of claims 1 to 5 includes the following steps: Incubate the multi-legged DNA walker, auxiliary strand A1, and blocking strand B1 to form a blocked multi-legged DNA walker; Incubate the gold electrode with several substrate strands S attached to its surface, auxiliary strand A2, and blocking strand B2 to form a blocked gold electrode; Mix and react the blocked multi-legged DNA walker, fuel strand F, and the nucleic acid of the target to be detected. After the reaction, mix the reaction solution with the labeled DNA and then drop it onto the surface of the blocked gold electrode for incubation. Detect the electrochemical signal after incubation.

8. The method for detecting nucleic acid according to claim 7, characterized in that, The incubation time of the multi-legged DNA walker, auxiliary strand A1, and blocking strand B1 is 70 - 90 min; Or, the incubation time of the gold electrode with several substrate strands S attached to its surface, auxiliary strand A2, and blocking strand B2 is 70 - 90 min; Or, the mixing and reaction time of the blocked multi-legged DNA walker, fuel strand F, and the nucleic acid of the target to be detected is 70 - 90 min; Or, the incubation time after mixing the reaction solution with the labeled DNA and dropping it onto the surface of the blocked gold electrode is 50 - 70 min.

9. A nucleic acid detection kit, characterized in that, A biosensor and a buffer solution comprising the dynamic DNA nanosystem based on strand displacement reaction driving according to any one of claims 1 - 5.

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