Programmable DNA nanotube mediated molecular machine and exosome nucleic acid detection method thereof

Abstract

The invention discloses a molecular machine based on programmable DNA nanotube mediation and a detection method of exosome nucleic acid of the molecular machine, and the preparation method of the molecular machine based on programmable DNA nanotube mediation is characterized by comprising the following steps: (1) complementarily pairing target exosome nucleic acid miR-193b and a template sequence Probe A to obtain a product extension chain; (2) carrying out catalytic extension and cutting on an extension chain by using Klenow polymerase, i.e., carrying out multiple rounds of polymerization and cutting on the product in the step a to generate a large amount of Probe B for subsequent reaction; and (3) enabling S1, S2, S3-DNAzyme, S4 and S5 to react in a TM buffer solution at 37 DEG C for 1 hour, so as to prepare the blocked molecular machine based on the programmable DNA nanotube mediation. The technology realizes high-sensitivity and high-selectivity nucleic acid detection, and has important clinical diagnostic value.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a molecular machine mediated by programmable DNA nanotubes and a method for detecting exosome nucleic acid thereof. Background Art

[0002] With the deepening of biomedical research, the detection of exosomal nucleic acids (such as miRNA) has received widespread attention due to its important role in disease diagnosis and treatment monitoring. Exosomal nucleic acids are small molecule nucleic acids present in extracellular vesicles. They play a key role in a variety of physiological and pathological processes, including intercellular communication, gene expression regulation, and the occurrence and development of diseases. Therefore, the development of a highly sensitive and specific exosomal nucleic acid detection method is of great significance for early diagnosis, efficacy evaluation, and prognosis judgment.

[0003] Traditional nucleic acid detection methods, such as PCR and Northern blot, have high sensitivity and specificity, but these methods usually require complex operation procedures, long detection time and professional experimental equipment, which limits their application in clinical rapid detection. In addition, these methods often require a high sample volume, while the extraction amount of exosome nucleic acid is usually low, which further limits their use in exosome nucleic acid detection.

[0004] To overcome these limitations, researchers have developed new detection platforms based on nanomaterials and biomolecular engineering. These platforms use the high specific surface area of ​​nanomaterials and the specific recognition ability of biomolecules to achieve high-sensitivity detection of target nucleic acids. However, existing detection platforms still face problems such as low signal amplification efficiency, high detection limit and insufficient specificity.

[0005] Currently, there is a lack of a detection method based on programmable DNA nanotube-mediated molecular machines and their exosomal nucleic acids for accurately distinguishing Alzheimer's patients from normal people. Summary of the invention

[0006] The purpose of the present invention is to provide a method for accurately distinguishing Alzheimer's disease patients from normal people based on programmable DNA nanotube-mediated molecular machines and their exosomal nucleic acid detection methods.

[0007] In order to solve the problems of the prior art, the present invention provides the following technical solutions:

[0008] In a first aspect, the present application provides a method for preparing a molecular machine mediated by programmable DNA nanotubes.

[0009] In a second aspect, the present application provides a molecular machine mediated by programmable DNA nanotubes prepared by a preparation method.

[0010] In a third aspect, the present application provides an ultra-sensitive method for detecting exosomal nucleic acids based on programmable DNA nanotube-mediated molecular machines.

[0011] The first aspect of the present application provides a method for preparing a molecular machine mediated by programmable DNA nanotubes, which is characterized by comprising the following steps:

[0012] (1) Complementarily pairing the target exosomal nucleic acid miR-193b with the template sequence Probe A to obtain a product extension chain;

[0013] (2) using Klenow polymerase to catalyze the extension and cleavage of the extended chain, i.e., the product in step (1), to undergo multiple rounds of polymerization and cleavage to generate a large amount of Probe B for subsequent reactions;

[0014] (3) S1, S2, S3-DNAzyme, S4, and S5 were reacted in TM buffer at a temperature of 37° C. for a reaction time of 0.5-2 h to obtain a blocked molecular machine mediated by programmable DNA nanotubes.

[0015] Further, in step (1), the nucleotide sequence of miR-193b is shown as SEQ ID NO.6, and the nucleotide sequence of Probe A is shown as SEQ ID NO.7; in step (3), the nucleotide sequence of S1 is shown as SEQ ID NO.1; the nucleotide sequence of S2 is shown as SEQ ID NO.2; the nucleotide sequence of S3 is shown as SEQ ID NO.3; the nucleotide sequence of S4 is shown as SEQ ID NO.4; the nucleotide sequence of S5 is shown as SEQ ID NO.5; and the nucleotide sequence of Probe B is shown as SEQ ID NO.8.

[0016] Further, in step (3), Probe B is mixed with the blocked DNA nanotubes, and Probe B opens the ribozyme blocked on the DNA nanotubes to obtain activated DNA nanotubes.

[0017] Furthermore, the second aspect of the present application provides a molecular machine mediated by programmable DNA nanotubes prepared by a preparation method.

[0018] The third aspect of the present application provides a method for detecting exosomal nucleic acids using an ultrasensitive molecular machine, comprising the following steps: placing a DNA nanotube-mediated molecular machine in the presence of a suitable cofactor Mg 2+ In the presence of ferrocene, it can roll on the gold electrode surface and cleave the substrate chain Probe C with a ferrocene Fc signal tag, generating an amplified electrochemical signal.

[0019] Further, the gold electrode was cleaned and then modified: the bare electrode was soaked in a piranha solution for 10 min, the ratio of the piranha solution was 98% H2SO4:30% H2O2 was 3:1, and then polished with 1μm and 0.3μm aluminum pastes respectively. Subsequently, the electrode was ultrasonically treated in anhydrous ethanol, double distilled water and 50% HNO3 for 5 min; finally, the electrode was activated in a 0.5M H2SO4 solution. In order to reduce the disulfide bonds, the probe was incubated in 100mM TCEP at room temperature for 1h, and then 1μM Probe C was incubated with the bare electrode for 8h. Subsequently, the impurities on the electrode surface were rinsed with pure water, and finally blocked with 1mM MCH solution for 30min for use.

[0020] Further, the detection of the electrochemical signal is performed by electrochemical impedance spectroscopy analysis EIS, cyclic voltammetry analysis CV or square wave voltammetry SWV. The detection limit of the method is 5.7fM. The method is specific to the exosomal nucleic acid miR-193b, and only the presence of the exosomal nucleic acid miR-193b will cause a significant change in the electrochemical signal compared with other miRNAs.

[0021] Further, the probe was modified by incubating the probe in 100 mM TCEP for 1 hour to reduce disulfide bonds; incubating the bare electrode with 1 μM Probe C for 8 hours; washing the impurities on the electrode surface with pure water; and blocking with 1 mM MCH solution for 30 minutes.

[0022] Furthermore, the nucleotide sequence of ProbeC is shown in SEQ ID NO.9.

[0023] Beneficial effects: The present invention provides an efficient, sensitive, specific and cost-effective molecular detection platform. The method of the present invention utilizes the synergistic effect of strand displacement amplification (SDA) and DNA nanotubes to achieve ultrasensitive detection of exosomal nucleic acids. By precisely designing DNA probes and nanostructures, this method can detect target nucleic acids at low concentrations while maintaining high specificity and rapid response, providing a new technical means for the clinical detection of exosomal nucleic acids. It has important application value for biomedical research and clinical diagnosis.

[0024] Compared with the prior art, the present invention has the following advantages: (1) Ultrasensitive detection: The present invention uses programmable DNA nanotube-mediated molecular machines to achieve ultrasensitive detection of exosomal nucleic acid miR-193b. This detection method has high sensitivity and can detect very low concentrations of miR-193b, which is of great significance for early disease diagnosis and treatment monitoring. Signal amplification: Through strand displacement amplification (SDA) and DNA nanotube-mediated molecular machines, the present invention can generate amplified electrochemical signals, which helps to improve the sensitivity and accuracy of detection.

[0025] (2) High selectivity: The present invention demonstrates high selectivity for the specific exosomal nucleic acid miR-193b, which means that it can accurately detect the target molecule in complex biological samples without interference from other similar molecules. Easy operation: The experimental steps are clear, including the selection of materials and instruments, PAGE analysis, preparation of SDA and blocked DNA nanotubes, preparation of working electrodes, and electrochemical measurement, which ensure the repeatability and reliability of the experiment.

[0026] (3) Cost-effectiveness: The materials and instruments used are common laboratory items, easy to obtain, and the preparation process is relatively simple, which helps to reduce the cost of detection. Rapid response: Through electrochemical methods such as cyclic voltammetry (CV) and square wave voltammetry (SWV), the present invention can quickly respond to and detect target molecules, which is very useful for situations that require rapid diagnosis.

[0027] (4) Stability and repeatability: The present invention exhibits good stability and repeatability as determined by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), which is critical to ensuring the reliability of the test results. Low detection limit: According to the signal-to-noise ratio calculated by the 3σ method, the detection limit (LOD) is 5.7fM, which indicates that the present invention has extremely high sensitivity and can detect extremely low concentrations of exosomal nucleic acid miR-193b. Wide application: Due to the role of exosomal nucleic acid miR-193b in a variety of diseases, the detection method of the present invention can be applied to the early diagnosis and treatment monitoring of a variety of diseases, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application.

[0029] Figure 1 This is a diagram showing the preparation of the DNA nanotube-mediated molecular machine of the present invention and its effective operation on a gold electrode. Figure 1 A is the probe A of the present invention, which is combined with miR-193b and then extended and cut by Klenow fragment polymerase to generate Probe B. Figure 1 B is the Probe B of the present invention, which opens the ribozyme blocked on the DNA nanotube to form an activated DNA nanotube. Figure 1 C is the DNA nanotube of the present invention cutting Probe C with Fc on the surface of the gold electrode to generate an amplified electrochemical signal.

[0030] Figure 2 The figure is a diagram of PAGE verifying the assembly of SDA and blocked nanotubes and the ability of ribozyme to cleave substrates of the present invention. Figure 2A is the SDA verification diagram of the present invention. Lane 1: exosomal nucleic acid miR-193b; Lane 2: Probe A; Lane 3: mixed miR-193b and Probe A; Lane 4: mixed miR-193b, Probe A and Klenow. Lane 5: mixed miR-193b, Probe A, Klenow and Nb.BbvCI. Figure 2 B is a synthetic diagram of the blocked DNA nanotubes of the present invention. Lane 1: S1; Lane 2: S2; Lane 3: S3-DNAzyme; Lane 4: S4; Lane 5: S5; Lane 6: a mixture of S1, S2, S3-DNAzyme, S4, and S5. Figure 2 C is a graph of the present invention verifying the ability of the ribozyme to cleave the substrate. Lane 1: Probe B; Lane 2: S3-DNAzyme; Lane 3: Probe C; Lane 4: a mixture of Probe B and S3-DNAzyme; Lane 5: a mixture of S3-DNAzyme and Probe C; Lane 6: Probe B, S3-DNAzyme, Probe C and cofactor Mg 2+ Lane 7: No Mg 2+ Negative control.

[0031] Figure 3 This is a diagram of the assembly of DNA on a gold electrode and the operation of a DNA nanotube according to the present invention. Figure 3 A is the EIS diagram of the assembly of DNA on gold electrode and the operation of DNA nanotubes of the present invention; Figure 3 B is the CV diagram of the assembly of DNA on gold electrode and the operation of DNA nanotubes: I: bare electrode (GE). II: GE+Probe C. III: GE+Probe C+activated DNA nanotubes. IV: GE+Probe C+activated DNA nanotubes+Mg 2+ .

[0032] Figure 4 This is a performance diagram of the DNA nanotube-mediated molecular machine of the present invention. Figure 4 A is the SWV curve of the present invention in response to miR-193b (300 nM). Figure 4 B is the incubation of different concentrations of miR-193b (1, 10, 100, 300, 1×10 3 , 1×10 4 , 1×10 5 , 3×10 5 and 1×10 6 pM) SWV curve diagram. Figure 4 C is a linear relationship diagram of the SWV peak current and miR-193b concentration of the present invention. Figure 4 D is the selective response of the method of the present invention to miR-193b (300 nM) and several other miRNAs (1 μM). Target value, SD, n=3. DETAILED DESCRIPTION

[0033] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0034] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0035] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0036] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0037] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0038] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0039] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0040] The first aspect of the present application provides a method for preparing a molecular machine mediated by programmable DNA nanotubes, which is characterized by comprising the following steps:

[0041] (1) Complementarily pairing the target exosomal nucleic acid miR-193b with the template sequence Probe A to obtain a product extension chain;

[0042] (2) using Klenow polymerase to catalyze the extension and cleavage of the extended chain, i.e., the product in step (1), to undergo multiple rounds of polymerization and cleavage to generate a large amount of Probe B for subsequent reactions;

[0043] (3) S1, S2, S3-DNAzyme, S4, and S5 are reacted in TM buffer at 37°C for 0.5-2 hours to obtain a blocked molecular machine based on programmable DNA nanotubes. Probe B is mixed with the blocked DNA nanotubes, and Probe B opens the ribozyme blocked on the DNA nanotubes to obtain activated DNA nanotubes.

[0044] Further, in step (1), the nucleotide sequence of miR-193b is shown as SEQ ID NO.6, and the nucleotide sequence of Probe A is shown as SEQ ID NO.7; in step (3), the nucleotide sequence of S1 is shown as SEQ ID NO.1; the nucleotide sequence of S2 is shown as SEQ ID NO.2; the nucleotide sequence of S3 is shown as SEQ ID NO.3; the nucleotide sequence of S4 is shown as SEQ ID NO.4; the nucleotide sequence of S5 is shown as SEQ ID NO.5; and the nucleotide sequence of Probe B is shown as SEQ ID NO.8.

[0045] A second aspect of an embodiment of the present application provides a molecular machine mediated by programmable DNA nanotubes prepared by a preparation method.

[0046] The third aspect of the present application provides a method for detecting exosomal nucleic acids using an ultrasensitive molecular machine, comprising the following steps: placing a DNA nanotube-mediated molecular machine in the presence of a suitable cofactor Mg 2+In the presence of ferrocene, it can roll on the gold electrode surface and cleave the substrate chain Probe C with a ferrocene Fc signal tag, generating an amplified electrochemical signal.

[0047] In some embodiments, the gold electrode is cleaned and then modified: the bare electrode is soaked in a piranha solution for 10 minutes, the piranha solution is 98% H2SO4:30% H2O2 in a ratio of 3:1, and then polished with 1μm and 0.3μm aluminum pastes, respectively. Subsequently, the electrode is ultrasonically treated in anhydrous ethanol, double distilled water and 50% HNO3 for 5 minutes; finally, the electrode is activated in a 0.5M H2SO4 solution. In order to reduce disulfide bonds, the probe is incubated in 100mM TCEP at room temperature for 1 hour, and then 1μM Probe C is incubated with the bare electrode for 8 hours. Subsequently, impurities on the electrode surface are rinsed with pure water, and finally, it is sealed with a 1mM MCH solution for 30 minutes for use.

[0048] In some embodiments, the detection of the electrochemical signal is performed by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), or square wave voltammetry (SWV). The detection limit of the method is 5.7 fM. The method is specific to exosomal nucleic acid miR-193b, and only the presence of exosomal nucleic acid miR-193b will cause a significant change in the electrochemical signal compared to other miRNAs.

[0049] In some embodiments, the probe is modified by incubating the probe in 100 mM TCEP for 1 hour to reduce disulfide bonds; incubating the bare electrode with 1 μM Probe C for 8 hours; washing impurities on the electrode surface with pure water; and blocking with 1 mM MCH solution for 30 minutes.

[0050] In some embodiments, the nucleotide sequence of ProbeC is shown as SEQ ID NO.9.

[0051] Example 1

[0052] A method for preparing a molecular machine mediated by programmable DNA nanotubes of the present invention comprises the following steps:

[0053] (1) The target exosomal nucleic acid miR-193b is complementary to the template sequence Probe A to obtain a product extension chain; the nucleotide sequence of miR-193b is shown in SEQ ID NO.6,

[0054] (2) using Klenow polymerase to catalyze the extension and cleavage of the extended chain, i.e., the product in step a, to undergo multiple rounds of polymerization and cleavage to generate a large amount of Probe B for subsequent reactions;

[0055] (3) S1, S2, S3-DNAzyme, S4, and S5 were placed in TM buffer at a reaction temperature of 37°C for a reaction time of 1.5 h to obtain a blocked molecular machine based on programmable DNA nanotubes. The nucleotide sequence of S1 is shown in SEQ ID NO.1; the nucleotide sequence of S2 is shown in SEQ ID NO.2; the nucleotide sequence of S3 is shown in SEQ ID NO.3; the nucleotide sequence of S4 is shown in SEQ ID NO.4; the nucleotide sequence of S5 is shown in SEQ ID NO.5; and the nucleotide sequence of Probe B is shown in SEQ ID NO.8. Probe B is mixed with the blocked DNA nanotubes, and Probe B opens the ribozyme blocked on the DNA nanotubes to obtain activated DNA nanotubes.

[0056] A molecular machine mediated by programmable DNA nanotubes is prepared by a preparation method of the present invention.

[0057] Example 2

[0058] The difference between Example 2 and Example 1 is that in step (3), S1, S2, S3-DNAzyme, S4, and S5 are reacted in TM buffer at a reaction temperature of 37° C. for a reaction time of 0.5 h to obtain a blocked molecular machine mediated by programmable DNA nanotubes.

[0059] Example 3

[0060] The difference between Example 3 and Example 1 is that in step (3), S1, S2, S3-DNAzyme, S4, and S5 are reacted in TM buffer at a reaction temperature of 37° C. for a reaction time of 2 h to obtain a blocked molecular machine mediated by programmable DNA nanotubes.

[0061] Example 4

[0062] The present invention provides a method for detecting exosomal nucleic acids using a molecular machine with ultra-sensitivity, comprising the following steps: placing a DNA nanotube-mediated molecular machine in the presence of a suitable cofactor Mg 2+ In the presence of ferrocene, it can roll on the gold electrode surface and cleave the substrate chain Probe C with a ferrocene Fc signal tag, generating an amplified electrochemical signal.

[0063] The gold electrode was cleaned and then modified: the bare electrode was soaked in a piranha solution for 10 minutes, the ratio of the piranha solution was 98% H2SO4:30% H2O2 was 3:1, and then polished with 1μm and 0.3μm aluminum pastes respectively, and then the electrode was ultrasonically treated in anhydrous ethanol, double distilled water and 50% HNO3 for 5 minutes; finally, the electrode was activated in a 0.5M H2SO4 solution, and in order to reduce the disulfide bond, the probe was placed in 100mM TCEP for 1 hour at room temperature, and then 1μM Probe C was incubated with the bare electrode for 8 hours, and then the impurities on the electrode surface were rinsed with pure water, and finally blocked with 1mM MCH solution for 30 minutes for use. The nucleotide sequence of ProbeC is shown in SEQ ID NO.9.

[0064] The detection of electrochemical signals was performed by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) or square wave voltammetry (SWV). Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) of a three-electrode system (reference electrode, counter electrode and working electrode) were performed using a CHI 760E electrochemical workstation. The frequency range of EIS was 0.01-10kHz and the amplitude was 5mV. CV was performed between 0.6V and -0.2V with a scan rate of 0.05V / s. Square wave voltammetry (SWV) was performed in a 20mM Tris-HCl solution with a step potential of 4mV, a frequency of 15Hz, an amplitude of 25mV, and a scan potential from -0.2V to 0.8V. The detection limit of the method was 5.7fM. The method was specific for exosomal nucleic acid miR-193b, and only the presence of exosomal nucleic acid miR-193b resulted in significant changes in the electrochemical signal compared with other miRNAs.

[0065] Probe modification: The probe was incubated in 100 mM TCEP for 1 hour to reduce disulfide bonds; 1 μM Probe C was incubated with the bare electrode for 8 hours; impurities on the electrode surface were rinsed with pure water; and the electrode was blocked with 1 mM MCH solution for 30 minutes.

[0066] Test Example 1

[0067] 1. Experimental part

[0068] 1.1 Materials and Instruments

[0069] Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 6-mercapto-1-hexanol (MCH), and ultrapure water were provided by Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Nb.BbvCI and DNA polymerase I (Klenow) were provided by New England Biotechnology Co., Ltd. (Beijing, China). All oligonucleotides used in this work were synthesized and purified by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Gel imaging was performed on a ChemiScope 6200 (Shanghai, China). Electrochemical measurements were performed using a CHI 760E electrochemical workstation (CH Instruments, China).

[0070] 1.2PAGE

[0071] 10% PAGE was performed using 1×TBE buffer (89 mM Tris-HCl, 2 mM EDTA, pH 8.0). The loading sample was prepared by mixing 10 μL of nucleic acid sample, 1 μL of gel red dye, and 2 μL of 6× loading buffer. Electrophoresis was performed at 100 V for 45 min, followed by exposure in an imaging system.

[0072] 1.3 Strand displacement amplification (SDA) and blocked DNA nanotube preparation

[0073] First, miR-193b was complementary to the template sequence (Probe A) and extended using Klenow polymerase, followed by further introduction of the cleavage extension chain, multiple rounds of polymerization and cleavage to generate a large amount of Probe B for subsequent reactions. Secondly, S1, S2, S3-DNAzyme, S4, and S5 were reacted in TM buffer at 37°C for 1 h to prepare blocked DNA nanotubes.

[0074] 1.4 Preparation of working electrode

[0075] The gold electrode was first cleaned and then modified. In short, the bare electrode was soaked in a piranha solution (98% H2SO4: 30% H2O2 = 3:1) for 10 min and then polished with 1μm and 0.3μm aluminum pastes, respectively. Subsequently, the electrode was ultrasonically treated in anhydrous ethanol, double distilled water, and 50% HNO3 for 5 min. Finally, the electrode was activated in a H2SO4 (0.5M) solution. In order to reduce disulfide bonds, the probe was incubated in 100mM TCEP for 1h at room temperature, and then 1μM Probe C was incubated with the bare electrode for 8h. Subsequently, the impurities on the electrode surface were rinsed with pure water, and finally blocked with MCH (1mM) solution for 30min and set aside.

[0076] 1.5 Electrochemical measurements

[0077] Electrochemical measurements were performed on a CHI 760E electrochemical workstation (CH Instruments, China). The three-electrode system included a reference electrode, a counter electrode, and a working electrode. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were performed in PBS containing 5 mM Fe(CN)63- / 4- solution (0.1 M KCl). The frequency range of EIS was 0.01-10 kHz with an amplitude of 5 mV. CV was performed between 0.6 V and -0.2 V with a scan rate of 0.05 V / s. Square wave voltammetry (SWV) was performed in 20 mM Tris-HCl solution with a step potential of 4 mV, a frequency of 15 Hz, an amplitude of 25 mV, and a scanning potential from -0.2 V to 0.8 V.

[0078] 2. Results

[0079] 2.1 Working Mechanism

[0080] The schematic diagram of strand displacement amplification SDA and DNA nanotube-mediated molecular machine rolling at the gold electrode interface is shown in Scheme 1. First, miR-193b and Probe A complementarily pair to generate a large amount of Probe B under the action of Klenow polymerase catalytic extension and cleavage. Then, Probe B is used to unlock the blocked DNA nanotubes pre-formed in TM buffer. Finally, the activated DNA nanotubes are activated in the presence of appropriate cofactors (Mg 2+ ) rolls on the electrode surface in the presence of , cleaving the substrate chain (Probe C) with a ferrocene (Fc) signal tag, generating an amplified electrochemical signal.

[0081] Strand Displacement Amplification (SDA) is a nucleic acid amplification technique that uses the strand displacement ability of DNA polymerase to amplify the target nucleic acid sequence. The SDA process usually includes the following steps:

[0082] Initialization: A primer that is partially complementary to the target nucleic acid sequence (usually called the initialization primer) is used to hybridize with the target sequence, and then DNA polymerase starts to synthesize a new DNA chain from the primer.

[0083] Strand displacement: When the newly synthesized DNA chain extends to the region complementary to another primer (called the displacement primer), due to the strand displacement activity of the DNA polymerase, the new chain can replace the original template chain, thereby releasing a single-stranded DNA region.

[0084] Cycle: The above process is repeated over and over again, and each cycle produces more single-stranded DNA regions, which can serve as new templates to further hybridize with primers and continue the amplification process.

[0085] Signal amplification: During the SDA process, specific primers and probes can be designed so that the amplified products can trigger the generation of signals, such as fluorescent signals, electrochemical signals, etc., thereby realizing the detection of target nucleic acids.

[0086] The advantages of SDA technology include high sensitivity, rapidity, no need for thermal cycling equipment, and can be designed as an isothermal reaction, which is suitable for rapid on-site detection and nucleic acid detection in low-resource environments. In the present invention, SDA is used to generate a large amount of ProbeB, which is a key step in the subsequent unlocking of DNA nanotubes and signal amplification.

[0087] Figure 1 .( Figure 1 A) Probe A binds to miR-193b and is extended and cleaved by Klenow fragment polymerase to produce Probe B. ( Figure 1 B) Probe B opens the ribozyme blocked on the DNA nanotube to form an activated DNA nanotube. Figure 1 C) DNA nanotubes cleave Probe C with Fc on the surface of the gold electrode, generating an amplified electrochemical signal.

[0088] 2.2 SDA verification and assembly of blocked DNA nanotubes

[0089] First, 10% PAGE analysis was performed to verify SDA. Figure 2 As shown in A, lanes 1 and 2 are miR-193b and Probe A, respectively. Lane 3 forms a bright band at a higher position, proving that miR-193b and Probe A are successfully complementary. Klenow polymerase and Nb.BbvCI are added to lane 5 to produce Probe B. Nb.BbvCI is not added to lane 4. Then, the assembly of blocked DNA nanotubes is verified. Figure 2 As shown in B, lanes 1, 2, 3, 4, and 5 are S1, S2, S3-DNAzyme, S4, and S5, respectively. The high bright band formed by mixing them in lane 6 proves that the blocked DNA nanotubes are successfully assembled. Finally, the ability of the ribozyme to cut the substrate chain is verified. Figure 2 C. Lanes 1, 2, and 3 are Probe B, S3-DNAzyme, and Probe C, respectively. The blocked ribozyme in lane 4 was successfully opened by ProbeB, and the blocked ribozyme in lane 5 could not cleave the substrate. Lane 6 was blocked by the cofactor Mg 2+ In the presence of Mg, the substrate chain is cleaved by the unlocked ribozyme and the substrate chain is consumed. Lane 7 is the case without the addition of Mg 2+ Negative control.

[0090] Figure 2This is a diagram of the PAGE verification of the assembly of SDA and blocked nanotubes and the ability of ribozyme to cleave substrates. Figure 2 C) SDA validation: Lane 1: miR-193b; Lane 2: Probe A; Lane 3: Mixed miR-193b and Probe A; Lane 4: Mixed miR-193b, Probe A and Klenow. Lane 5: Mixed miR-193b, Probe A, Klenow and Nb.BbvCI. ( Figure 2 B) Blockade of DNA nanotube synthesis. Lane 1: S1; Lane 2: S2; Lane 3: S3-DNAzyme; Lane 4: S4; Lane 5: S5; Lane 6: a mixture of S1, S2, S3-DNAzyme, S4, and S5. Figure 2 C) Verify the ability of ribozymes to cleave substrates. Lane 1: Probe B; Lane 2: S3-DNAzyme; Lane 3: Probe C; Lane 4: Mixture of Probe B and S3-DNAzyme; Lane 5: Mixture of S3-DNAzyme and Probe C; Lane 6: Probe B, S3-DNAzyme, Probe C, and cofactor Mg 2+ Lane 7: No Mg 2+ Negative control.

[0091] 2.3 Assembly of electrode interface

[0092] The present invention characterizes the assembly process of the electrode by electrochemical methods. Figure 3 As shown in A, compared with the bare electrode (curve Ⅰ), the electrode charge transfer resistance (Rct) of the assembled Probe C (curve Ⅱ) increased, indicating successful modification. Subsequently, DNA nanotube assembly increased Rct (curve Ⅲ), indicating successful assembly to the gold electrode interface. 2+ , the semicircle diameter becomes smaller due to the cutting of the substrate chain on the electrode surface (curve IV). Figure 3 The CV shows a similar trend as shown in B. The peak current decreases as more DNA chains are loaded on the electrode.

[0093] Figure 3 ( Figure 3 A) EIS and (B) CV graphs: I: bare electrode (GE). II: GE+Probe C. III: GE+Probe C+activated DNA nanotubes. IV: GE+Probe C+activated DNA nanotubes+Mg 2+ .

[0094] 2.4 Performance of DNA Nanomachines

[0095] The present invention further utilizes SWV to verify the performance of the proposed sensor. Figure 4 As shown in A, in the presence of miR-193b, Probe C on the gold electrode surface is cleaved, generating a lower current signal. In the absence of miR-193b, the current signal remains unchanged. Subsequently, the peak current of SWV was analyzed to investigate the sensitivity of the proposed electrochemical method. By preparing different concentrations of miR-193b (1pg / mL–1μg / mL), the electrochemical curves showed that the SWV peak current decreased with the increase of miR-193b concentration ( Figure 4 B). From the linear curve, it can be seen that there is a good linear correlation between the SWV peak current and the logarithm of the miR-193b concentration. The correlation coefficient is 0.99. The regression equation is y=-0.14x+1.16, where y represents the SWV peak current and x represents different concentrations of miR-193b. According to the signal-to-noise ratio calculated by the 3σ method, the detection limit (LOD) is 5.7fM. Finally, to verify the specificity of the DNA nanomachine, only after the addition of miR-193b, the current decreased significantly, and the SWV signal of the other four miRNAs did not change significantly ( Figure 4 D).

[0096] Figure 4 (A) SWV curve in response to miR-193b (300 nM). Figure 4 B) Incubation with different concentrations of miR-193b (1, 10, 100, 300, 1×10 3 , 1×10 4 , 1×10 5 , 3×10 5 and 1×10 6 pM) SWV curve. Figure 4 C) Linear relationship between SWV peak current and miR-193b concentration. Figure 4 D) The selective response of the method to miR-193b (300 nM) and several other miRNAs (1 μM). Target value, SD, n=3.

[0097] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. A method for preparing a molecular machine mediated by programmable DNA nanotubes, characterized in that The steps include: (1) Complementarily pairing the target exosomal nucleic acid miR-193b with the template sequence Probe A to obtain a product extension chain; (2) Klenow polymerase is used to catalyze the extension, and then the nicking enzyme is used to cut the extended chain, i.e., the product in step (1), and multiple rounds of polymerization and cutting are carried out to generate a large amount of Probe B for subsequent reactions; (3) S1, S2, S3-DNAzyme, S4, and S5 were reacted in TM buffer at a temperature of 37° C. for a reaction time of 0.5-2 h to obtain a blocked molecular machine mediated by programmable DNA nanotubes.

2. The method for preparing a molecular machine mediated by programmable DNA nanotubes according to claim 1, characterized in that: In step (1), the nucleotide sequence of miR-193b is shown as SEQ ID NO.6, and the nucleotide sequence of Probe A is shown as SEQ ID NO.7; in step (3), the nucleotide sequence of S1 is shown as SEQ ID NO.1; the nucleotide sequence of S2 is shown as SEQ ID NO.2; the nucleotide sequence of S3 is shown as SEQ ID NO.3; the nucleotide sequence of S4 is shown as SEQ ID NO.4; the nucleotide sequence of S5 is shown as SEQ ID NO.5; and the nucleotide sequence of Probe B is shown as SEQ ID NO.

8.

3. The method for preparing a molecular machine mediated by programmable DNA nanotubes according to claim 1, characterized in that: In step (3), Probe B is mixed with the blocked DNA nanotubes, and Probe B opens the ribozyme blocked on the DNA nanotubes to produce activated DNA nanotubes.

4. The molecular machine mediated by programmable DNA nanotubes prepared by the preparation method of claim 1.

5. A method for detecting ultrasensitive exosomal nucleic acids using the molecular machine of claim 2, characterized in that The method comprises the following steps: the DNA nanotube-mediated molecular machine of claim 1 is allowed to roll on the surface of a gold electrode in the presence of a suitable cofactor Mg2+, and cuts a substrate chain Probe C with a ferrocene Fc signal tag to generate an amplified electrochemical signal.

6. The method according to claim 5, characterized in that: The gold electrode was cleaned and then modified: the bare electrode was soaked in a piranha solution for 10 minutes, the molar ratio of the piranha solution was 98% H2SO4:30% H2O2 was 3:1, and then polished with 1μm and 0.3μm aluminum pastes respectively. Subsequently, the electrode was ultrasonically treated in anhydrous ethanol, double distilled water and 50% HNO3 for 5 minutes; finally, the electrode was activated in a 0.5M H2SO4 solution. In order to reduce the disulfide bond, the probe was incubated in 100mM TCEP at room temperature for 1h, and then 1μM Probe C was incubated with the bare electrode for 8h. Subsequently, the impurities on the electrode surface were rinsed with pure water, and finally blocked with 1mM MCH solution for 30min for use.

7. The method according to claim 5, characterized in that: The electrochemical signals were detected by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) or square wave voltammetry (SWV).

8. The method according to claim 6, characterized in that: Probe modification: The probe was placed in 100 mM TCEP and incubated for 1 hour to reduce disulfide bonds; 1 μM Probe C was incubated with the bare electrode for 8 hours; impurities on the electrode surface were rinsed with pure water; and the electrode was blocked with 1 mM MCH solution for 30 minutes.

9. The method according to claim 4, characterized in that: The nucleotide sequence of ProbeC is shown in SEQ ID NO.9.

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