Electrochemical biomolecular sensor based on DNA molecular pendulum and construction method

Abstract

The invention discloses an electrochemical biomolecular sensor based on a DNA molecular pendulum and a construction method. The electrochemical biomolecular sensor comprises a buffer solution, an electrode and a DNA double-chain swing rod, wherein the electrode and the DNA double-chain swing rod are arranged in the buffer solution; a step voltage reduction signal molecule is inserted into a phosphoric acid skeleton of the DNA double-stranded oscillating bar; one end of the DNA double-chain oscillating bar is connected to the electrode, and a target aptamer is connected to a single chain at the other end of the DNA double-chain oscillating bar. The invention has the advantages of wide detection range, high sensitivity and low detection limit, and has important significance for detecting proteins, viruses, nucleic acids, small molecular substances and the like.

Description

Technical Field

[0001] The invention relates to a preparation method and application of an electrochemical biomolecular sensor, belonging to the technical field of electrochemical biosensors. Background Art

[0002] Disease markers (also called biomarkers or markers) are biological molecules that can indicate the presence or status of a specific disease or changes in molecules or cells. These markers can be proteins (antigens, antibodies), hormones, enzymes, specific cell types or other molecules, which can be detected in body fluids or tissues such as blood, urine, and tissue samples. Disease markers have a wide range of applications, including early diagnosis of diseases, disease monitoring, efficacy evaluation, and prognosis judgment. With the advancement of medical research, more and more disease markers have been discovered and applied in clinical practice, but some instruments are expensive and complicated to operate, so it is very important to develop an economical and practical detection method with high sensitivity and low detection limit.

[0003] Electrochemical biosensors have developed rapidly in recent years. They are highly sensitive and can detect very low concentrations of analytes. They have a fast response and can provide measurement results in a short time. They are highly selective and portable. Electrochemical sensors can be designed to be small and lightweight, suitable for field detection and portable applications. They are simple to operate, and the manufacturing and operating costs of electrochemical sensors are generally lower than other sensing technologies. These advantages have made electrochemical sensors widely used in many fields such as environmental monitoring, food safety, and medical diagnosis. Summary of the invention

[0004] Purpose of the invention: To address the problems of complex operation and high cost of current marker detection, the present invention provides an electrochemical biomolecular sensor based on a DNA molecular pendulum.

[0005] Technical solution: To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] An electrochemical biomolecule sensor based on a DNA molecular pendulum comprises a buffer, an electrode, and a DNA double-stranded pendulum, wherein the electrode and the DNA double-stranded pendulum are arranged in the buffer. A step voltage reduction signal molecule is inserted into the phosphate backbone of the DNA double-stranded pendulum. One end of the DNA double-stranded pendulum is connected to the electrode, and a target aptamer is connected to a single strand at the other end of the DNA double-stranded pendulum.

[0007] Preferably: the step voltage reduction signal molecule is [Ru(NH3)6] 3+ Signal reduction molecules.

[0008] Preferably, the electrode is a gold electrode, a silver electrode, a silver chloride electrode, or a platinum electrode.

[0009] Preferably, the buffer is a Tris-HCl solution or a phosphate buffer solution (PBS), or a mixture of two or more thereof.

[0010] Preferably, the target aptamer is CEA, PSA, RNA, or ATP with a target.

[0011] Another object of the present invention is to provide a detection method of an electrochemical biomolecule sensor based on a DNA molecular pendulum. In the chronoamperometric CA mode, the DNA double-stranded pendulum swings toward the electrode under the action of an applied step negative electric field, and the step voltage reduction signal molecule is reduced under the step voltage to generate a detectable Faraday current. When the target aptamer on the DNA double-stranded pendulum binds to the target marker, its hydrodynamic diameter becomes larger, the time required for swinging becomes longer, and the current becomes smaller. By detecting the change in current, it is possible to determine whether a target marker binds to the target aptamer on the DNA double-stranded pendulum, thereby realizing the detection of the target marker.

[0012] Another object of the present invention is to provide a method for constructing an electrochemical biomolecular sensor based on a DNA molecular pendulum, comprising the following steps:

[0013] Step S1, subjecting the electrode to ultrasonic treatment in ultrapure water, and then polishing it, and then washing it with ultrapure water.

[0014] Step S2, chemically cleaning and activating the polished and cleaned electrode.

[0015] Step S3, incubate the DNA double-stranded pendulum with the target aptamer on the cleaned and activated electrode, take out the electrode, blow it clean, and use [Ru(NH3)6] 3+ The electrode is incubated with signal reduction molecules to modify the DNA molecules, and then the electrode is taken out, blown clean, and incubated with MCH to shield the non-specific binding sites.

[0016] Step S4, the electrode that has completed the MCH incubation to shield the non-specific binding sites is rinsed with a buffer solution and stored in the buffer solution to obtain an electrochemical biomolecular sensor based on a DNA molecular pendulum.

[0017] Preferably, in step S2, the polished and cleaned electrode is activated in H2SO4 using cyclic voltammetry, and CV scanning is performed continuously at 0.03-0.7V / s in a potential range of -0.2 to 1.6V.

[0018] Preferably: in step S3, 4.0-6.0 μL of 8-12 μM DNA double-stranded pendulum with target aptamer is incubated on the cleaned and activated electrode, and then placed in a -15-25°C refrigerator for incubation for 0.5-1.5 h.

[0019] Preferably: in step S3, [Ru(NH3)6] 3+ The electrode was incubated with signal reduction molecules to modify DNA molecules and placed in an environment of -5 to -3°C for 0.5 to 1.5 hours. The electrode was incubated with MCH to shield nonspecific binding sites and placed in an environment of -5 to -3°C for 0.5 to 1.5 hours.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The inventors of the present invention incubated the double-stranded DNA with the aptamer on the gold electrode, and the ruthenium redox signal molecule ([Ru(NH4)]6 3+ ) is used as a modification material in the preparation of electrochemical biomolecular sensors. Based on the specific recognition of the aptamer and the target substance, the hydrodynamic diameter of the molecule becomes larger after binding to the target.

[0022] After the electric field is applied, the DNA molecules take longer to swing, thereby generating a regular electrical signal related to the target concentration for detection. The sensor is simple to construct, sensitive, and can regenerate detection.

[0023] (2) The electrochemical biomolecular sensor prepared by the present invention is used for the detection of disease markers CEA, PSA, RNA, and ATP. The sensor has high stability, good reproducibility, high sensitivity, and a wide linear range, and can achieve rapid, highly sensitive, and reproducible continuous detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the detection of electrochemical biomolecular sensor based on DNA molecular pendulum.

[0025] Figure 2 Schematic diagram of the electrochemical DNA molecular pendulum sensing platform for the detection of disease markers of different concentrations: (a) CEA, (b) mRNA21, and (c) ATP. DETAILED DESCRIPTION

[0026] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0027] Example 1

[0028] An electrochemical biomolecular sensor based on a DNA molecular pendulum can be used for the detection of disease marker proteins or other marker proteins, such as Figure 1As shown, it includes a buffer, an electrode, and a double-stranded DNA pendulum. In this embodiment, the electrode is a gold electrode. In another embodiment, the electrode is a silver electrode. In another embodiment, the electrode is a silver chloride electrode. In another embodiment, the electrode is a platinum electrode. The electrode and the double-stranded DNA pendulum are set in a buffer solution, and the buffer solution is a Tris-HCl solution. A step voltage reduction signal molecule is inserted into the phosphate backbone of the double-stranded DNA pendulum. In this embodiment, the step voltage reduction signal molecule is [Ru(NH3)6] 3+ In another embodiment, the step voltage reduction signal molecule is hexaammonium ruthenium chloride ([Ru(NH3)6] 3 Cl3) signal reduction molecule. One end of the double-stranded DNA swing rod is connected to the electrode, and a single strand at the other end of the double-stranded DNA swing rod is connected to a target aptamer.

[0029] We used DNA duplex with aptamers as the working basis of molecular pendulum, and ruthenium redox signal molecule ([Ru(NH3)6] 3+ ) can interact electrostatically with the phosphate backbone of the double-stranded DNA part, insert into the DNA backbone to make the DNA positively charged. In the chronoamperometry (CA) mode, the DNA swings toward the Au electrode under the action of the applied step negative electric field (-0.5~0V), and then the signal molecule is reduced under the step voltage to generate a detectable Faraday current. When the aptamer on the DNA molecule pendulum binds to the target marker, its hydrodynamic diameter becomes larger and the time required for swinging becomes longer. According to the Cottrell equation, the longer the time, the smaller the current. Based on this principle, the detection of disease markers can be achieved. The test results show that the electrochemical sensor has high sensitivity, low detection limit and good stability.

[0030] This embodiment is based on a double-stranded DNA pendulum, in which a single-stranded extended target aptamer can recognize specific biomolecules. In the chronoamperometric mode, the electron transfer process can be accurately recorded, thereby realizing the detection of target substances. Electrochemical MS sensing is based on active, strong electric field-mediated electrical signal transmission to detect biorecognition events. The signal molecule of the sensor is based on [Ru(NH3)6] 3+ to [Ru(NH3)6] 2+ Therefore, the sensor's detection ability can be restored by reverse oxidation or slow natural oxidation (sensor regeneration). Compared with sensors that detect once, it can continuously detect multiple times, has a longer working time, is more suitable for long-term detection, and significantly improves the working efficiency of the sensor.

[0031] The existing MS sensors driven by positive electric field (oxidation signal molecules) are interfered by reducing substances in the biological environment (such as glutathione, ascorbic acid, uric acid, dopamine, hydrogen peroxide, etc.). 3+ Modification of DNA, [Ru(NH3)6] 3+ By inserting into the phosphate backbone of DNA through electrostatic interaction, the charge amount and molecular position on DNA can be adjusted by controlling the modified chain and the concentration of the modified molecule. 3+ Reduction to [Ru(NH3)6] 2+ Compared with the CA method using positive potential, it can avoid the interference of reducing substances, has better anti-interference ability, makes the experimental results more accurate, and is more suitable for the detection and analysis of biological molecules in body fluids.

[0032] Example 2

[0033] This embodiment provides a detection method for an electrochemical biomolecule sensor based on a DNA molecular pendulum. In the chronoamperometric CA mode, the DNA double-stranded pendulum swings toward the electrode under the action of an applied step negative electric field, and the step voltage reduction signal molecule is reduced under the step voltage to generate a detectable Faraday current. When the target aptamer on the DNA double-stranded pendulum binds to the target marker, its hydrodynamic diameter becomes larger, the time required for swinging becomes longer, and the current becomes smaller. By detecting the change in current, it is determined whether there is a target marker binding to the target aptamer on the DNA double-stranded pendulum, and then the detection of the target marker is achieved. If there is a target marker binding to the target aptamer on the DNA double-stranded pendulum, the current gradually decreases. Therefore, if the current gradually decreases, it means that there is a target marker binding to the target aptamer on the DNA double-stranded pendulum, indicating that the target marker is detected.

[0034] Example 3

[0035] This embodiment provides a method for constructing an electrochemical biomolecule sensor based on a DNA molecular pendulum, comprising the following steps:

[0036] Step S1, subjecting the electrode to ultrasonic treatment in ultrapure water, and then polishing it, and then washing it with ultrapure water.

[0037] Step S2, chemically cleaning and activating the polished and cleaned electrode.

[0038] In another embodiment, the polished and cleaned electrode is activated in H2SO4 using cyclic voltammetry, and CV scanning is performed continuously at 0.03-0.7V / s in the potential range of -0.2 to 1.6V.

[0039] Step S3, incubate the DNA double-stranded pendulum with the target aptamer on the cleaned and activated electrode, take out the electrode, blow it clean, and use [Ru(NH3)6] 3+ The electrode is incubated with signal reduction molecules to modify the DNA molecules, and then the electrode is taken out, blown clean, and incubated with MCH to shield the non-specific binding sites.

[0040] In another embodiment, 4.0-6.0 μL of 8-12 μM DNA double-stranded pendulum with target aptamer is incubated on the cleaned and activated electrode, and then placed in a -15-25° C. refrigerator for incubation for 0.5-1.5 h.

[0041] In another embodiment, [Ru(NH3)6] 3+ The electrode was incubated with signal reduction molecules to modify DNA molecules and placed in an environment of -5 to -3°C for 0.5 to 1.5 hours. The electrode was incubated with MCH to shield nonspecific binding sites and placed in an environment of -5 to -3°C for 0.5 to 1.5 hours.

[0042] Step S4, the electrode that has completed the MCH incubation to shield the non-specific binding sites is rinsed with a buffer solution and stored in the buffer solution to obtain an electrochemical biomolecular sensor based on a DNA molecular pendulum.

[0043] Example 4

[0044] This embodiment provides a method for constructing a biomarker protein electrochemical biomolecular sensor based on a DNA molecular pendulum, comprising the following steps:

[0045] (1) First, place a gold electrode (S = 0.2 mm 2 ) was ultrasonically treated in ultrapure water for 3 minutes, and then polished with 0.3μm and 0.05μm aluminum oxide polishing powder on the suede, each polishing step (5 minutes), and then washed with ultrapure water.

[0046] (2) The polished electrode requires further electrochemical cleaning and activation to remove surface contaminants. Cyclic voltammetry (CV) was performed in 0.5 M H2SO4 at 0.05 V / s for 10 consecutive CV scans in the potential range of -0.2 to 1.6 V (vsAg / AgCl).

[0047] (3) Incubate 5.0 μL of 10 μM DNA double-strand solution with CEA (carcinoembryonic antigen) aptamer on the cleaned and activated gold electrode, and then place it in a -20°C refrigerator for 1 hour. Take out the electrode, blow it clean, and use [Ru(NH3)6] 3+The electrode was incubated with a signal reduction molecule to modify the DNA molecule and placed in a -4°C environment for 1 hour. The gold electrode was then taken out and blown clean, and then incubated with MCH to shield the nonspecific binding sites, and placed in a -4°C incubation for 1 hour. Finally, the gold electrode was rinsed with Tris-HCl buffer and stored in 10mM Tris-HCl buffer before use to prepare an electrochemical CEA sensor.

[0048] Example 5

[0049] This embodiment provides a method for constructing an RNA electrochemical biomolecular sensor based on a pendulum in a DNA molecule, comprising the following steps:

[0050] (1) First, place a gold electrode (S = 0.2 mm 2 ) were ultrasonically treated in ultrapure water for 3 min and then polished on a micro-cloth with 0.3 μm and 0.05 μm alumina polishing powders and rinsed with ultrapure water after each polishing step (5 min).

[0051] (2) The polished electrode requires further electrochemical cleaning and activation to remove thiols / organic contaminants: Cyclic voltammetry (CV) was performed in 0.5 M H2SO4 at 0.05 V / s for 10 consecutive CV scans in the potential range of -0.2 to 1.6 V (vsAg / AgCl).

[0052] (3) Incubate 5.0 μL of 10 μM double-stranded DNA solution with RNA aptamer on the cleaned and activated gold electrode, and incubate it at -20°C for 4 hours. Take out the electrode and blow it clean, incubate the gold electrode with ruthenium signal reduction molecules to modify the DNA molecules, and place it at -4°C for 55 minutes. Take out the gold electrode and blow it clean, incubate it with MCH, and incubate it at -20°C for 4 hours. Finally, rinse the gold electrode with Tris-HCl buffer and store it in 10 mM Tris-HCl buffer before use to prepare an RNA molecule pendulum electrochemical biosensor.

[0053] Example 6

[0054] This embodiment provides a method for constructing an electrochemical bio-small molecule sensor based on a DNA molecular pendulum, comprising the following steps:

[0055] 1) First, place the gold electrode (S = 0.2 mm 2 ) was ultrasonically treated in ultrapure water for 3 minutes, and then polished with 0.3μm and 0.05μm aluminum oxide polishing powder on the suede, each polishing step (5 minutes), and then washed with ultrapure water.

[0056] (2) The polished electrode requires further electrochemical cleaning and activation to remove surface contaminants. Cyclic voltammetry (CV) was performed in 0.5 M H2SO4 at 0.05 V / s for 10 consecutive CV scans in the potential range of -0.2 to 1.6 V (vsAg / AgCl).

[0057] (3) Incubate 5.0 μL of 10 μM DNA double-strand solution with ATP (adenosine triphosphate) aptamer on the cleaned and activated gold electrode, and then place it in a -20°C refrigerator for 1 hour. Take out the electrode, blow it clean, and use [Ru(NH3)6] 3+ The electrode was incubated with a signal reduction molecule to modify the DNA molecule and placed in a -4°C environment for 1 hour. The gold electrode was then taken out and blown clean, and then incubated with MCH to shield the nonspecific binding sites, and placed in a -4°C incubation for 1 hour. Finally, the gold electrode was rinsed with Tris-HCl buffer and stored in 10mM Tris-HCl buffer before use to prepare an electrochemical ATP sensor.

[0058] Example 7

[0059] This embodiment provides a method for detecting a disease marker protein (CEA), comprising the following steps:

[0060] (1) An electrochemical workstation was used for testing with a three-electrode system. The Ag / AgCl electrode was used as the reference electrode, the platinum wire electrode was used as the auxiliary electrode, and the prepared biomolecular sensor was used as the working electrode. The working electrode was incubated with 5 μL of target molecule and carcinoembryonic antigen (CEA) with concentrations of 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1 μg / mL for 2 h and then placed in 10 mL of 10 mM Tris-HCl buffer at pH 7.4 for detection.

[0061] (2) The chronoamperometry method was used to detect the sample, with the potential range set to -0.1 to -0.5 V and the running time set to 0.2 s.

[0062] (3) After the electrodes are placed, they are tested every 2 seconds, the current is recorded, and the working curve is drawn.

[0063] Example 8

[0064] This embodiment provides a method for detecting a biomolecule (RNA), comprising the following steps:

[0065] (1) An electrochemical workstation was used for testing with a three-electrode system. A saturated silver chloride electrode was used as the reference electrode, a platinum wire electrode was used as the auxiliary electrode, and the prepared biomolecule sensor was used as the working electrode. After incubating 5 μL of nucleotides (RNA) with concentrations of 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM for 2 h, the working electrode was placed in 10 mL of 10 mM Tris-HCl buffer with a pH of 7.4 for detection.

[0066] (2) The chronoamperometry method was used to detect the sample, with the potential range set to -0.1 to -0.5 V and the running time set to 0.2 s.

[0067] (3) After the electrodes are placed, they are tested every 2 seconds, the current is recorded, and the working curve is drawn.

[0068] Example 9

[0069] This embodiment provides a method for detecting a biomolecule (ATP), comprising the following steps:

[0070] (1) An electrochemical workstation was used to perform the test with a three-electrode system. A saturated silver chloride electrode was used as the reference electrode, a platinum wire electrode was used as the auxiliary electrode, and the prepared biomolecule sensor was used as the working electrode. After incubation for 2 h with 5 μL of adenosine triphosphate (ATP) at concentrations of 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, and 100 μM, the working electrode was placed in 10 mL of 10 mM Tris-HCl buffer at pH 7.4 for detection.

[0071] (2) The chronoamperometry method was used to detect the sample, with the potential range set to -0.1 to -0.5 V and the running time set to 0.2 s.

[0072] (3) After the electrodes are placed, they are tested every 2 seconds, the current is recorded, and the working curve is drawn.

[0073] Based on the electrochemical biomolecular sensor of DNA molecular pendulum, we selected protein (carcinoembryonic antigen, CEA), nucleotide (mRNA21) and small molecule (adenosine triphosphate, ATP) as target substances for detection. Figure 2 As shown, the chronocurrent signal gradually decreases with the increase of concentration, which is consistent with our theory and proves the successful construction of the sensing platform. The present invention realizes the construction of the electrochemical biomolecular sensor and effectively detects biomolecules, including disease marker proteins, small molecules, viruses, etc., and performs accurate quantitative analysis.

[0074] The present invention is based on the specific recognition and matching of biological molecules (disease marker proteins, RNA, etc.) and aptamers, using a double-stranded DNA with an aptamer as the substrate material, and using a ruthenium complex to modify the DNA molecular pendulum and then fix the Au electrode. In the chronoamperometric mode, an electric field opposite to the DNA molecular pendulum is applied to make it swing and generate an electric signal, thereby realizing the detection of the target molecule. The present invention realizes the construction of the electrochemical biomolecular sensor and effectively detects biomolecules, including disease marker proteins, small molecules, viruses, etc., and performs accurate quantitative analysis. The constructed electrochemical biomolecular sensor has a wide detection range, is simple to construct, and has high sensitivity and low detection limit, which is of great significance for the detection of proteins, viruses, nucleic acids, and some small molecules.

[0075] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An electrochemical biomolecular sensor based on a DNA molecular pendulum, characterized in that: The invention comprises a buffer, an electrode and a double-stranded DNA pendulum, wherein the electrode and the double-stranded DNA pendulum are arranged in the buffer; a step voltage reduction signal molecule is inserted into the phosphate backbone of the double-stranded DNA pendulum; one end of the double-stranded DNA pendulum is connected to the electrode, and a target aptamer is connected to a single strand at the other end of the double-stranded DNA pendulum.

2. The electrochemical biomolecular sensor based on the DNA molecular pendulum according to claim 1, characterized in that: The step voltage reduction signal molecule is [Ru(NH3)6] 3+ Signal reduction molecules.

3. The electrochemical biomolecular sensor based on the DNA molecular pendulum according to claim 1, characterized in that: The electrode is a gold electrode, a silver electrode, a silver chloride electrode, or a platinum electrode.

4. The electrochemical biomolecular sensor based on the DNA molecular pendulum according to claim 2, characterized in that: The buffer is a Tris-HCl solution or a phosphate buffer solution PBS or a mixture of two or more thereof.

5. The electrochemical biomolecular sensor based on DNA molecular pendulum according to claim 2, characterized in that: The target aptamer is CEA, PSA, RNA, or ATP with a target.

6. A detection method of an electrochemical biomolecule sensor based on a DNA molecular pendulum, characterized in that: In the chronoamperometric mode, the DNA double-stranded pendulum swings toward the electrode under the action of the applied step negative electric field, and the step voltage reduction signal molecule is reduced under the step voltage to generate a detectable Faraday current; when the target aptamer on the DNA double-stranded pendulum binds to the target marker, its hydrodynamic diameter becomes larger, the time required for swinging becomes longer, and the current becomes smaller; by detecting the current change, it can be determined whether there is a target marker binding to the target aptamer on the DNA double-stranded pendulum, thereby realizing the detection of the target marker.

7. A method for constructing an electrochemical biomolecular sensor based on a DNA molecular pendulum, comprising the following steps: Step S1, subjecting the electrode to ultrasonic treatment in ultrapure water, and then polishing with aluminum oxide nanoparticles, and then washing with ultrapure water; Step S2, chemically cleaning and activating the polished and cleaned electrode; Step S3, incubate the DNA double-stranded pendulum with the target aptamer on the cleaned and activated electrode, take out the electrode, blow it clean, and use [Ru(NH3)6] 3+ The electrode is incubated with signal reduction molecules to modify the DNA molecules; the electrode is then taken out and blown clean, and then incubated with MCH to shield the non-specific binding sites; Step S4, the electrode that has completed the MCH incubation to shield the non-specific binding sites is rinsed with a buffer solution and stored in the buffer solution to obtain an electrochemical biomolecular sensor based on a DNA molecular pendulum.

8. The method for constructing an electrochemical biomolecular sensor based on a DNA molecular pendulum according to claim 5, characterized in that: In step S2, the polished and cleaned electrode is activated in H2SO4 using cyclic voltammetry, and CV scanning is performed continuously at 0.03-0.7V / s in the potential range of -0.2 to 1.6V.

9. The method for constructing an electrochemical biomolecular sensor based on a DNA molecular pendulum according to claim 6, characterized in that: In step S3, 4.0 to 6.0 μL of 8 to 12 μM DNA double-stranded pendulum with target aptamer is incubated on the cleaned and activated electrode, and then placed in a -15 to 25° C. refrigerator for incubation for 0.5 to 1.5 h.

10. The method for constructing an electrochemical biomolecular sensor based on a DNA molecular pendulum according to claim 7, characterized in that: In step S3, [Ru(NH3)6] 3+ The electrode was incubated with signal reduction molecules to modify DNA molecules and placed in a -5 to -3°C environment for 0.5 to 1.5 hours; the electrode was incubated with MCH to shield nonspecific binding sites and placed in a -5 to 4°C environment for 0.5 to 1.5 hours.

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