A method for detecting charge transport in long-distance DNA molecular wires

Abstract

The present invention belongs to the technical field of molecular electronics optical detection, and relates to a method for detecting charge transport in long-distance DNA molecular wires. By using DNA self-assembly technology, an interface of gold nanoparticles (AuNP) / DNA molecular wire / electrode is prepared. Combining with a plasmon-based electrochemical current imaging system, the charge transport process through the long-distance DNA molecular wire between interfaces can be detected in an aqueous environment. By using the quantitative relationship between the optical image intensity and the redox reaction occurring on a single particle, the transport current in the DNA molecular wire between a single AuNP / electrode interface can be quantitatively analyzed in real time. The method of the present invention has the characteristics of high sensitivity and high spatial resolution, and can quantitatively measure the transport current through the DNA molecular wire at a single nanoparticle / interface without interference and in-situ online, and study the influence of the structure and size of the DNA molecular wire on the charge transport behavior, providing an effective tool for the research of DNA-based molecular electronics.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular electronics optical detection, and relates to a plasmon-based electrochemical current imaging technology, in particular to a method for detecting charge transport in long-distance DNA molecular wires. Background Art

[0002] DNA molecular wires, as electrical conduction bridges for electrons, provide a powerful tool for studying biological processes involving electron conduction. Charge transport can occur along DNA through the stacking between the π-orbitals of heterocyclic base pairs. DNA molecules also have precise coding and addressing capabilities, and can fabricate DNA self-assembled structures with specific sizes and shapes from bottom to top at the nanoscale, which is of guiding significance for the future applications of molecular wires and the development of molecular circuits.

[0003] Plasmonic-based Electrochemical Current imaging (P-ECi) technology is an analytical method based on physical optics, which combines the advantages of label-free detection, real-time online, sensitive and high-resolution analysis, and is widely used in the research of charge transport processes at the nanoscale. This technology records the surface plasmon resonance (SPR) scattering signals on individual nanoparticles through a charge-coupled device (CCD) camera, can observe multiple nanoparticles simultaneously and provide rich spatial information; by converting the local redox signal into an optical signal, the precise information of the electrochemical reaction process at the single-particle level can be further obtained. It has a sensitive response to the refractive index changes caused by tiny phase changes on the particles within a scale of 200 nanometers, and there is a quantitative relationship between the obtained optical image and charge transport.

[0004] Most of the existing studies on charge transport in DNA molecular wires at the single-molecule level focus on scales less than 5 nanometers, and cannot effectively provide information on long-distance charge transport; their experiments are usually carried out under dry and vacuum conditions, causing the DNA molecular wires to deviate from the natural B-type conformation. At the level of electrochemical methods, the existing electrochemical methods based on DNA monolayers can obtain the molecular transport current on the entire electrode area, but need to introduce redox molecules as labels and lack spatial resolution, and can only obtain the average result of DNA charge transport on the electrode, making it difficult to study the charge transport process at the single-particle and single-molecule levels in situ and quantitatively. Summary of the Invention

[0005] The present invention solves the above-mentioned technical problems existing in the prior art, and prepares a gold nanoparticle (AuNP) / DNA molecular wire / electrode interface by using DNA self-assembly technology. Combining with a plasmon-based electrochemical current imaging system, the charge transfer process between interfaces through long-distance DNA molecular wires can be detected in an aqueous environment. Utilizing the quantitative relationship between the optical image intensity and the redox reaction occurring on a single particle, the transport current in the DNA molecular wire between a single AuNP / electrode interface is quantitatively analyzed in real time.

[0006] The technical solution of the present invention is as follows:

[0007] A method for detecting charge transfer in a long-distance DNA molecular wire, comprising the following steps:

[0008] (1) Selection of the DNA molecular wire main body: The DNA molecular wire main body is formed by self-assembly of single-stranded DNA, and sticky ends extend from both ends thereof; the sticky end at one end of the DNA molecular wire main body is complementary to the thiol DNA sequence 1, and the sticky end at the other end is complementary to the thiol DNA sequence 2;

[0009] (2) Preparation of AuNP-DNA molecular wire nanoclusters: Modify the thiol DNA sequence 1 on AuNP, and assemble the DNA molecular wire main body onto the AuNP surface through hybridization; the thiol DNA sequence 1 is modified on AuNP through Au-S bonds;

[0010] (3) Construction of the working electrode: Modify the thiol DNA sequence 2 on the gold film substrate interface, and anchor the AuNP-DNA molecular wire nanoclusters obtained in step (2) onto the gold film substrate interface through DNA hybridization;

[0011] (4) Detection of the transport current in the DNA molecular wire: Use the working electrode constructed in step (3) as the working electrode of the nanoelectrochemical system, and observe the change Δθ(t) of the plasmon optical image intensity of a single AuNP-DNA molecular wire nanocluster anchored on the gold film substrate surface under linear cyclic voltammetry scanning by using a surface plasmon resonance microscope, so as to calculate the transport current i CT (t) passing through the DNA molecular wire during the observation time.

[0012] Preferably, the transport current i CT (t) passing through the DNA molecular wire during the observation time is calculated by the following formula:

[0013]

[0014] Where:

[0015] t and t' respectively represent the time and the sampling interval time of the image sequence;

[0016] e -d / l is a correction term for the height of the nanoparticle, d represents the gap distance between the nanoparticle and the electrode, and l represents the attenuation constant of the surface plasmon wave;

[0017] A = B(α R D R -1 / 2 -α O D O -1 / 2 )(nFπ 1 / 2 ) -1

[0018] Where:

[0019] For the electrocatalytic hydrogen evolution reaction (HER), the constant A is calibrated to 0.0336 mdeg s 1 / 2 C -1 ;

[0020] B represents a constant used to measure the sensitivity of the SPR angle to the refractive index change, which can be calibrated by a given optical device and reaction species;

[0021] α R and α O represent the refractive index change of the solution caused by the unit concentration change of the reducing molecule and the oxidizing molecule;

[0022] D R and D O represent the diffusion coefficients of the reducing molecule and the oxidizing molecule;

[0023] n represents the number of electrons transferred per reaction, and F represents the Faraday constant.

[0024] When a single AuNP-DNA molecular wire nanocluster is anchored on the surface of the gold film substrate, under the condition of negative potential sweep, along the DNA molecular wire path, the electrode injects electrons onto the AuNP, resulting in the HER reaction occurring on the AuNP surface. The generated hydrogen gas causes a decrease in the refractive index near the nanoparticle, thereby causing a decrease in the intensity of the plasmonic optical image.

[0025] Preferably, the DNA molecular wire is a six-helix-bundle (6HB) linear nanostructure.

[0026] Preferably, the length of the DNA molecular wire is 30 - 180 nm; more preferably, the length of the DNA molecular wire is 30 - 120 nm.

[0027] Preferably, the DNA molecular wire main body is self-assembled from single-stranded DNA with the sequence of SEQ No. 24-122 (as shown in Figure 1 ) or self-assembled from single-stranded DNA with the sequence of SEQ No. 1-21 (as shown in Figure 6 ).

[0028] Preferably, the sequence of the DNA molecular wire is designed and synthesized by using structural DNA nanotechnology, specifically designed by caDNAno software (downloaded from http: / / cadnano.org / ).

[0029] Preferably, the preparation method of the DNA molecular wire main body in the step (1) is to mix 100 μM component DNA strands in a buffer solution containing 12 mM magnesium acetate, 1 mM ethylenediaminetetraacetic acid and 40 mM tris(hydroxymethyl)aminomethane acetate buffer, and then anneal the mixed solution according to the following annealing program: 90 °C, constant temperature for 10 min; 60 °C to 25 °C, cooling rate of -0.1 °C / 5 min; 25 °C to 20 °C, cooling rate of -1 °C / 5 min; keep at 4 °C for constant temperature.

[0030] Preferably, in the step (2), the diameter of the AuNP is 10-500 nm; more preferably, the diameter of the AuNP is 30-200 nm.

[0031] Preferably, the specific steps of the step (2) are: mix 400 μL of 65 nm AuNPs solution with 8 μL of aqueous solution of thiol DNA sequence 1 (200 μM), freeze at -20 °C for 2 hours, and after thawing, remove the excess oligonucleotide chains by centrifugation 3 times, the centrifugation conditions are 3000 rpm, 5 minutes, and redisperse in 250 μL of secondary water.

[0032] Preferably, in the step (2), mix 25 μL of DNA-functionalized AuNP solution with 10 μL of DNA molecular wire main body (100 nM); anneal the mixed solution according to the following annealing program: 40 °C, constant temperature for 10 min; 40 °C to 25 °C, cooling rate of -0.1 °C / 6 min; 25 °C to 20 °C, cooling rate of -1 °C / 5 min; keep at 4 °C for constant temperature; after annealing, centrifuge the mixed solution 2 times to remove the excess DNA wires, the centrifugation conditions are 3000 rpm, 3 minutes, and redisperse with 12 mM magnesium acetate solution.

[0033] Preferably, in the step (3), after modifying the thiol DNA sequence 2 on the gold film substrate interface, passivate the exposed electrode area with a thiol small molecule backfill agent to obtain a stable DNA monolayer.

[0034] Preferably, the thiol small molecule backfill agent is any one or a combination of several of thiol-functionalized methoxypolyethylene glycol (mPEG-SH, MW 350 Da), 6-mercaptohexanol, and 8-mercapto-1-octanol.

[0035] Preferably, the preparation method of the gold film substrate electrode is as follows: using electron beam evaporation coating technology, on the surface of a BK7 glass sheet with a thickness of 0.17 mm, first evaporate 2 nm of Cr as an adhesion substrate, and then evaporate 47 nm of Au as a surface plasmon resonance substrate; before each experiment, the gold film is rinsed with ethanol and deionized water.

[0036] Preferably, the preparation steps of step (3) are as follows:

[0037] Step (3.1), incubate an aqueous solution of 200 μM thiol DNA sequence 2 with tris(2-carboxyethyl)phosphine at a ratio of 1:100; the mixed solution is kept in an ice bath for 1.5 hours, and then purified using a size exclusion column (G-25, GE Healthcare).

[0038] Step (3.2), install a polydimethylsiloxane (PDMS) chamber on the gold film substrate electrode; inject a 60 μM solution of 3'-thiol-modified oligonucleotide containing 100 mM magnesium acetate, which has been treated in step (3.1), into the chamber; after incubating for 18 to 24 hours in a humid environment, rinse the gold film substrate 4 times with deionized water; then, backfill the chamber with an aqueous solution of 2 mM thiol-functionalized methoxypolyethylene glycol (mPEG-SH, MW 350 Da) and incubate for 2 hours; finally, rinse the modified electrode 4 times with the electrolyte solution and keep it in the solution environment.

[0039] Step (3.3), add the AuNP-DNA molecular wire nanoclusters prepared in step (2) into the PDMS chamber on the gold film substrate, and let it stand for 50 minutes to ensure that the nanoclusters settle on the modified electrode and are effectively anchored through DNA hybridization.

[0040] Preferably, the reference electrode and the counter electrode of the nanoelectrochemical system in step (4) are an Ag / AgCl reference electrode and a platinum wire counter electrode, respectively; the electrolyte solution is a mixed solution containing 250 mM phosphate buffer (pH 7.0) and 12 mM magnesium acetate.

[0041] Compared with the prior art, the advantages of the present invention are as follows:

[0042] The present invention quantitatively detects the transmission current of long-distance DNA molecular wires between single nanoparticles / electrode interfaces, and can simultaneously detect the electrochemical reaction processes occurring on multiple AuNP-DNA molecular wire nanoclusters in the field of view.

[0043] The present invention is based on the surface plasmon resonance imaging method, which is a label-free method. Compared with the current electrochemical technology that modifies a DNA monolayer on the electrode interface to introduce redox molecular markers, it can quantitatively obtain the transmission current through the DNA molecular wire at a single nanoparticle / interface without interference and in-situ online, study the influence of the DNA molecular wire structure and size on the charge transport behavior, and provide an effective tool for the research of DNA-based molecular electronics.

[0044] The method of the present invention has the characteristics of high sensitivity and high spatial resolution, and can observe DNA molecular wires within a nanoscale of 200 nm. Compared with the current single-molecule current detection technology for directly measuring DNA molecular wires, it can measure the DNA charge transport process over a long distance (30 - 180 nm) in a solution environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a design diagram of the main body of the 6HB molecular wire with a length of 120 nm in Example 1.

[0046] Figure 2 It is an example of a transmission electron microscope image of a nanocluster formed by the composite of a single gold nanoparticle and a 120-nm 6HB molecular wire in Example 1.

[0047] Figure 3 It is a schematic diagram of the detection method of the present invention.

[0048] Figure 4 It is the plasmonic optical image sequence and intensity change of a single AuNP-6HB molecular wire nanocluster in the linear cyclic voltammetry scan of Example 1.

[0049] Figure 5 A shows the relationship between the plasmonic intensity change of a single AuNP-6HB molecular wire nanocluster in Example 2 and the voltage at the corresponding moment, and the inset shows its plasmonic optical image.

[0050] Figure 5 B shows the relationship between the transmission current through the DNA molecular wire and the voltage at the corresponding moment in the linear cyclic voltammetry scan calculated using the relational expression in Example 2.

[0051] Figure 5 C shows the cyclic voltammogram of a three-electrode electrochemical system recorded by a potentiostat in Example 2.

[0052] Figure 6 It is a design diagram of the main body of the 6HB molecular wire with a length of 30 nm in Example 3.

[0053] Figure 7A shows the relationship between the change in the plasmon intensity of a single AuNP-6HB molecular wire nanocluster in Example 3 and the voltage at the corresponding time. The inset shows its plasmon optical image.

[0054] Figure 7 B shows the relationship between the transport current through the DNA molecular wire in the linear cyclic voltammetry scan calculated using the relationship and the voltage at the corresponding time in Example 3. Detailed implementation manners

[0055] The plasmon-based electrochemical current imaging system adopted in the present invention is referred to Xiaonan Shan, Urmez Patel, Shaopeng Wang, Rodrigo Iglesias, and Nongjian Tao, Science 2010, 327(5971), 1363 - 1366; Ruihong Liu, Xiaonan Shan, Hui Wang, and Nongjian Tao, J. Am. Chem. Soc. 2019, 141(29), 11694 - 11699, and includes a light source emission module, a surface plasmon resonance excitation module, an electrochemical analysis module, a focusing control module, and an imaging acquisition module. The light source emission module includes a laser monochromatic light source and a position adjustment component for adjusting the beam direction. The surface plasmon resonance excitation module is based on an inverted microscope and mainly includes a p-polarizer, an objective lens, and a gold film substrate. The electrochemical analysis module is based on a potentiostat instrument and is constructed in a three-electrode mode. The focusing control module includes an electric microscope focusing controller. The imaging acquisition module includes an image sensor for acquiring imaging signals.

[0056] Example 1:

[0057] A single-particle / electrode interface is constructed using 65-nm gold nanoparticles to detect the decrease in the intensity of the plasmon optical image caused by the charge transport in the DNA molecular wire between the interfaces. The selectable range of the diameter of the gold nanoparticles is 10 - 500 nm; the optimal range is 30 - 200 nm.

[0058] The preparation method of the AuNP-DNA molecular wire nanocluster is as follows: The 6HB molecular wire main body with a length designed by the caDNAno software is used, which is composed of 99 single-stranded DNAs, and the design diagram is as Figure 1 shown.

[0059] 100 μM of the component DNA strands (sequences shown in SEQ No. 24 - 122) were mixed in a buffer solution containing 12 mM magnesium acetate, 1 mM ethylenediaminetetraacetic acid, and 40 mM tris(hydroxymethyl)aminomethane acetate buffer. Then, the mixed solution was annealed according to the following annealing program: 90 °C for 10 min at a constant temperature; from 60 °C to 25 °C with a cooling rate of -0.1 °C / 5 min; from 25 °C to 20 °C with a cooling rate of -1 °C / 5 min; and stored at 4 °C at a constant temperature. 400 μL of a 65 nm AuNPs solution was mixed with 8 μL of an aqueous solution of 200 μM 5'-thiol-modified oligonucleotide strand (sequence shown in SEQ No. 22), frozen at -20 °C for 2 hours, thawed, and centrifuged 3 times under the conditions of 3000 rpm for 5 minutes, and then redispersed in 250 μL of secondary water. 25 μL of the DNA-functionalized AuNP solution was mixed with 10 μL of the DNA molecular wire (100 nM), and slowly annealed from 40 °C to 20 °C for 20 h, and stored at 4 °C at a constant temperature. After annealing, the mixed solution was centrifuged 2 times under the conditions of 3000 rpm for 3 minutes and redispersed with a 12 mM magnesium acetate solution.

[0060] The preparation method of the gold film substrate as the imaging interface is as follows: on the surface of a 0.17 mm thick BK7 glass sheet, by means of electron beam evaporation, first 2 nm of Cr was evaporated as an adhesion substrate, and then 47 nm of Au was evaporated as a substrate for plasma resonance. Before use, the gold film was repeatedly rinsed clean with ethanol and deionized water. The method for DNA modification and nanocluster anchoring on the electrode surface is as follows: 200 μM of an aqueous solution of 3'-thiol-modified oligonucleotide (sequence shown in SEQ No. 23) was incubated with tris(2-carboxyethyl)phosphine at a ratio of 1:100. The mixed solution was kept in an ice bath for 1.5 hours and then purified using a G25 size exclusion column. The PDMS chamber was mounted on the gold film substrate, and the treated 60 μM 3'-thiol-modified oligonucleotide solution containing 100 mM magnesium acetate was injected into the chamber. After incubating for 18 to 24 hours in a humid environment, the gold film substrate was rinsed 4 times with deionized water. Then, 2 mM of an aqueous solution of mPEG-SH (MW 350 Da) was backfilled into the chamber and incubated for 2 hours. The modified electrode was finally rinsed 4 times with the electrolyte solution and kept in the solution environment. The prepared AuNP-DNA molecular wire nanoclusters were added to the PDMS chamber on the gold film substrate, and left to stand and incubate for 50 minutes to ensure the sedimentation of the nanoclusters on the modified electrode and effective anchoring through DNA hybridization.

[0061] Figure 2Several scattered images are given here for the nanoclusters formed by the composite of single gold nanoparticles and 120nm 6HB molecular wires. The 120nm 6HB molecular wires were assembled on 65nm AuNPs, confirming the successful preparation of the nanoclusters.

[0062] like Figure 3 , the optical imaging system of this embodiment adopts a surface plasmon resonance imaging system. The light source is a superluminescent diode with a wavelength of 670nm. The inverted microscope objective is a high numerical aperture objective (60×, NA=1.49), used with immersion oil that matches the refractive index of the glass sheet. The p-polarized light beam is incident at a specific angle to excite surface plasmon resonance. The imaging acquisition module uses a fast CCD camera to record the intensity of the plasma optical image. The electrochemical analysis system of this embodiment adopts a three-electrode system, the working electrode is a gold film substrate, the reference electrode and the counter electrode are Ag / AgCl reference electrode and platinum wire counter electrode, respectively. The cyclic voltammetry experiment was automatically measured and recorded by a constant potential instrument at room temperature, and the potential scanning rate was 0.1V s -1 The electrolyte solution contained 250 mM phosphate buffer (pH 7.0) and 12 mM magnesium acetate.

[0063] During the linear voltammetric scan, by scanning the potential from 0V to -1.2V (relative to Ag / AgCl), the electrons ejected from the electrode are transmitted along the on-state 6HB molecular wire to the AuNP, resulting in an electrocatalytic hydrogen evolution reaction (HER) on the AuNP surface. The hydrogen produced by HER causes the refractive index near the nanoparticles to decrease, thereby causing a decrease in the intensity of the plasma optical image. The plasma optical image sequence of a single nanoparticle / electrode interface under the scanning voltage is obtained by synchronous acquisition with a fast CCD camera. The change in the plasma intensity of a single AuNP-6HB molecular wire nanocluster is shown in Figure 2. Figure 4 As shown; the inset represents the image at the corresponding time point minus the first image, and the dark color indicates the decrease in plasma intensity.

[0064] Embodiment 2:

[0065] Same as Example 1, except that an electric microscope focus controller is used to avoid focus drift during long-term continuous acquisition. Processing of the collected plasma optical image sequence: The image of a single nanocluster is subtracted from the background signal of the nearby blank area, and the brightness change of the local dark pattern indicates the decrease of plasma intensity to varying degrees.

[0066] Using the change in the intensity of the plasma optical image Δθ(t), the transmission current i through the DNA molecular wire is calculated using the following relationship: CT (t):

[0067]

[0068] Wherein

[0069] t and t' respectively represent the time and the sampling interval time of the image sequence; e -d / l is the correction term of the nanoparticle height, d represents the gap distance between the nanoparticle and the electrode, and l represents the attenuation constant of the surface plasmon wave;

[0070] A = B(α R D R -1 / 2 -α O D O -1 / 2 )(nFπ 1 / 2 ) -1

[0071] For the HER reaction, the constant A is calibrated to 0.0336 mdeg s 1 / 2 C -1 , where:

[0072] B represents a constant used to measure the sensitivity of the SPR angle to the refractive index change, which can be calibrated by a given optical device and reaction species;

[0073] α R and α O represent the local refractive index changes per unit concentration of the reduced and oxidized molecules;

[0074] D R and D O represent the diffusion coefficients of the reduced and oxidized molecules;

[0075] n represents the number of electrons transferred per reaction, and F represents the Faraday constant.

[0076] Figure 5 A shows the change in the plasmonic optical image of a single nanocluster during a linear cyclic voltammetry scan (voltage range: 0 V to -1.2 V). The relationship between the transport current and voltage quantitatively calculated by the above formula is as Figure 5 shown in B, and its shape is similar to the cyclic voltammogram recorded by a potentiostat ( Figure 5 C).

[0077] Based on the above experiments, the surface plasmon resonance imaging method for detecting the charge transport properties of long DNA sequences in a solution environment by the present invention, which combines DNA molecule self-assembly and local electrochemical optical imaging technology at the single-particle level, can obtain the charge transport properties of long DNA sequences in a solution environment.

[0078] Example 3:

[0079] Same as Example 2, except that the 6HB molecular wire main body is composed of 21 single-stranded DNAs (sequences are shown in SEQ No. 1-21), and the design diagram is as Figure 6 shown; the nucleotide sequence of the aqueous solution of the 5'-thiol-modified oligonucleotide chain is HS-C6-CGTCGTATTCGATAGCTTAG (sequence is shown in SEQ No. 22); the nucleotide sequence of the aqueous solution of the 3'-thiol-modified oligonucleotide is GAAGTGATGGATGAT-C3-SH (sequence is shown in SEQ No. 23).

[0080] Figure 7 A shows the change of the plasmonic optical image of a single nanocluster during the linear cyclic voltammetry scan (voltage range: 0V to -1.2V). The relationship between the transfer current and voltage quantitatively calculated by the above formula is as Figure 7 shown in B.

[0081] Based on the above experiments, the surface plasmon resonance imaging method for detecting the charge transfer between single particles / electrode interfaces through DNA molecular wires in the present invention, combined with DNA molecular self-assembly and local electrochemical optical imaging technology at the single-particle level, can obtain the charge transfer properties of long DNA sequences in a solution environment.

[0082] Nucleotide Sequence List

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] It should be noted that the above embodiments are only the preferred embodiments of the present invention and do not limit the protection scope of the present invention. Equivalent replacements or substitutions made on the above basis all belong to the protection scope of the present invention.

Claims

1. A method for detecting charge transport in a long - distance DNA molecular wire, characterized in that, It includes the following steps: (1) Selection of the DNA molecular wire main body: The DNA molecular wire main body is self-assembled by single-stranded DNA, and sticky ends extend from both ends thereof; the sticky end at one end of the DNA molecular wire main body is complementary to the thiol DNA sequence 1, and the sticky end at the other end is complementary to the thiol DNA sequence 2; The DNA molecular wire main body is a six-helix bundle linear nanostructure; The length of the DNA molecular wire main body is 30 - 180 nm; (2) Preparation of the AuNP-DNA molecular wire nanocluster: Modify the thiol DNA sequence 1 on the AuNP, and assemble the DNA molecular wire main body onto the AuNP surface through hybridization; the thiol DNA sequence 1 is modified on the AuNP through Au-S bonds; (3) Construction of the working electrode: Modify the thiol DNA sequence 2 on the gold film substrate electrode interface, and anchor the AuNP-DNA molecular wire nanocluster obtained in step (2) onto the gold film substrate electrode interface through DNA hybridization; (4) Detection of the transmitted current in the DNA molecular wire: Using the working electrode constructed in step (3) as the working electrode of the nanoelectrochemical system, the change Δθ(t) in the intensity of the plasmonic optical image of a single AuNP-DNA molecular wire nanocluster anchored on the surface of the gold film substrate under linear cyclic voltammetry scanning is observed using a surface plasmon resonance microscope, so as to calculate the transmitted current i CT (t) passing through the DNA molecular wire during the observation time; During the linear cyclic voltammetry scanning process, the electrons ejected from the working electrode are transmitted along the conductive DNA molecular wire main body to the AuNP, resulting in an electrocatalytic hydrogen evolution reaction occurring on the AuNP surface.

2. The method according to claim 1, wherein The transmitted current i through the DNA molecular wire during the observation time CT (t) is calculated by the following formula: Wherein: t and t' respectively represent time and the sampling interval time of the image sequence; e -d / l is a correction term for the height of the nanoparticle, d represents the gap distance between the nanoparticle and the electrode, and l represents the attenuation constant of the surface plasmon wave; A = B(α R D R -1 / 2 -α O D O -1 / 2 )(nFπ 1 / 2 ) -1 Wherein: B represents a constant used to measure the sensitivity of the SPR angle to the refractive index change, and is calibrated through a given optical device and reaction species; α R and α O represent the change in the refractive index of the solution caused by the change in the unit concentration of the reduced molecule and the oxidized molecule; D R and D O represent the diffusion coefficients of the reduced and oxidized molecules; n represents the number of electrons transferred in each reaction, and F represents the Faraday constant.

3. The method according to claim 1, characterized in that The DNA molecular wire main body is self-assembled by single-stranded DNA with the sequence of SEQ No. 24 - 122 or self-assembled by single-stranded DNA with the sequence of SEQ No. 1 - 21.

4. The method according to claim 1, wherein the preparation method of the DNA molecular wire main body in step (1) is to anneal the component single-stranded DNA to form the DNA molecular wire main body, and the annealing program is: 90 °C, constant temperature for 10 min; 60 °C - 25 °C, the cooling rate is -0.1 °C / 5 min; 25 °C - 20 °C, the cooling rate is -1 °C / 5 min; The specific steps of step (2) are: Mix the AuNPs solution with the aqueous solution of the thiol DNA sequence 1 and freeze to obtain the DNA-functionalized AuNP solution; mix the DNA-functionalized AuNP solution with the DNA molecular wire main body; anneal the mixed solution according to the following annealing program: 40 °C, constant temperature for 10 min; 40 °C - 25 °C, the cooling rate is -0.1 °C / 6 min; 25 °C - 20 °C, the cooling rate is -1 °C / 5 min; In step (3), after the thiol DNA sequence 2 is modified on the gold film substrate electrode interface, the bare electrode area is passivated with a thiol small molecule backfill agent to obtain a stable DNA monolayer.

5. The method according to claim 4, wherein The thiol small molecule backfill agent is any one or a combination of several of thiol-functionalized methoxypolyethylene glycol, 6-mercaptohexanol, and 8-mercapto-1-octanol.

6. The method according to claim 1, wherein The preparation method of the gold film substrate electrode is as follows: Using electron beam evaporation coating technology, on the surface of a BK7 glass sheet with a thickness of 0.17 mm, first deposit 2 nm of Cr as an adhesion substrate, and then deposit 47 nm of Au as a substrate for surface plasmon resonance.

7. The method according to claim 1, characterized in that The preparation steps of step (3) are as follows: Step (3.1), incubate and purify the aqueous solution of thiol DNA sequence 2 with tris(2-carboxyethyl)phosphine; Step (3.2), install the PDMS chamber on the gold film substrate electrode; inject the solution containing thiol DNA sequence 2 processed in step (3.1) into the chamber; after incubation, backfill the thiol small molecule backfill agent into the chamber and incubate; Step (3.3), add the AuNP-DNA molecular wire nanoclusters prepared in step (2) into the PDMS chamber in step (3.2), and complete after standing.

8. The method according to claim 1, wherein In step (4), the reference electrode and the counter electrode of the nanoelectrochemical system are an Ag / AgCl reference electrode and a platinum wire counter electrode respectively.

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