Preparation method and application of framework nucleic acid synergistically enhanced click chemistry and DNAzyme catalytic electrode

Abstract

The present invention discloses a preparation method and application of a framework nucleic acid synergistically enhanced click chemistry and DNAzyme catalytic electrode. The present invention assembles a split G-quadruplex fragment A modified by an azide group onto the double-stranded DNA (dsDNA) edge of a DNA tetrahedron through Hoogsteen hydrogen bonds, so that the reactants achieve a reasonable spatial distribution and improve the efficiency of click chemistry reactions on the interface. Under Cu(I) catalytic conditions, the split G-quadruplex fragment B modified by an alkyne group and the fragment A assembled on the tetrahedral scaffold form a complete G-quadruplex through a click chemistry reaction. Further compounded with Hemin, the formed G-quadruplex (G-quadruplex) / hemin complex has peroxidase-like activity. At the same time, the nucleic acid framework with strong electronegativity further enhances the response current of the catalytic oxidation of TMB, achieving Cu(I) 2+ and sensitive detection of alkaline phosphatase.

Description

Technical Field

[0001] The present invention relates to a preparation method and application of framework nucleic acid synergistically enhanced click chemistry and DNAzyme catalytic electrodes, belonging to the field of biosensor technology. Background Art

[0002] The copper (I)-catalyzed azide-alkyne cycloaddition reaction (click chemistry) can realize the directional double chain of two molecules by forming a triazole ring structure. Due to the high reaction efficiency and mild conditions, it is conducive to the site-directed modification and double chain of oligonucleotides, and is widely used in the field of sensing as a signal conversion tool. However, compared with the click reaction in the homogeneous solution, the single-stranded nucleic acid probe directly modified on the electrode interface often presents unfavorable conformations such as lodging, interchain entanglement, and accumulation, which reduces the click reaction efficiency on the electrode interface and also affects the recognition specificity of the probe. At the same time, due to the efficiency of the click reaction, the concentration of electrochemical signal molecules loaded on the electrode surface is low, resulting in poor detection sensitivity. In addition, the signal reporter molecules introduced by click chemistry are often fixed to the electrode surface by covalent bonds, and the differences between different modified electrodes cause the final detection signal deviation to be large. Summary of the Invention

[0003] The purpose of the present invention is to address the deficiencies of the existing technology and provide a preparation method and application of a framework nucleic acid synergistically enhanced click chemistry and DNAzyme catalytic electrode. The present invention assembles the split G-quadruplex A used for click chemistry reaction onto three edges of the DNA tetrahedral framework through Hoogsteen hydrogen bonds to prepare an electrode, thereby constructing an efficient click chemistry reaction interface and realizing ultrasensitive detection applications of copper ions and alkaline phosphatase (ALP).

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The first aspect of the present invention provides a method for preparing a framework nucleic acid enhanced click chemistry and DNAzyme catalytic electrode, comprising the following steps:

[0006] Step 1): Pre-processing the gold electrode by polishing;

[0007] Step 2): co-incubating the DNA tetrahedron with the gold electrode obtained in step 1), and then co-incubating with MCH to obtain a DNA tetrahedron-modified gold electrode Au-TDN;

[0008] Step 3): The gold electrode Au-TDN modified with the DNA tetrahedron obtained in step 2) is incubated with a triplex probe (TE-G1) with a split G-quadruplex A in a neutral buffer to obtain a catalytic electrode Au-TDN-TE-G1; the triplex probe comprises a thymine-rich anchor region, a polythymine spacer region, and a split G-quadruplex A fragment from the 5' end to the 3' end, and the 3' end is modified with an azide group; wherein the thymine-rich anchor region is rich in thymine and, under neutral pH conditions, assembles onto the double-stranded DNA (dsDNA) edge of the DNA tetrahedron through Hoogsteen hydrogen bonds, thereby forming a triplex helical structure; the sequence of the split G-quadruplex A fragment is GTGG.

[0009] Optionally, the buffer is TM buffer.

[0010] Optionally, the polishing pretreatment in step 1) specifically includes: polishing the gold electrode, ultrasonically treating it in ethanol and Milli-Q water respectively, then soaking it in piranha solution, and then ultrasonically treating it in Milli-Q water; finally, cleaning it by CV scanning in alkaline electrolyte and acid electrolyte in sequence, rinsing it with Milli-Q water, and drying it for later use.

[0011] Optionally, the DNA tetrahedron in step 2) is self-assembled by four single strands having sequences as shown in SEQ ID NOs: 1 to 4 through an annealing process.

[0012] Optionally, the sequence of the triplex probe with the split G-quadruplex A in step 3) is as shown in SEQ ID NO: 11, and the 3' end is modified with an azide group.

[0013] The second aspect of the present invention provides a catalytic electrode prepared by the preparation method of the first aspect of the present invention for detecting copper ions (Cu 2+ ) and alkaline phosphatase.

[0014] Optionally, the method for detecting copper ions comprises: incubating the prepared catalytic electrode Au-TDN-TE-G1 with a sample to be tested, adding sodium ascorbate and alkyne-modified split G-quadruplex B (G2), and after the click chemistry reaction is completed, reacting with hemin and K + Co-incubation is performed, and the copper ions in the sample are detected by electrochemically measuring the change in current intensity; the sequence of the alkyne-modified split G-quadruplex B is shown in SEQ ID NO: 12;

[0015] The principle of the detection is as follows: if the sample to be tested contains copper ions, the divalent copper ions are reduced to monovalent copper ions under the action of sodium ascorbate. Under the catalysis of monovalent copper ions, a click chemistry reaction occurs between the probe-modified azide group in the catalytic electrode Au-TDN-TE-G1 and the alkyne-modified split G-quadruplex B, thereby achieving covalent connection of the nucleic acid fragments. The formed complete G-quadruplex is complexed with Hemin in the presence of potassium ions. The formed G-quadruplex / hemin complex has peroxidase-like activity. At the same time, with the help of the electronegative characteristics of the nucleic acid framework (further enhancing the intensity of the oxidized TMB peak current), the current intensity changes. If the sample to be tested does not contain copper ions, the current intensity in the system will not change.

[0016] Optionally, the method for detecting alkaline phosphatase comprises: pre-reacting the sample to be tested with sodium ascorbic acid phosphate to generate ascorbic acid, then adding a catalytic electrode Au-TDN-TE-G1, an alkyne-modified split G-quadruplex B (G2) and a certain concentration of copper ion solution for mixed reaction, and after the click chemistry reaction is completed, reacting with hemin and K + Co-incubation, and detection of alkaline phosphatase in the sample by electrochemically measuring the change in current intensity; the sequence of the alkyne-modified split G-quadruplex B is shown in SEQ ID NO: 12;

[0017] The principle of the detection is as follows: if the sample to be tested contains alkaline phosphatase, alkaline phosphatase reacts with sodium ascorbyl phosphate in advance to generate ascorbic acid. Under the action of ascorbic acid, divalent copper ions are reduced to monovalent copper ions. Under the catalysis of monovalent copper ions, a click chemistry reaction occurs between the azide group modified with the probe in the catalytic electrode Au-TDN-TE-G1 and the alkyne-modified split G-quadruplex B, thereby achieving covalent connection of the nucleic acid fragment. The formed complete G-quadruplex is complexed with Hemin in the presence of potassium ions. The formed G-quadruplex / hemin complex has peroxidase-like activity. At the same time, the electronegative characteristics of the nucleic acid framework (further enhancing the intensity of the oxidized TMB peak current) lead to a change in current intensity. If the sample to be tested does not contain alkaline phosphatase, the current intensity in the system will not change.

[0018] Optionally, the detection includes quantitative detection, and the method of the quantitative detection includes: adding different concentrations of copper ions (Cu 2+) standard solution or alkaline phosphatase standard solution is co-incubated with the catalytic electrode Au-TDN-TE-G1, and a linear relationship is established between the current difference before and after detection and the standard solution of different concentrations, that is, a standard curve is established; by detecting the change in current intensity before and after incubation of the sample to be tested with Au-TDN-TE-G1, it is substituted into the standard curve to obtain the concentration of copper ions or alkaline phosphatase in the sample to be tested, thereby achieving quantitative detection of copper ions or alkaline phosphatase.

[0019] In the present invention, the catalytic electrode Au-TDN-TE-G1 can be regenerated. The regeneration method comprises: incubating the electrode after measurement with a dissociation buffer solution, and then incubating it again with a solution of a triplex probe (TE-G1) containing a split G-quadruplex A. The dissociation buffer solution is an alkaline TM buffer solution; more preferably, the alkaline TM buffer solution has a pH of 10.

[0020] The present invention assembles a thymine-rich triplex probe (with a split G-quadruplex fragment A at the tail) onto the double-stranded DNA (dsDNA) edge of the DNA tetrahedron through Hoogsteen hydrogen bonds, so that the reactants achieve a reasonable spatial distribution and improve the efficiency of the click chemistry reaction on the interface. Under Cu(I) catalytic conditions, the split G-quadruplex fragment B modified with the alkyne group and the fragment A assembled on the tetrahedral scaffold form a complete G-quadruplex through a click chemistry reaction. It is further complexed with Hemin, and the formed G-quadruplex (G-quadruplex) / hemin complex has peroxidase-like activity. At the same time, the nucleic acid framework with strong electronegativity further enhances the response current of the catalytic oxidation of TMB, achieving Cu(I) 2+ Furthermore, by utilizing the unique pH-responsive properties of Hoogsteen hydrogen bonds, the click chemistry products can be dissociated from the nucleic acid framework by adjusting the ambient pH, enabling the reuse and regeneration of the click chemistry reaction interface and reducing the deviation in detection signals between different modified electrodes. More importantly, combined with the dephosphorylation effect of alkaline phosphatase, the modified electrode constructed can be expanded to sensitive detection of alkaline phosphatase (ALP).

[0021] In the present invention, the four single strands used to assemble the DNA tetrahedron and the probe sequences involved are shown in Table 1, wherein the "AAAGAAAAGA" and " TCTTTTCTTT " can form a triplex helical structure with "TTTCTTTTCT" in the TE sequence.

[0022] Table 1 Sequence Listing

[0023]

[0024]

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. This invention constructs an ordered nucleic acid nanostructure interface to optimize the spatial configuration of reactants on the interface, thereby improving the efficiency of interfacial click chemistry reactions. At the same time, the electronegativity of the nucleic acid framework is utilized to enhance the peroxidase-like activity of the G-quadruplex-Hemin complex, achieving highly sensitive detection.

[0027] 2. The present invention can dissociate the click chemistry product from the nucleic acid framework by regulating the environmental pH value, thereby achieving the reuse and regeneration of the click chemistry reaction interface and reducing the detection signal deviation between different modified electrodes.

[0028] 3. The modified electrode constructed in the present invention can be expanded to detect other click chemistry reaction components. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the principle of the present invention.

[0030] Figure 2 Cyclic voltammetry scans of bare gold electrode a, TDN-modified gold electrode b, and TDN-TE c.

[0031] Figure 3 To detect the electrocatalytic signals of G-quadruplex formed by DNA tetrahedron and triplex probe TE-PS2.M with intact G-quadruplex / thiol-modified intact G-quadruplex probe SH-PS2.M using chronoamperometry (A) and differential pulse voltammetry (B), respectively, and to detect the electrocatalytic signals of G-quadruplex formed by DNA tetrahedron and triplex probe TE-PS2.M, TE-T30695, TE-AA, TE-PW17, TE-Human telomere (22AG), TE-TBATE-G1 with intact G-quadruplex / thiol-modified intact G-quadruplex probe SH-PS2.M, SH-T30695, SH-AA, SH-PW17, SH-Human telomere (22AG) and SH-TBATE-G1 can enhance the statistical comparison of the current detection signal changes of the formed G-quadruplex (C), and the results show that the signal enhancement has no sequence dependence; the principle of enhancing DNAzyme catalysis is: the G-quadruplex on the nucleic acid scaffold of the DNA tetrahedron, after the addition of potassium ions and Hemin, the formed G-quadruplex (G-quadruplex) / hemin complex has peroxidase-like activity, and the electronegativity characteristics of the nucleic acid framework further enhance the intensity of the oxidized TMB peak current, thereby causing the current intensity to change; wherein, the complete G-quadruplex is rationally split to form split G-quadruplex fragments, which provide the subsequent copper ion (Cu 2+) and alkaline phosphatase detection provide the basis for signal amplification;

[0032] Figure 4 The signal differences between the triplex probe TE-G1 with split G-quadruplex A and the single-stranded split G-quadruplex probe SH-G1 modified with sulfhydryl groups and the different concentrations of copper ions Cu 2+ The linear relationship diagram (A) and the Cu 2+ selectivity (B).

[0033] Figure 5 Figure 2: Click chemistry time curves using different probes (triplet probe TE-G1 with split G-quadruplex A and thiol-modified single-stranded split G-quadruplex A probe SH-G1) (A) and a schematic diagram of multiple regenerations of the catalytic electrode recognition interface of the triplex probe TE-G1 with split G-quadruplex A by adjusting the pH (B).

[0034] Figure 6 The linear relationship diagram (A) and the comparison results of the detection of different detection substrates using the triplex probe TE-G1 with split G-quadruplex A when the target is changed to ALP show its good selectivity for the detection of ALP (B). DETAILED DESCRIPTION

[0035] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0036] In the following examples, the experimental methods without specific conditions were carried out according to conventional methods and conditions, or selected according to the product specifications; the materials and reagents used were all conventional commercially available products unless otherwise specified.

[0037] Example 1

[0038] Preparation method of framework nucleic acid synergistically enhanced click chemistry and DNAzyme catalytic electrode:

[0039] 1) Polishing of gold electrodes and preparation of DNA tetrahedrons

[0040] Gold electrodes were polished for 3 minutes in 0.3 and 0.05 μm alumina, respectively. Next, the electrodes were ultrasonicated in ethanol and Milli-Q water for 3 minutes, respectively. After soaking in piranha solution (a mixture of concentrated sulfuric acid and 30% hydrogen peroxide (7:3)) for 10 minutes, they were finally ultrasonicated in Milli-Q water for 5 minutes. The electrodes were then cleaned by CV scans in 0.5 M NaOH and 0.5 M H₂SO₄. Finally, the gold electrode surface was rinsed with Milli-Q water and dried with nitrogen gas before use.

[0041] 2) Preparation of DNA Tetrahedrons

[0042] The four single-stranded Probes TA, TB, TC, and TD (sequences shown in Table 1) were mixed at a ratio of 1:1, and then 30 mM TCEP (Tris(2-carboxyethyl)phosphine) was added to make the final DNA concentration 1 μM. Then, a slow annealing operation was performed using a PCR instrument, and the mixture was cooled to 4°C and stored until use.

[0043] 3) Synergistic enhancement of click chemistry by framework nucleic acids and preparation of DNAzyme-catalyzed electrodes

[0044] The prepared DNA tetrahedrons (1 μM) were incubated with the polished electrode at 4°C overnight. The modified gold electrode was then immersed in 2 mM MCH (6-mercaptohexanol) for 1 hour and then incubated in TE-G1 solution (TM buffer, pH 7.0) at 25°C for 90 minutes. This yielded the catalytic electrode, Au-TDN-TE-G1. The TM buffer primarily consisted of Tris-HCl and magnesium chloride.

[0045] Example 2

[0046] Cu 2+ Detection method:

[0047] The resulting catalytic electrode, Au-TDN-TE-G1, was reacted with sodium ascorbate, alkyne-modified split G-quadruplex B (G2), and copper ion solutions of varying concentrations for 60 minutes. After the click chemistry reaction was complete, potassium ions and 10 μM hemin were added and incubated for 1 hour before electrochemical measurements were performed.

[0048] Example 3

[0049] Regeneration and repeated measurements of the identification interface:

[0050] After the measurement, the electrode was incubated with dissociation buffer (TM buffer, pH 10.0) for 90 minutes, then incubated with TE-G1 solution at 25°C for 90 minutes, and then mixed with sodium ascorbate, alkyne-modified split G-quadruplex B (G2) and different concentrations of copper ion solutions for 60 minutes. Finally, potassium ions were added and incubated with hemin for 1 hour before electrochemical measurement.

[0051] Example 4

[0052] Alkaline phosphatase detection method:

[0053] Afterwards, alkaline phosphatase was pre-reacted with sodium ascorbyl phosphate at 37°C for 30 minutes. Alkyne-modified split G-quadruplex B (G2) and a certain concentration of copper ion solution were then added. The mixture was then mixed with the catalytic electrode Au-TDN-TE-G1 prepared in Example 1 and reacted for 60 minutes. After the click chemistry reaction was complete, potassium ions were added and incubated with 10 μM hemin for 1 hour before electrochemical measurements were performed.

[0054] Figure 1 This is a schematic diagram of the electrode preparation and detection principle of the present invention. The principle of the present invention is: the partial DNA sequence that makes up the tetrahedron can form a "TAT" triple DNA structure (i.e., a triplex helical structure) with the probe TE. In the probe TE, the first part is a thymine-rich sequence that can be inserted into the double-stranded DNA (dsDNA) edge of the DNA tetrahedron under neutral pH conditions, thereby forming a triplex helical structure; the second part is a T sequence that is used to separate the triplex anchor sequence and the sequence used for click chemistry reaction; the tail part is a split G-quadruplex probe A, which is modified with an azide group at the 3' end. In Cu 2+ In the presence of sodium ascorbate and an oligonucleotide modified with an alkyne group, a click chemistry reaction can occur, achieving directional coupling of the two molecules. After the click reaction is completed, a complete G-quadruplex is formed on the nucleic acid scaffold. With the help of the electronegativity characteristics of the nucleic acid framework, the intensity of the oxidized TMB peak current is further enhanced. At the same time, due to the unique pH dependence of triple DNA (i.e., triplex helix structure), the complete G-quadruplex formed after the reaction can be dissociated from the DNA tetrahedron scaffold by adjusting the environmental pH value, thereby achieving regeneration of the modified electrode interface.

[0055] Figure 2 The current intensity of (a) bare gold electrode, (b) TDN-modified gold electrode and (c) TDN-TE~G1 gradually decreases with DNA modification, which to some extent indicates the successful preparation of TDN-TE~G1.

[0056] Figure 3 It is shown that at the same complete G-quadruplex concentration, chronoamperometry (Figure A) and differential pulse voltammetry (Figure B) show that the electrocatalytic signal obtained by the triplex probe fixed on the DNA tetrahedron (triplex probe with complete G-quadruplex) is higher than the signal obtained by the single-stranded complete G-quadruplex probe (thiol-modified complete G-quadruplex probe); Figure C shows that the use of triplex probe with complete G-quadruplex can have a strong universality for the G-quadruplex sequence, that is, the signal enhancement is not sequence-dependent. Therefore, by rationally splitting the complete G-quadruplex to form split G-quadruplex fragments, the subsequent copper ion (Cu) 2+ ) and alkaline phosphatase detection provide the basis for signal amplification.

[0057] Figure 4 It was shown that within a certain concentration range of copper ion solution, the signal differences obtained by the triplex probe (with split G-quadruplex) TE-G1 (SEQ ID NO: 11) on the DNA tetrahedron and the thiol-modified single-strand split G-quadruplex probe SH-G1 (SEQ ID NO: 19) were linearly correlated with the copper ion concentration, and the higher detection limit obtained was 200 times higher than the lower detection limit, and the recognition effect was improved. Figure 4 As shown in A; at the same time, it shows good selectivity for copper ion detection, such as Figure 4 As shown in B.

[0058] Figure 5 Panel A in the middle shows a click chemistry reaction between the triplex probe (with a split G-quadruplex) TE-G1 (SEQ ID NO: 11) bound to a DNA tetrahedron and the thiol-modified single-stranded split G-quadruplex probe SH-G1 (SEQ ID NO: 19). The triplex probe (red line) completes the reaction significantly faster than the thiol-modified single-stranded split G-quadruplex probe (black line), indicating that the DNA tetrahedron significantly accelerates the click chemistry reaction rate. Figure 5 Figure B shows that the catalytic electrodes combined with different probes can reconstruct the click chemistry reaction interface after 5 elutions and reassembly, indicating that the proposed construction strategy can achieve the regeneration of the modified electrode interface and improve the repeatability of the modified electrode.

[0059] Figure 6 It shows that after changing the target to alkaline phosphatase (ALP), the current response signal still has a good linear relationship with the concentration of ALP solution ( Figure 6 A), indicating that the proposed identification strategy can be applied to ALP detection; at the same time, the selectivity is good ( Figure 6 B).

[0060] The above description is only a preferred embodiment of the present invention and does not constitute any formal or substantial limitation to the present invention. It should be noted that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A catalytic electrode for detecting copper ions (Cu 2+ ) and alkaline phosphatase, characterized in that, The preparation method of the catalytic electrode comprises the following steps: Step 1): Pre-grind the gold electrode; Step 2): The DNA tetrahedron is co-incubated with the gold electrode obtained in step 1), and then co-incubated with MCH to obtain a DNA tetrahedron-modified gold electrode Au-TDN; Step 3): The gold electrode Au-TDN modified with the DNA tetrahedron obtained in step 2) is incubated with a triplex probe with a split G-quadruplex A in a neutral buffer to obtain a catalytic electrode Au-TDN-TE-G1; the triplex probe comprises a thymine-rich anchor region, a polythymine spacer region, and a G-quadruplex A fragment from the 5' end to the 3' end, and the 3' end is modified with an azide group; wherein the thymine-rich anchor region is rich in thymine and assembles onto the double-stranded DNA (dsDNA) edge of the DNA tetrahedron through Hoogsteen hydrogen bonds under neutral pH conditions, thereby forming a triplex helical structure; the sequence of the split G-quadruplex A fragment is GTGG; The method for detecting copper ions comprises: incubating the prepared catalytic electrode Au-TDN-TE-G1 with a sample to be tested, adding sodium ascorbate and alkyne-modified split G-quadruplex B (G2), and after the click chemistry reaction is completed, reacting with hemin and K + Co-incubation is performed, and the copper ions in the sample are detected by electrochemically measuring the change in current intensity; the sequence of the alkyne-modified split G-quadruplex B is shown in SEQ ID NO: 12; The detection principle is as follows: if the sample to be tested contains copper ions, the divalent copper ions are reduced to monovalent copper ions under the action of sodium ascorbate. Under the catalysis of the monovalent copper ions, a click chemistry reaction occurs between the probe-modified azide group in the catalytic electrode Au-TDN-TE-G1 and the alkyne-modified split G-quadruplex B, achieving covalent connection of the nucleic acid fragments. The formed complete G-quadruplex complexes with Hemin in the presence of potassium ions. The formed G-quadruplex / hemin complex has peroxidase-like activity. At the same time, the electronegativity characteristics of the nucleic acid framework further enhance the intensity of the oxidized TMB peak current, resulting in a change in the current intensity. If the sample to be tested does not contain copper ions, the current intensity in the system will not change. The method for detecting alkaline phosphatase comprises: reacting a sample to be tested with sodium ascorbyl phosphate to generate ascorbic acid in advance, then adding a catalytic electrode Au-TDN-TE-G1, an alkyne-modified split G-quadruplex B (G2) and a certain concentration of copper ion solution for mixed reaction, and after the click chemistry reaction is completed, reacting with hemin and K + Co-incubation, and detecting copper ions in the sample by electrochemically measuring the change in current intensity; the sequence of the alkyne-modified split G-quadruplex B is shown in SEQ ID NO: 12; The detection principle is as follows: if the sample to be tested contains copper ions, the divalent copper ions are reduced to monovalent copper ions under the action of sodium ascorbate. Under the catalysis of monovalent copper ions, a click chemistry reaction occurs between the probe-modified azide group in the catalytic electrode Au-TDN-TE-G1 and the alkyne-modified split G-quadruplex B, thereby achieving covalent connection of the nucleic acid fragments. The formed complete G-quadruplex complexes with Hemin in the presence of potassium ions. The formed G-quadruplex / hemin complex has peroxidase-like activity. At the same time, the electronegativity characteristics of the nucleic acid framework further enhance the intensity of the oxidized TMB peak current, thereby causing the current intensity to change. If the sample to be tested does not contain alkaline phosphatase, the current intensity in the system will not change.

2. The use according to claim 1, characterized in that The polishing pretreatment in step 1) specifically includes: polishing the gold electrode, ultrasonically treating it in ethanol and Milli-Q water respectively, then soaking it in piranha solution, and then ultrasonically treating it in Milli-Q water; finally, cleaning it by CV scanning in alkaline electrolyte and acid electrolyte, rinsing it with Milli-Q water, and drying it for later use.

3. The use according to claim 1, characterized in that The DNA tetrahedron in step 2) is self-assembled by four single strands with sequences as shown in SEQ ID NOs: 1 to 4 through an annealing process.

4. The use according to claim 1, characterized in that The sequence of the triplex probe with the split G-quadruplex A in step 3) is shown in SEQ ID NO: 11, and the 3' end is modified with an azide group.

5. The use according to claim 1, characterized in that The detection includes quantitative detection, and the method of the quantitative detection includes: incubating copper ion or alkaline phosphatase standard solutions of different concentrations with the catalytic electrode Au-TDN-TE-G1, detecting the change in current intensity before and after incubation, establishing a linear relationship between the standard solutions of different concentrations and the change in current intensity, that is, establishing a standard curve; by detecting the change in current intensity after incubation of the sample to be tested with Au-TDN-TE-G1, substituting it into the standard curve, that is, obtaining the concentration of copper ions or alkaline phosphatase in the sample to be tested, thereby achieving quantitative detection of copper ions or alkaline phosphatase.

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