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

Abstract

The invention discloses a preparation method and application of a framework nucleic acid synergistic enhanced click chemistry and DNAzyme catalytic electrode. According to the present invention, the azide group modified split G-quadruplex fragment A is assembled on the double-stranded DNA (dsDNA) edge of the DNA tetrahedron through the Hoogsteen hydrogen bond, such that the reasonable spatial distribution of the reactants is achieved, and the click chemical reaction efficiency on the interface is improved; under the catalytic condition of Cu (I), the alkyne group modified split G-quadruplex fragment B and the fragment A assembled on the tetrahedral scaffold form a complete G-quadruplex through a click chemical reaction. And further, the compound is compounded with Hemin, so that the formed G-quadruplex / hemin compound has peroxidase-like activity. Meanwhile, the nucleic acid framework with the strong electronegativity characteristic further enhances the response current of catalytic oxidation TMB, and sensitive detection of Cu < 2 + > and alkaline phosphatase is achieved.

Description

Technical Field

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

[0002] The copper (I)-catalyzed azide-alkyne cycloaddition reaction (click chemistry) can achieve directional coupling of two molecules by forming a triazole ring structure. Due to the high reaction efficiency and mild conditions, it is conducive to the site-specific modification and coupling of oligonucleotides, and is widely used as a signal conversion tool in the field of sensing. However, compared with the click reaction in homogeneous solution, the single-stranded nucleic acid probe directly modified on the electrode interface often presents unfavorable conformations such as lodging, interchain entanglement, and stacking, which will reduce the efficiency of the click reaction on the electrode interface and also affect the recognition specificity of the probe. At the same time, limited by 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 lead to large deviations in the final detection signal. Summary of the invention

[0003] The purpose of the present invention is: to address the deficiencies of the prior art, the present invention provides 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 A used for click chemistry reaction onto three edges of a 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 a DNAzyme catalytic electrode, comprising the following steps:

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

[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 is assembled to 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.

[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, 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 with sequences as shown in SEQ ID NOs: 1 to 4 through an annealing process.

[0012] Optionally, the sequence of the triplex probe with 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 according to 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, and detecting the copper ions in the sample to be tested 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, divalent copper ions are reduced to monovalent copper ions under the action of sodium ascorbate, and under the catalysis of monovalent copper ions, a click chemistry reaction is carried out between the probe-modified azide group in the catalytic electrode Au-TDN-TE-G1 and the alkyne-modified split G-quadruplex B to achieve covalent connection of the nucleic acid fragments, and the formed complete G-quadruplex is complexed with Hemin in the presence of potassium ions. The formed G-quadruplex (G-quadruplex) / hemin complex has peroxidase-like activity, and 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: reacting the 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-incubate, and detect 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, and divalent copper ions are reduced to monovalent copper ions under the action of ascorbic acid. Under the catalysis of monovalent copper ions, a click chemical reaction occurs between the probe-modified azide group in the catalytic electrode Au-TDN-TE-G1 and the alkyne-modified split G-quadruplex B to achieve 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 (G-quadruplex) / hemin complex has peroxidase-like activity, and 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 alkaline phosphatase, the current intensity in the system will not change.

[0018] Optionally, the detection includes quantitative detection, and the method of quantitative detection includes: mixing 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 value before and after the incubation and the standard solution of different concentrations, that is, a standard curve is established; by detecting the change value of the current intensity before and after the 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 realizing the quantitative detection of copper ions or alkaline phosphatase.

[0019] In the present invention, the catalytic electrode Au-TDN-TE-G1 can be regenerated, and the regeneration method includes: incubating the electrode after the measurement with a dissociation buffer solution, and then incubating it again with a triplex probe (TE-G1) solution with a split G-quadruplex A. The dissociation buffer solution is an alkaline TM buffer solution; more preferably, the pH value of the alkaline TM buffer solution is 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 a DNA tetrahedron through Hoogsteen hydrogen bonds, so that the reactants can 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 with an alkyne group and the fragment A assembled on the tetrahedral scaffold form a complete G-quadruplex through a click chemistry reaction. Further complexed 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+ In addition, by utilizing the unique pH response characteristics of Hoogsteen hydrogen bonds, the click chemistry products can be dissociated from the nucleic acid framework by adjusting the environmental pH value, achieving the reuse and regeneration of the click chemistry reaction interface and reducing the detection signal deviation between different modified electrodes. More importantly, combined with the dephosphorylation effect of alkaline phosphatase, the modified electrode constructed by the institute can be expanded for 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 triple 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. The present invention constructs an ordered nucleic acid nanostructure interface to optimize the spatial configuration of reactants on the interface, thereby improving the efficiency of the interfacial click chemistry reaction; at the same time, the electronegative properties of the nucleic acid framework are utilized to enhance the peroxidase-like activity of the G-quadruplex-Hemin complex to achieve 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 by 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-quadruplexes 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-quadruplexes formed by DNA tetrahedron and triplex probe TE-PS2.M, TE-T30695, TE-AA, TE-PW17, TE-Human telomere(22AG), TE-TBATE-G1, thiol-modified intact G-quadruplex probe SH-PS2.M, SH-T30695, SH-AA, SH-PW17, SH-Human The statistical comparison diagram of the changes in the current detection signal of the G-quadruplex formed by telomere (22AG) and SH-TBATE-G1 (C) shows that the signal enhancement has no sequence dependence; the principle of enhancing DNAzyme catalysis is that after the addition of potassium ions and Hemin, the G-quadruplex / hemin complex formed on the nucleic acid scaffold of the DNA tetrahedron has peroxidase-like activity, and the electronegative characteristics of the nucleic acid framework further enhance the intensity of the oxidized TMB peak current, thereby causing the current intensity to change; the complete G-quadruplex is reasonably split to form split G-quadruplex fragments, which provide a basis for the subsequent copper ion (Cu 2+) and alkaline phosphatase detection provide the basis for signal amplification;

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

[0033] Figure 5 The 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 the schematic diagram of the multiple regeneration 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 detection comparison results of different detection substrates when the triplex probe TE-G1 with split G-quadruplex A was used and the target was changed to ALP, showing that it has good selectivity for the detection of ALP (B). DETAILED DESCRIPTION

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

[0036] The experimental methods in the following examples without specifying specific conditions were carried out according to conventional methods and conditions, or selected according to the product instructions; the materials, reagents, etc. used were 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] The gold electrode was polished in 0.3 and 0.05 μm alumina for 3 minutes. Next, the electrode was ultrasonicated in ethanol and Milli-Q water for 3 minutes, soaked in piranha solution (a mixture of concentrated sulfuric acid and 30% hydrogen peroxide (7:3)) for 10 minutes, and finally ultrasonicated in Milli-Q water for 5 minutes. The electrode was then cleaned by CV scanning 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 for use.

[0041] 2) Preparation of DNA Tetrahedron

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

[0043] 3) Framework nucleic acid synergistically enhanced click chemistry and preparation of DNAzyme catalytic electrodes

[0044] The prepared DNA tetrahedron (1 μM) was 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 immersed in TE-G1 solution (TM buffer, pH 7.0) at 25°C for 90 minutes. The catalytic electrode Au-TDN-TE-G1 was obtained. Among them, TM buffer is mainly composed of Tris-HCl and magnesium chloride.

[0045] Example 2

[0046] Cu 2+ Detection method:

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

[0048] Example 3

[0049] Regeneration and repeated measurements of identification interfaces:

[0050] After the measurement, the electrode was incubated with dissociation buffer solution (TM buffer, pH 10.0) for 90 minutes, and 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 copper ion solutions of different concentrations 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] After that, alkaline phosphatase and sodium ascorbyl phosphate were pre-reacted at 37°C for 30 minutes, and then alkyne-modified split G-quadruplex B (G2) and a certain concentration of copper ion solution were added and mixed with the catalytic electrode Au-TDN-TE-G1 prepared in Example 1 for 60 minutes. After the click chemistry reaction was completed, potassium ions were added and incubated with 10 μM hemin for 1 hour, and finally electrochemical measurement was performed.

[0054] Figure 1 This is the principle diagram of the electrode preparation and detection of the present invention. The principle of the present invention is that the partial DNA sequence constituting the tetrahedron can form a triple DNA structure of "TAT" (i.e., a triple helical structure) with the probe TE. In the probe TE, the first part is a thymine-rich sequence, which can be inserted into the double-stranded DNA (dsDNA) edge of the DNA tetrahedron under neutral pH conditions to form a triple helical structure; the second part is a T sequence 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 oligonucleotides modified with alkyne groups, a click chemistry reaction can occur to achieve directional coupling of 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 tetrahedral 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 The results show that, at the same complete G-quadruplex concentration, the 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 (complete G-quadruplex probe modified with thiol); Figure C shows that the use of triplex probe with complete G-quadruplex can have strong universality for G-quadruplex sequence, that is, signal enhancement is not sequence-dependent. Therefore, by reasonably splitting the complete G-quadruplex to form split G-quadruplex fragments, the subsequent copper ion (Cu) is generated. 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 detected 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 Figure A in the middle shows that when the triplex probe (with split G-quadruplex) TE-G1 (SEQ ID NO: 11) bound to the DNA tetrahedron and the thiol-modified single-stranded split G-quadruplex probe SH-G1 (SEQ ID NO: 19) undergo click chemistry reaction, the triplex probe (red line) has a significantly faster reaction completion time 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 is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a framework nucleic acid synergistically enhanced click chemistry and DNAzyme catalytic electrode, characterized in that: The following steps are involved: Step 1): Pre-processing the gold electrode by grinding; Step 2): co-incubate the DNA tetrahedron with the gold electrode obtained in step 1), and then co-incubate 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 is assembled to 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.

2. The preparation method 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 preparation method 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 preparation method according to claim 1, characterized in that: The sequence of the triplex probe with 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 catalytic electrode prepared by the preparation method according to any one of claims 1 to 4 is effective in detecting copper ions (Cu 2+ ) and alkaline phosphatase.

6. The use according to claim 5, characterized in that: 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, and detecting the copper ions in the sample to be tested 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 principle of the detection is as follows: if the sample to be tested contains copper ions, divalent copper ions are reduced to monovalent copper ions under the action of sodium ascorbate, and under the catalysis of monovalent copper ions, a click chemistry reaction is carried out between the probe-modified azide group in the catalytic electrode Au-TDN-TE-G1 and the alkyne-modified split G-quadruplex B to achieve covalent connection of the nucleic acid fragments, and the formed complete G-quadruplex is complexed with Hemin in the presence of potassium ions. The formed G-quadruplex (G-quadruplex) / hemin complex has peroxidase-like activity, and at the same time, the electronegativity characteristics of the nucleic acid framework are used to 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 copper ions, the current intensity in the system will not change.

7. The use according to claim 5, characterized in that: 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 copper ion solution of a certain concentration for mixed reaction, and after the click chemical reaction is completed, reacting with hemin and K + Co-incubation, and detecting the copper ions in the sample to be tested 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 principle of the detection is as follows: if the sample to be tested contains copper ions, divalent copper ions are reduced to monovalent copper ions under the action of sodium ascorbate, and under the catalysis of monovalent copper ions, a click chemistry reaction is carried out between the probe-modified azide group in the catalytic electrode Au-TDN-TE-G1 and the alkyne-modified split G-quadruplex B to achieve covalent connection of the nucleic acid fragments, and the formed complete G-quadruplex is complexed with Hemin in the presence of potassium ions. The formed G-quadruplex (G-quadruplex) / hemin complex has peroxidase-like activity, and at the same time, the electronegativity characteristics of the nucleic acid framework are used to 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.

8. The use according to claim 5, characterized in that: The detection includes quantitative detection, and the method of 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 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 the sample to be tested is incubated with Au-TDN-TE-G1, substituting it into the standard curve, the concentration of copper ions or alkaline phosphatase in the sample to be tested is obtained, and quantitative detection of copper ions or alkaline phosphatase is achieved.

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