A kit and method for selectively recognizing and detecting tumor cells

Abstract

This invention provides a kit and method for selectively identifying and detecting tumor cells. The kit includes: a triaptamer logic gate complex, a Toehold-driven strand displacement reaction complex, an F-chain, DNA nanoflowers, a metal electrode, and a DNA polyhedral framework structure. This invention uses the constructed triaptamer logic gate complex to recognize tumor cells. The resulting T-chain further triggers a Toehold-mediated strand displacement reaction, amplifying the signal. The SP-chain product of the strand displacement reaction further triggers the capture of DNA nanoflowers modified with electrochemical signal tags by an electrode modified with a DNA tetrahedral framework structure, thereby generating an electrochemical signal. The recognition and amplification systems constructed in this invention are simple, rapid, safe, environmentally friendly, and low-cost, enabling rapid identification and detection of target tumor cells, providing a new approach for the efficient and specific detection of circulating tumor cells.

Description

Technical Field

[0001] This invention relates to the field of tumor cell detection technology, and in particular to a kit and detection method for selectively identifying and detecting tumor cells. Background Technology

[0002] Cancer is a global health problem with serious impacts on individuals and society, and is one of the most serious threats to public health. The concept of liquid biopsy was introduced in 2010 and has quickly become a very promising early cancer diagnostic technique. Compared with tissue biopsy, this method has advantages such as being less invasive, convenient for sampling, and providing comprehensive information, facilitating early diagnosis, decision-making, and prognostic monitoring for cancer. Circulating tumor cells (CTCs) are tumor markers, which are tumor cells shed from primary or secondary tumor sites and enter the peripheral blood circulation. They are closely related to cancer metastasis and recurrence. CTCs can be detected in the peripheral blood of patients with advanced solid tumors in most types, and their presence may indicate tumor metastasis. However, the number of CTCs in peripheral blood is very rare, averaging only 10 per 1000 cells. 6 ~10 7 One tumor cell-mediated cytokine (CTC) is found in only one white blood cell. DNA aptamers, as inexpensive short DNA chains, can specifically bind to target tumor cells through aptamer screening technology, serving as a rapid and efficient recognition probe. However, the target factor of a single DNA aptamer is often expressed in multiple tumor cells, meaning that using a single aptamer can only distinguish between normal cells and tumor cells, but cannot accurately identify which type of tumor cell it is.

[0003] Electrochemical analysis is a class of instrumental analytical methods based on the electrochemical properties of substances in solution. It is based on the electrochemical properties and changes of substances in solution, and relies on the quantitative relationships between electrical quantities such as potential, conductance, current, and charge and certain quantities of the analyte to perform qualitative and quantitative analysis of components. Utilizing the specific binding of DNA molecules to biomolecules, electrodes are modified with DNA molecules to achieve the detection of various biomolecules—this is known as electrochemical biosensors. Currently, electrochemical analysis methods are widely used for the detection of various biomolecules. However, methods specifically targeting tumor cells are still rare. Summary of the Invention

[0004] Based on this, the present invention provides a kit for selectively identifying and detecting tumor cells that can simultaneously and accurately identify three target factors with high sensitivity.

[0005] A kit for selectively identifying and detecting tumor cells includes: a triaptamer logic gate complex, a Toehold driven chain displacement reaction complex, an F chain, DNA nanoflowers, a metal electrode, and a DNA polyhedral framework structure.

[0006] The three-aptamer logic gate complex includes the EpCAM aptamer, sgc8c aptamer, XQ-2d aptamer, and T chain, with nucleic acid sequences as shown in SEQ ID NO: 1-4, respectively; the Toehold-driven chain displacement reaction complex includes the AP chain, SP chain, and L chain, with nucleic acid sequences as shown in SEQ ID NO: 5-7, respectively; the nucleic acid sequence of the F chain is shown in SEQ ID NO: 8; the DNA nanoflower is modified with an electrochemical signal tag; the bottom end of the DNA polyhedral framework structure is modified with a thiol group, the top end is modified with a DNase, and the sides are modified with stem-loop structures. The activity of the DNase is masked by the complementary strand. The SP chain can bind to the complementary strand to expose the activity of the DNase to cleave the stem-loop structure. The sticky ends formed after the stem-loop structure is cleaved by the DNase can bind to the DNA nanoflower.

[0007] In one embodiment, the DNA nanoflower comprises an S1 chain, an S2 chain, an S3 chain, and an S4 chain, with nucleic acid sequences as shown in SEQ ID NO: 9-12, respectively, and the S4 chain is modified with the electrochemical signal tag.

[0008] In one embodiment, the DNA polyhedral framework structure includes A1 chain, A2 chain, A3 chain and A4 chain, and the nucleic acid sequences are shown in SEQ ID NO: 13-16, respectively. The A1 chain, A3 chain and A4 chain are modified with thiol groups at the 5' end.

[0009] In one embodiment, the nucleic acid sequence of the DNA enzyme is shown in SEQ ID NO: 17, the nucleic acid sequence of the stem-loop structure is shown in SEQ ID NO: 18, and the nucleic acid sequence of the complementary strand is shown in SEQ ID NO: 19.

[0010] In one embodiment, the electrochemical signal tag is selected from one or more of ferrocene, methylene blue, thionine, and neutral red.

[0011] In one embodiment, the metal electrode is a gold electrode.

[0012] In one embodiment, the preparation method of the triaptamer logic gate complex includes the following steps: dispersing equal amounts of the EpCAM aptamer, sgc8c aptamer, XQ-2d aptamer and T chain in PBS buffer, heating at 95°C for 5 min, and cooling at room temperature for 30 min.

[0013] In one embodiment, the preparation method of the Toehold driven chain displacement reaction complex includes the following steps: dispersing equal amounts of the AP chain, SP chain and L chain in PBS buffer, heating at 95°C for 5 min, and cooling at room temperature for 30 min.

[0014] In one embodiment, the DNA polyhedral framework structure is bound to the metal electrode via the thiol groups.

[0015] This invention also provides a method for detecting tumor cells for purposes other than disease diagnosis and treatment, which uses the above-mentioned kit and includes the following steps:

[0016] Obtain cell samples;

[0017] The cell sample was mixed with the triaptamer logic gate complex, incubated, and then centrifuged to collect the supernatant.

[0018] The supernatant was mixed with the Toehold-driven chain displacement reaction complex, F chain, DNA nanoflowers and the metal electrode modified with the DNA polyhedral framework structure. After incubation, the metal electrode was removed for electrochemical measurement.

[0019] The above-described solution of the present invention has the following beneficial effects:

[0020] This invention is the first to prepare a triaptamer logic gate complex for tumor cell recognition. This structure is simple, rapid, inexpensive, and environmentally friendly to prepare, enabling highly efficient recognition. This invention breaks through conventional approaches and barriers in tumor cell detection, by designing and realizing a triaptamer-based AND-type logic gate structure for the first time. Utilizing the principle that different aptamers bind to different proteins, it identifies target cells from a variety of tumor cells. Combined with DNA strand amplification technology and electrochemical biotechnology, it enables rapid identification and measurement of tumor cells. This method is simple, safe, reliable, and can efficiently and rapidly identify target tumor cells. Furthermore, in the signal processing of this invention, the signal is amplified twice, especially in cases of low circulating tumor concentration, further enhancing the practical application capability of the invention. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the detection principle of a reagent kit according to an embodiment of the present invention;

[0022] Figure 2 This is a graph showing the SWV results of electrochemical measurement of normal human liver cells L02 in Example 1 of the present invention;

[0023] Figure 3 This is a graph showing the SWV results of electrochemical measurements on human breast tumor cells MCF-7 in Example 2 of this invention;

[0024] Figure 4 This is a graph showing the SWV results of electrochemical measurement of HepG2 human liver tumor cells in Example 3 of the present invention;

[0025] Figure 5 This refers to the SWV results of electrochemical measurements of human lung tumor cells A549 in Example 4 of this invention;

[0026] Figure 6 This is the SWV result of electrochemical measurement of human lung tumor cells A549 in Comparative Example 1 of this invention. Detailed Implementation

[0027] To make the technical problems, solutions, and advantages of this invention clearer, a detailed description will be provided below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0028] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Unless otherwise specified, the reagents and instruments used in the embodiments are conventional choices in the art. Experimental methods not specifying specific conditions in the embodiments are implemented according to conventional conditions, such as those described in literature, books, or methods recommended by the manufacturer.

[0029] An embodiment of the present invention provides a kit for selectively identifying and detecting tumor cells, comprising: a triaptamer logic gate complex, a Toehold driving chain displacement reaction complex, an F chain, DNA nanoflowers, a metal electrode, and a DNA polyhedral framework structure.

[0030] The aforementioned three-aptamer logic gate complex includes the EpCAM aptamer, sgc8c aptamer, XQ-2d aptamer, and T chain, with nucleic acid sequences shown in SEQ ID NO: 1–4, respectively. The aforementioned Toehold-driven chain substitution reaction complex includes the AP chain, SP chain, and L chain, with nucleic acid sequences shown in SEQ ID NO: 5–7, respectively. The nucleic acid sequence of the aforementioned F chain is shown in SEQ ID NO: 8. The aforementioned DNA nanoflowers are modified with electrochemical signal tags. The bottom end of the DNA polyhedral framework structure is modified with thiol groups, the top end with DNase, and the sides with stem-loop structures. The DNase activity is masked by the complementary strand. The SP chain can bind to the complementary strand, thereby exposing the DNase activity to cleave the stem-loop structure. The sticky ends formed after the stem-loop structure is cleaved by the DNase can bind to the DNA nanoflowers.

[0031] This invention introduces specific base sequences into the EpCAM, sgc8c, and XQ-2d aptamers, making them complementary to the T chain. The T chain and the three aptamer chains, after annealing in a buffer solution, can form an AND-type logic gate structure. The L, AP, and SP chains, after annealing in a buffer solution, can pair to form the basic structure for the Toehold chain substitution reaction. The DNA polyhedral framework structure can be modified by binding to the metal electrode via thiol groups. Figure 1 As shown, when detecting cell samples, under the condition that all three antigens are present, the three aptamers competitively leave, releasing the T chain. The T chain then attacks the Toehold-driven chain displacement reaction complex formed by the AP / SP / L chains, and completes the entire chain displacement reaction with the participation of the fuel chain F chain, achieving cyclic amplification of the T chain. The competitively leaving SP chain binds to the complementary strand of the DNase, thereby exposing the DNase chain and initiating the cleavage of the stem-loop structure, exposing its sticky ends. The DNA nanoflowers then bind to the sticky ends, and the electrochemical signal tag is captured by the DNA polyhedral framework structure and brought close to the metal electrode, thereby generating an electrical signal. The presence of target tumor cells in the sample can be determined based on this electrical signal.

[0032] This invention presents the first kit to prepare a triaptamer logic gate complex for tumor cell recognition. This structure is simple, rapid, inexpensive, and environmentally friendly, enabling highly efficient recognition. This invention breaks through conventional approaches and barriers in tumor cell detection, designing and realizing for the first time a triaptamer-based AND-type logic gate structure. Utilizing the principle that different aptamers bind to different proteins, it identifies target cells from a variety of tumor cells. Combined with DNA strand amplification and electrochemical biotechnology, it enables rapid identification and measurement of tumor cells. This method is simple, safe, reliable, and efficiently identifies target tumor cells. Furthermore, in the signal processing of this invention, the signal is amplified twice, especially in cases of low circulating tumor concentration, further enhancing the practical application capability of the invention.

[0033] In one specific example, the DNA nanoflower comprises an S1 chain, an S2 chain, an S3 chain, and an S4 chain, with nucleic acid sequences as shown in SEQ ID NO: 9–12, respectively, and the S4 chain is modified with an electrochemical signal tag.

[0034] In a specific example, the DNA polyhedral framework structure includes A1, A2, A3, and A4 chains, with nucleic acid sequences shown in SEQ ID NO: 13–16, respectively. The A1, A3, and A4 chains are modified with thiol groups at their 5' ends. This DNA polyhedral framework structure provides better fixation and support, facilitating interfacial reactions and promoting the DNAwalker reaction to capture DNA nanoflowers. After the DNA enzyme chain binds to the A1 chain, it extends outwards along the 5' end to the tetrahedral vertices. The stem-loop structure (enzyme substrate) binds to the A1, A3, and A4 chains, extending outwards along the three lateral edges of the tetrahedron.

[0035] In a specific example, the nucleic acid sequence of the DNase is shown in SEQ ID NO: 17, the nucleic acid sequence of the stem-loop structure is shown in SEQ ID NO: 18, and the nucleic acid sequence of the complementary strand is shown in SEQ ID NO: 19.

[0036] In a specific example, the electrochemical signal tag is selected from one or more of ferrocene, methylene blue, thionine, and neutral red, and can be selected as needed, preferably ferrocene (Fc).

[0037] In a specific example, the metal electrode is a gold electrode, but other commonly used metal electrodes can be selected as needed.

[0038] In a specific example, the preparation method of the triaptamer logic gate complex includes the following steps: equal amounts of EpCAM aptamer, sgc8c aptamer, XQ-2d aptamer, and T chain are dispersed in PBS buffer, heated at 95°C for 5 min, cooled to room temperature for 30 min, and the resulting triaptamer logic gate complex is stored at 4°C for later use. Preferably, the pH of the PBS buffer is 7.2. Preferably, the concentration of the three aptamers and the T chain in the PBS buffer is 100 μM. It is understood that the triaptamer logic gate complex in the kit can exist in a bound state or as a single-chain component.

[0039] In a specific example, the preparation method of the Toehold driven chain displacement reaction complex includes the following steps: equal amounts of AP, SP, and L chains are dispersed in PBS buffer, heated at 95°C for 5 min, cooled to room temperature for 30 min, and the resulting Toehold driven chain displacement reaction complex is stored at 4°C for later use. Preferably, the pH of the PBS buffer is 7.2. Preferably, the concentration of AP, SP, and L chains in the PBS buffer is 100 μM. It is understood that the Toehold driven chain displacement reaction complex in the kit can exist in a bound state or as a single-chain component.

[0040] In a specific example, the preparation method of DNA nanoflowers includes the following steps: equal amounts of S1, S2, S3, and S4 chains are dispersed in PBS buffer, heated at 95°C for 5 min, cooled to room temperature for 30 min, and the resulting DNA nanoflowers are stored at 4°C for later use. Preferably, the pH of the PBS buffer is 7.2. Preferably, the concentration of S1, S2, S3, and S4 chains in the PBS buffer is 100 μM. It is understood that the DNA nanoflowers in the kit can exist in a bound state or as single-stranded components.

[0041] In a specific example, the preparation method of the DNA polyhedral framework structure includes the following steps: equal amounts of A1, A2, A3, and A4 chains are dispersed in TM buffer, heated at 95°C for 10 min, cooled at 4°C for 30 min, and the resulting DNA polyhedral framework structure is stored at 4°C for later use. Preferably, the TM buffer contains 20 mM Tris and 50 mM Mg. 2+ The pH is 7.2. Preferably, the concentrations of A1, A2, A3, and A4 chains in the TM buffer are all 100 μM. It is understood that the DNA polyhedral framework structure in the kit can exist in a bound state or as a single-stranded component.

[0042] In one specific example, the DNA polyhedral framework structure is bound to the metal electrode via thiol groups. Furthermore, the nucleic acid strands constituting the DNA polyhedral framework require reduction of the modified thiol groups using 200 equivalents of TCEP; that is, before annealing, the thiol groups are reduced using 200 equivalents of TCEP, meaning that a final concentration of 600 μM TCEP is added to the mixed strands, and the reaction is carried out at room temperature for 30 min.

[0043] Optionally, the kit may provide the metal electrode and DNA polyhedral framework structure separately, or it may provide the metal electrode pre-modified with the DNA polyhedral framework structure. Optionally, the method for modifying the metal electrode with the DNA polyhedral framework structure includes the following steps: drop-coating the DNA polyhedral framework structure (TDN) onto the surface of the metal electrode, modifying overnight, rinsing the metal electrode surface with PBS buffer, blocking with 1 mM mercaptohexanol for 30 min, rinsing the metal electrode surface with PBS buffer, storing in PBS buffer at 4°C, and drying before use.

[0044] A method for detecting tumor cells according to an embodiment of the present invention includes the following steps:

[0045] S1. Obtain cell samples;

[0046] S2. Mix the cell sample with the triaptamer logic gate complex, incubate, and then centrifuge to collect the supernatant.

[0047] S3. Mix the supernatant with the Toehold driving chain displacement reaction complex, F chain, DNA nanoflowers and a metal electrode modified with a DNA polyhedral framework structure, incubate, and then remove the metal electrode for electrochemical measurement.

[0048] Optionally, the cell sample preparation method includes the following steps: digesting cells with trypsin for 3 min, adding DMEM complete medium, pipetting and transferring the cells to a centrifuge tube; centrifuging at 1500 rpm for 5 min, discarding the supernatant; washing with PBS, and then redispersing the cells in binding buffer (containing 5 mM MgCl₂). 2+ Cell samples were obtained in PBS (25 mM glucose, 1 mg / mL BSA, pH 7.2).

[0049] Optionally, the working concentrations of the triaptamer logic gate complex, the Toehold-driven chain displacement reaction complex, the F chain, and the DNA nanoflower are 200 nM.

[0050] Optionally, the electrochemical measurement method is square wave voltammetry, with a scan rate of 0.1 V / s, a scan range of 0-0.5 V, a voltage drop of 0.001 V, and an amplitude of 0.05 V.

[0051] The following are specific examples.

[0052] Example 1

[0053] Triadaptor logic gate structure for detection in normal human hepatocytes (L02).

[0054]

[0055]

[0056] 1. DNA Logic Circuit Construction: Equal amounts of three aptamers (EpCAM, sgc8c, XQ-2d) and DNA strands (T strand) were dispersed in PBS buffer (pH = 7.2) to prepare a 100 μM mixed solution. The solution was heated at 95 °C for 5 min, followed by cooling at room temperature for 30 min to obtain the triaptamer logic gate complex (Apts / T). Equal amounts of three DNA strands (AP, SP, L strands) were dispersed in PBS buffer (pH = 7.2) to prepare a 100 μM mixed solution. The solution was heated at 95 °C for 5 min, followed by cooling at room temperature for 30 min to obtain the Toehold driven chain displacement reaction complex (AP / SP / L). Equal amounts of four DNA strands (S1-4 strands) were dispersed in PBS buffer (pH = 7.2) to prepare a 100 μM mixed solution. The solution was heated at 95 °C for 5 min, followed by cooling at room temperature for 30 min to obtain ferrocene-modified DNA nanoflowers (DFs). Disperse equal amounts of the four DNA strands (A1-4 strands) in TM buffer (20mM Tris, 50mM Mg). 2+ A 100 μM mixed solution was prepared by heating the solution at 95 °C for 10 min, followed by cooling at 4 °C for 30 min to obtain the DNA polyhedral framework structure (TDN). (pH = 7.2)

[0057] 2. Electrode modification: 3 μL of DNA framework structure (TDN) was drop-coated onto the gold electrode surface and left to modify overnight. The electrode surface was rinsed twice with PBS and blocked with 1 mM mercaptohexanol for 30 min. The electrode surface was rinsed twice with PBS, stored in PBS buffer at 4°C, and dried before use.

[0058] 3. Sample Preparation: Digest cells with 500 μL trypsin for 3 min, add 1 mL of DMEM complete medium, pipette and transfer cells to centrifuge tubes. Centrifuge at 1500 rpm for 5 min, discard the supernatant. Wash twice with PBS, and redisperse cells in binding buffer (containing 5 mM MgCl₂). 2+ L02 cell solutions of different concentrations were prepared in PBS (25 mM glucose, 1 mg / mL BSA, pH 7.2).

[0059] 4. Cell incubation: Take 200 μL of cell solution and add Apts / T to bring the logic gate structure concentration to 200 nM. Incubate the solution at 37°C for 90 min. Centrifuge at 1500 rpm for 3 min and collect the supernatant. Add the DNA strand complex (AP / SP / L), DNA nanoflower structures (DFs), and F strand to the supernatant to a final concentration of 200 nM. Immerse the electrode in the solution and incubate at 37°C for 2 h.

[0060] 5. Electrochemical measurement: Remove the electrode, rinse the electrode surface twice with PBS, dry it with nitrogen, and then place it in PBS buffer for electrochemical measurement.

[0061] Electrochemical measurements were performed using a Chenhua CH660E electrode, with an Ag / AgCl reference electrode and a Pt counter electrode. Electrochemical method: SWV, scan rate: 0.1 V / s, scan range: 0-0.5 V. Voltage drop: 0.001 V, amplitude: 0.05 V.

[0062] Since three conditions must be met for the logic gate to trigger the next reaction, and normal liver cells do not meet these conditions, the result is as follows: Figure 2 As shown (cell concentration of 5000 cells per milliliter), almost no electrochemical signal was generated during the measurement in this example.

[0063] Example 2:

[0064] The assay was performed using human breast cancer cells (MCF-7), and the remaining steps were the same as in Example 1.

[0065] When only one or two aptamers respond, the logic gate structure can effectively filter them, and the entire DNA signaling circuit will not be triggered, resulting in... Figure 3 As shown (cell concentration of 5000 cells per milliliter), the detection results in this example did not exhibit the characteristic electrochemical signal of ferrocene.

[0066] Example 3:

[0067] The assay was performed using human liver cancer cells (HepG2), and the remaining steps were the same as in Example 1.

[0068] Similar to Example 2, the entire DNA signaling circuit is not triggered when only one or two aptamers respond; only cells that show positivity for all three aptamers can generate an electrochemical signal, as shown in the following results. Figure 4 As shown (cell concentration of 5000 cells per milliliter), the detection results in this example did not exhibit the characteristic electrochemical signal of ferrocene.

[0069] Example 4:

[0070] The assay was performed using human lung cancer cells (A549), with the remaining steps being the same as in Example 1.

[0071] Human lung cancer cells (A549) simultaneously express EpCAM, sgc8c, and XQ-2d; therefore, the kit can effectively detect the target tumor (A549). The triaptamer logic gate structure enables efficient and accurate cell recognition, and subsequent electrochemical biological methods can efficiently measure A549 tumor cells. The results are as follows: Figure 5As shown (cell concentration of 5000 cells / mL), the detection results in this example exhibited a clear characteristic electrochemical signal of ferrocene. Further measurements were performed using different concentrations of A549 cells, with the detection limit as low as 34 cells / mL.

[0072] Comparative Example 1

[0073] The assay was performed using human lung cancer cells (A549). This comparative example added the binding site between the T chain and the aptamer; the remaining steps were the same as in Example 1. The T chain sequence used in this comparative example is shown in SEQ ID NO: 20.

[0074] When the binding sites between the T chain and the aptamer are increased, the triaptamer logic gate structure becomes more stable. Stronger binding hinders the competitive binding of the aptamer to the target protein, leading to difficulty in aptamer dissociation and T chain release. When measured on tumor cells A549, although the results showed characteristic electrochemical signals of ferrocene, under identical conditions, such as... Figure 6 As shown, its electrochemical signal is significantly lower than before the increase.

[0075] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A kit for selectively identifying and detecting tumor cells, characterized in that, include: Triaptor logic gate complex, Toehold driven chain substitution reaction complex, F chain, DNA nanoflower, metal electrode and DNA polyhedral framework structure; The three-aptamer logic gate complex includes the EpCAM aptamer, sgc8c aptamer, XQ-2d aptamer, and T chain, with nucleic acid sequences as shown in SEQ ID NO: 1-4, respectively; the Toehold-driven chain displacement reaction complex includes the AP chain, SP chain, and L chain, with nucleic acid sequences as shown in SEQ ID NO: 5-7, respectively; the nucleic acid sequence of the F chain is shown in SEQ ID NO: 8; the DNA nanoflower is modified with an electrochemical signal tag; the bottom end of the DNA polyhedral framework structure is modified with a thiol group, the top end is modified with a DNase, and the sides are modified with a stem-loop structure; the activity of the DNase is masked by the complementary strand; the SP chain can bind to the complementary strand to expose the activity of the DNase to cleave the stem-loop structure; the sticky ends formed after the stem-loop structure is cleaved by the DNase can bind to the DNA nanoflower; The DNA nanoflower comprises an S1 chain, an S2 chain, an S3 chain, and an S4 chain, with nucleic acid sequences as shown in SEQ ID NO: 9~12, respectively. The S4 chain is modified with the electrochemical signal tag. The DNA polyhedral framework structure includes A1 chain, A2 chain, A3 chain and A4 chain, and the nucleic acid sequences are shown in SEQ ID NO: 13~16, respectively. The A1 chain, A3 chain and A4 chain are modified with thiol groups at the 5' end. The nucleic acid sequence of the DNA enzyme is shown in SEQ ID NO: 17, the nucleic acid sequence of the stem-loop structure is shown in SEQ ID NO: 18, and the nucleic acid sequence of the complementary strand is shown in SEQ ID NO:

19.

2. The reagent kit according to claim 1, characterized in that, The electrochemical signal tag is selected from one or more of ferrocene, methylene blue, thionine, and neutral red.

3. The reagent kit according to claim 1, characterized in that, The metal electrode is a gold electrode.

4. The reagent kit according to claim 1, characterized in that, The preparation method of the triaptamer logic gate complex includes the following steps: equal amounts of the EpCAM aptamer, sgc8c aptamer, XQ-2d aptamer and T chain are dispersed in PBS buffer, heated at 95°C for 5 min, and cooled at room temperature for 30 min.

5. The reagent kit according to claim 1, characterized in that, The preparation method of the Toehold driven chain displacement reaction complex includes the following steps: equal amounts of the AP chain, SP chain and L chain are dispersed in PBS buffer, heated at 95°C for 5 min, and cooled at room temperature for 30 min.

6. The reagent kit according to claim 1, characterized in that, The DNA polyhedral framework structure is bound to the metal electrode via the thiol groups.

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