Preparation of electrochemical biosensor based on efficient Ce / Tb metal organogel and rigid ordered chain DNA track

Abstract

The invention belongs to the technical field of immunoassay and biosensing, and provides a preparation method of an electrochemical biosensor based on efficient Ce / Tb metal organic gel and a rigid ordered chain DNA track. Ce / Tb metal organic gel of a three-dimensional network structure is adopted as a luminous body, the luminous body has the advantages of being mild in synthesis condition, easy to prepare, good in film-forming property and the like, the porous structure of the luminous body is more beneficial to full contact with a co-reaction reagent, and due to the ligand-metal antenna effect and dual sensitization of directional Ce < 3 + >-Tb < 3 + > energy transfer, the internal energy transfer efficiency is improved, and the luminous body is more stable in performance. Therefore, the ECL luminous efficiency is greatly improved. Besides, an enzyme-driven DNA walker based on the chain-shaped DNA track as a guide is designed, compared with a randomly constructed DNA track, the method has the advantages that the chain spacing can be accurately controlled, dynamic obstacles are reduced through rigid ordered arrangement of binding sites, and the rolling efficiency and speed of the DNA walker are remarkably improved. According to different electrochemical luminescence signal intensities of to-be-detected substances with different concentrations, the detection of the acute B lymphocytic leukemia marker Pax-5a gene is realized.

Description

Technical Field

[0001] The present invention relates to a method for preparing an electrochemical biosensor based on a highly efficient Ce / Tb metal organogel and rigid, ordered, chain-like DNA tracks. Specifically, the Ce / Tb metal organogel, which forms a three-dimensional network structure through ultrafast self-assembly at room temperature, is used as a luminescent material. Furthermore, an enzyme-driven DNA walker guided by the chain-like DNA tracks is designed to produce an electrochemical biosensor for detecting the Pax-5a gene, a marker for acute B-lymphocytic leukemia. This method belongs to the field of novel functional materials and biosensor detection technology. Background Art

[0002] Pax-5 is a key transcription factor that regulates multiple target genes and is crucial for the correct characterization and differentiation of cell lineages. Among the four Pax-5 isoforms, Pax-5a plays a key role in B-cell differentiation and development. Its abnormal expression is directly associated with the pathogenesis of acute B-lymphoblastic leukemia, thus establishing Pax-5a as a reliable biomarker for the detection of acute B-lymphoblastic leukemia. Furthermore, acute B-lymphoblastic leukemia develops rapidly and is associated with frequent high-risk complications. Without timely diagnosis and treatment, it can develop into a life-threatening disease. Therefore, the development of accurate, efficient, rapid, and sensitive nucleic acid detection methods is crucial for the early detection and timely treatment of acute B-lymphoblastic leukemia.

[0003] At present, the detection of acute B-lymphocytic leukemia markers mostly adopts cytogenetics, fluorescence in situ hybridization, etc., but these methods are still subject to the constraints of factors such as low sensitivity, unsatisfactory quantitative analysis and reliance on high-cost special instruments, and are difficult to meet the great demand for early diagnosis of acute B-lymphocytic leukemia. Among them, electrochemiluminescence has the advantages of high sensitivity, wide detection range, rapid response, good controllability, etc., and has a wide range of applications in the fields of biological imaging, medical diagnosis, and detection of disease markers. Compared with traditional analytical methods, the electrochemical biosensor adopted in the present invention has the advantages of high sensitivity, good selectivity, fast response speed, simple operation, and easy miniaturization. Therefore, the present invention prepares an electrochemical biosensor based on efficient Ce / Tb metal organic gel and rigid ordered chain DNA track to achieve sensitive detection of the acute B-lymphocytic leukemia marker Pax-5a gene.

[0004] The present invention uses 2,2':6',2''-terpyridine-4,4',4''-tricarboxylic acid as an organic ligand, Ce 3+ , Tb 3+ As a dual-center ion, it assembles into Ce / Tb metal organic gel at ultra-fast speed at room temperature. This material has a three-dimensional network structure, and its porous structure is more conducive to sufficient contact with the co-reactant, and because of the ligand-metal antenna effect and directional Ce 3+ to Tb3+ The dual sensitization of energy transfer improves the efficiency of internal energy transfer, thereby greatly improving the ECL luminescence efficiency. In addition, an enzyme-driven DNA walker based on a chain-shaped DNA track is designed. Compared with the randomly constructed DNA track, this method can accurately control the chain spacing, and the rigid and ordered arrangement of the binding sites reduces the kinetic barrier, significantly improving the rolling efficiency and rate of the DNA walker. The present invention prepares an electrochemical biosensor based on high-efficiency Ce / Tb metal organic gel and rigid and ordered chain DNA track to achieve quantitative detection of the Pax-5a gene, a marker for acute B-lymphocytic leukemia, with the advantages of high sensitivity, fast response speed, wide detection range, low detection limit, etc., and has good stability, selectivity and reproducibility. The construction of this electrochemical biosensor provides a new idea for studying the sensitive detection of the Pax-5a gene, a marker for acute B-lymphocytic leukemia. Summary of the Invention

[0005] One of the purposes of the present invention is to use 2,2':6',2''-terpyridine-4,4',4''-tricarboxylic acid as an organic ligand, Ce 3+ , Tb 3+ As a dual-center ion, it assembles ultra-rapidly at room temperature into a Ce / Tb metal organic gel. This material has a three-dimensional network structure, and its porous structure is more conducive to sufficient contact with the co-reactant, thus greatly improving the ECL luminescence efficiency.

[0006] The second purpose of the present invention is to select lanthanide elements Ce and Tb for testing and obtain Ce 3+ , Tb 3+ Co-doped MOGs and due to the ligand-metal antenna effect and directional Ce 3+ to Tb 3+ Dual sensitization of energy transfer improves internal energy transfer efficiency and has the strongest ECL response.

[0007] The third purpose of the present invention is to design an enzyme-driven DNA walker based on a chain-shaped DNA track. Compared with randomly constructed DNA tracks, this method can precisely control the distance between chains, and the rigid and orderly arrangement of binding sites reduces kinetic barriers, significantly improving the rolling efficiency and rate of the DNA walker.

[0008] The technical solutions of the present invention are as follows: The preparation method of the electrochemical biosensor based on the high-efficiency Ce / Tb metal organic gel and the rigid ordered chain DNA track comprises the following steps: (1) The GCE electrode surface was polished with 0.3 and 0.05 μm Al2O3 powders, ultrasonically treated with ethanol and ultrapure water, and dried with nitrogen to obtain a clean and smooth electrode surface; (2) Take 10 μL of 0.8 ~ 2.4 mg / mL Ce / Tb metal organic gel dispersion and drop it onto the electrode surface and let it dry at room temperature; (3) Add 10 μL of gold nanoparticles to the electrode surface to attach DNA and let it dry at room temperature; (4) Take 10 μL of DNA track solution with 100-300 mmol / L dopamine and drop it onto the electrode surface. Rinse the electrode surface with deionized water and incubate in a refrigerator at 4°C overnight. (5) Continue to take 10 μL of 50 ~ 150 mmol / L 1-hexanethiol and add it to the electrode surface to block the nonspecific active sites on the electrode surface. Rinse the electrode surface with phosphate buffer (pH = 7.38) and dry it in a refrigerator at 4°C. (6) Finally, 10 μL of DNA walker solution prepared from the acute B lymphoblastic leukemia marker Pax-5a gene at different concentrations of 0.00001 ~ 10 pmol / L was added to the electrode surface, and 10 μL of 10 ~ 50 U / 100 μL of ExoIII enzyme was added for shearing. The solution was incubated at room temperature for 1 ~ 5 hours, and the electrode surface was rinsed with phosphate buffer (pH = 7.38) to obtain an electrochemical biosensor based on high-efficiency Ce / Tb metal organic gel and rigid ordered chain DNA track. The solution was stored in a refrigerator at 4 °C for future use.

[0009] 2. The three-dimensional network structure of Ce / Tb metal organic gel dispersion is prepared as follows: (1) Preparation of Ce / Tb metal organic gel Dissolve 40.92 mg of 2,2':6',2''-terpyridine-4,4',4''-tricarboxylic acid in 1 mL of ultrapure water. Add 15 µL of triethylamine to deprotonate and promote dissolution during ultrasonic treatment. At the same time, weigh 24.32 mg of Ce(NO3)3 and 25.37 mg of Tb(NO3)3 and dissolve them in 1 mL of ultrapure water to obtain a central ion solution with the same concentration as the ligand. Next, quickly add the ligand solution to the mixed Ce solution. 3+ , Tb 3+ The initial product was obtained in the solution. The glass bottle was inverted to ensure successful gel formation. The product was then washed and centrifuged three times with ultrapure water. After freeze-drying, 0.8 to 2.4 mg of the dried powder was added to 1 mL of ultrapure water and sonicated for 5 minutes to produce a three-dimensional network-structured Ce / Tb metal organic gel dispersion. (2) Preparation of DNA walker First, DNA strand H1 was treated with TCEP for one hour, then annealed at 95°C to form a hairpin structure, which was then attached to gold nanoparticles via thiol groups. The structure was incubated overnight at 4°C, centrifuged at 10,000 rpm, and redispersed in TE buffer. A Pax-5a gene solution (0.00001 to 10 pmol / L) was then added to open the hairpin structure. Subsequently, 10 to 50 U / 100 µL of ExoIII enzyme was added at 37°C to cleave the DNA walker. The resulting solution was stored at 4°C until use. (3) Preparation of DNA tracks. First, DNA strand L2 was treated with TCEP for one hour. Then, DNA single strands L1, L2, and L3 were rapidly annealed. L1, L2, and L3 were mixed and hybridized at 37°C for two hours. In the hybridized track, L3 with -COOH modification was activated with carbodiimide (0.2 M) and N-hydroxysuccinimide (0.05 M) for one hour. 10–200 mmol / L dopamine was added and shaken at 4°C overnight. The resulting solution was stored at 4°C until use.

[0010] 3. Detection of the Pax-5a gene, a marker for acute B-lymphoblastic leukemia, comprising the following steps: (1) The MPI-E electrochemiluminescence analysis system was used for the test in a three-electrode system. The Ag / AgCl electrode was used as the reference electrode, the Pt electrode was used as the counter electrode, and the prepared electrochemical biosensor based on the high-efficiency Ce / Tb metal organic gel and the rigid ordered chain DNA track was used as the working electrode. The voltage of the photomultiplier tube was set to 600 V. The cyclic voltammetry test voltage was -2.0 ~ 0.0 V, and the scan rate was 200 mV / s. (2) In 10 mL of phosphate buffer solution containing 0.04 to 0.12 mol / L potassium persulfate and a pH of 6.47 to 8.04, the electrochemiluminescence signal generated by the antigen at different concentrations was detected and a working curve was drawn; (3) The sample solution to be tested is used instead of the Pax-5a gene solution, a marker of acute B-lymphocytic leukemia, for testing.

[0011] Beneficial results of the present invention (1) The present invention successfully synthesized Ce / Tb metal organic gel with a three-dimensional network structure. The material has the advantages of mild synthesis conditions, simple preparation, good film-forming properties, etc. Its porous structure is more conducive to sufficient contact with the co-reactant, and due to the ligand-metal antenna effect and directional Ce 3+ to Tb 3+ Energy transfer improves the internal energy transfer efficiency, thereby greatly improving the ECL luminescence efficiency.

[0012] (2) In the present invention, an enzyme-driven DNA walker guided by a chain-shaped DNA track is designed. Compared with randomly constructed DNA tracks, this method can precisely control the distance between chains, and the rigid and orderly arrangement of binding sites reduces the kinetic barriers, significantly improving the rolling efficiency and rate of the DNA walker.

[0013] (3) The electrochemical biosensor prepared by the present invention is used for the quantitative detection of the Pax-5a gene, a marker of acute B-lymphocytic leukemia. It has a fast response speed, a wide linear range, a low detection limit, good stability, selectivity and reproducibility, and can achieve simple, rapid, highly sensitive and specific detection. DETAILED DESCRIPTION

[0014] (The present invention will now be further described through specific embodiments, but is not limited thereto) Example 1. A method for preparing an electrochemical biosensor based on a high-efficiency Ce / Tb metal organic gel and a rigid ordered chain DNA track comprises the following steps: (1) The GCE electrode surface was polished with 0.3 and 0.05 μm Al2O3 powders, ultrasonically treated with ethanol and ultrapure water, and dried with nitrogen to obtain a clean and smooth electrode surface; (2) Take 10 μL of 0.8 mg / mL Ce / Tb metal organic gel dispersion and drop it onto the electrode surface and let it dry at room temperature; (3) Add 10 μL of gold nanoparticles to the electrode surface to attach DNA and let it dry at room temperature; (4) Take 10 μL of DNA track solution connected with 100 mmol / L dopamine and drop it onto the electrode surface. Rinse the electrode surface with deionized water and incubate in a refrigerator at 4°C overnight. (5) Continue to take 10 μL of 50 mmol / L 1-hexanethiol and add it dropwise to the electrode surface to block the nonspecific active sites on the electrode surface. Rinse the electrode surface with phosphate buffer (pH = 7.38) and dry it in a refrigerator at 4°C. (6) Finally, 10 μL of DNA walker solution prepared from the acute B lymphoblastic leukemia marker Pax-5a gene at different concentrations of 0.00001 ~ 10 pmol / L was added to the electrode surface, and 10 μL of 10 U / 100 μL ExoIII enzyme was added for shearing. The solution was incubated at room temperature for 1 hour, and the electrode surface was rinsed with phosphate buffer (pH = 7.38) to obtain an electrochemical biosensor based on high-efficiency Ce / Tb metal organic gel and rigid ordered chain DNA track. The solution was stored in a refrigerator at 4 °C for future use.

[0015] Example 2. A method for preparing an electrochemical biosensor based on a high-efficiency Ce / Tb metal organic gel and a rigid ordered chain DNA track comprises the following steps: (1) The GCE electrode surface was polished with 0.3 and 0.05 μm Al2O3 powders, ultrasonically treated with ethanol and ultrapure water, and dried with nitrogen to obtain a clean and smooth electrode surface; (2) Take 10 μL of 1.6 mg / mL Ce / Tb metal organic gel dispersion and drop it onto the electrode surface and let it dry at room temperature; (3) Add 10 μL of gold nanoparticles to the electrode surface to attach DNA and let it dry at room temperature; (4) Take 10 μL of DNA track solution connected with 200 mmol / L dopamine and drop it onto the electrode surface. Rinse the electrode surface with deionized water and incubate in a refrigerator at 4°C overnight. (5) Continue to take 10 μL of 100 mmol / L 1-hexanethiol and add it dropwise to the electrode surface to block the nonspecific active sites on the electrode surface. Rinse the electrode surface with phosphate buffer (pH = 7.38) and dry it in a refrigerator at 4°C. (6) Finally, 10 μL of DNA walker solution prepared from the acute B lymphoblastic leukemia marker Pax-5a gene at different concentrations of 0.00001 ~ 10 pmol / L was added to the electrode surface, and 10 μL of 30 U / 100 μL ExoIII enzyme was added for shearing. The solution was incubated at room temperature for 3 hours, and the electrode surface was rinsed with phosphate buffer (pH = 7.38) to obtain an electrochemical biosensor based on high-efficiency Ce / Tb metal organic gel and rigid ordered chain DNA track. The solution was stored in a refrigerator at 4 °C for future use.

[0016] Example 3. A method for preparing an electrochemical biosensor based on a high-efficiency Ce / Tb metal organic gel and a rigid ordered chain DNA track comprises the following steps: (1) The GCE electrode surface was polished with 0.3 and 0.05 μm Al2O3 powders, ultrasonically treated with ethanol and ultrapure water, and dried with nitrogen to obtain a clean and smooth electrode surface; (2) Take 10 μL of 2.4 mg / mL Ce / Tb metal organic gel dispersion and drop it onto the electrode surface and let it dry at room temperature; (3) Add 10 μL of gold nanoparticles to the electrode surface to attach DNA and let it dry at room temperature; (4) Take 10 μL of DNA track solution connected with 300 mmol / L dopamine and drop it onto the electrode surface. Rinse the electrode surface with deionized water and incubate in a refrigerator at 4°C overnight. (5) Continue to take 10 μL of 150 mmol / L 1-hexanethiol and add it dropwise to the electrode surface to block the nonspecific active sites on the electrode surface. Rinse the electrode surface with phosphate buffer (pH = 7.38) and dry it in a refrigerator at 4°C. (6) Finally, 10 μL of DNA walker solution prepared from the acute B lymphoblastic leukemia marker Pax-5a gene at different concentrations of 0.00001 ~ 10 pmol / L was added to the electrode surface, and 10 μL of 50 U / 100 μL ExoIII enzyme was added for shearing. The solution was incubated at room temperature for 5 hours, and the electrode surface was rinsed with phosphate buffer (pH = 7.38) to obtain an electrochemical biosensor based on high-efficiency Ce / Tb metal organic gel and rigid ordered chain DNA track. The solution was stored in a refrigerator at 4 °C for future use.

[0017] Example 4. The three-dimensional network structure of Ce / Tb metal organic gel dispersion, the preparation steps are as follows: (1) Preparation of Ce / Tb metal organic gel Dissolve 40.92 mg of 2,2':6',2''-terpyridine-4,4',4''-tricarboxylic acid in 1 mL of ultrapure water. Add 15 µL of triethylamine to deprotonate and promote dissolution during ultrasonic treatment. At the same time, weigh 24.32 mg of Ce(NO3)3 and 25.37 mg of Tb(NO3)3 and dissolve them in 1 mL of ultrapure water to obtain a central ion solution with the same concentration as the ligand. Next, quickly add the ligand solution to the mixed Ce solution. 3+ , Tb 3+ The initial product was obtained in the solution. The glass bottle was inverted to ensure the gel was successfully prepared. The product was then washed and centrifuged three times with ultrapure water. After freeze-drying, 0.8 mg of the dried powder was added to 1 mL of ultrapure water and sonicated for 5 minutes to obtain a three-dimensional network-structured Ce / Tb metal organic gel dispersion. (2) Preparation of DNA walker First, DNA strand H1 was treated with TCEP for one hour and then annealed at 95°C to form a hairpin structure. This structure was then attached to gold nanoparticles via thiol groups and incubated overnight at 4°C. After centrifugation at 10,000 rpm, the DNA strand was redispersed in TE buffer. A Pax-5a gene solution (0.00001–10 pmol / L) was then added to open the hairpin structure. Subsequently, 10 U / 100 µL of ExoIII enzyme was added at 37°C to cleave the DNA walker. The resulting solution was stored at 4°C until further use. (3) Preparation of DNA tracks. First, DNA strand L2 was treated with TCEP for one hour. Then, DNA single strands L1, L2, and L3 were rapidly annealed. L1, L2, and L3 were then mixed and hybridized at 37°C for two hours. In the hybridized track, L3 with -COOH modification was activated with carbodiimide (0.2 M) and N-hydroxysuccinimide (0.05 M) for one hour. 10 mmol / L dopamine was added and shaken at 4°C overnight. The resulting solution was stored at 4°C until use.

[0018] Example 5. The three-dimensional network structure of Ce / Tb metal organic gel dispersion, the preparation steps are as follows: (1) Preparation of Ce / Tb metal organic gel Dissolve 40.92 mg of 2,2':6',2''-terpyridine-4,4',4''-tricarboxylic acid in 1 mL of ultrapure water. Add 15 µL of triethylamine to deprotonate and promote dissolution during ultrasonic treatment. At the same time, weigh 24.32 mg of Ce(NO3)3 and 25.37 mg of Tb(NO3)3 and dissolve them in 1 mL of ultrapure water to obtain a central ion solution with the same concentration as the ligand. Next, quickly add the ligand solution to the mixed Ce solution. 3+ , Tb 3+ The initial product was obtained in the solution. The glass bottle was inverted to ensure the gel was successfully prepared. The product was then washed and centrifuged three times with ultrapure water. After freeze-drying, 1.6 mg of the dried powder was added to 1 mL of ultrapure water and sonicated for 5 minutes to obtain a Ce / Tb metal organic gel dispersion with a three-dimensional network structure. (2) Preparation of DNA walker First, DNA strand H1 was treated with TCEP for one hour and then annealed at 95°C to form a hairpin structure. This structure was then attached to gold nanoparticles via thiol groups and incubated overnight at 4°C. After centrifugation at 10,000 rpm, the DNA strand was redispersed in TE buffer. A Pax-5a gene solution (0.00001–10 pmol / L) was then added to open the hairpin structure. Subsequently, 30 U / 100 µL of ExoIII enzyme was added at 37°C to cleave the DNA walker. The resulting solution was stored at 4°C until further use. (3) Preparation of DNA tracks. First, DNA strand L2 was treated with TCEP for one hour. Then, DNA single strands L1, L2, and L3 were rapidly annealed. L1, L2, and L3 were mixed and hybridized at 37°C for two hours. In the hybridized track, L3 with -COOH modification was activated with carbodiimide (0.2 M) and N-hydroxysuccinimide (0.05 M) for one hour. 100 mmol / L dopamine was added and shaken at 4°C overnight. The resulting solution was stored at 4°C until use.

[0019] Example 6. The three-dimensional network structure of Ce / Tb metal organic gel dispersion, the preparation steps are as follows: (1) Preparation of Ce / Tb metal organic gel Dissolve 40.92 mg of 2,2':6',2''-terpyridine-4,4',4''-tricarboxylic acid in 1 mL of ultrapure water. Add 15 µL of triethylamine to deprotonate and promote dissolution during ultrasonic treatment. At the same time, weigh 24.32 mg of Ce(NO3)3 and 25.37 mg of Tb(NO3)3 and dissolve them in 1 mL of ultrapure water to obtain a central ion solution with the same concentration as the ligand. Next, quickly add the ligand solution to the mixed Ce solution. 3+ , Tb 3+ The initial product was obtained in the solution. The glass bottle was inverted to assess the success of the gel preparation. The gel was then washed and centrifuged three times with ultrapure water. After freeze-drying, 2.4 mg of the dried powder was added to 1 mL of ultrapure water and sonicated for 5 minutes to produce a three-dimensional network-structured Ce / Tb metal organic gel dispersion. (2) Preparation of DNA walker First, DNA strand H1 was treated with TCEP for one hour and then annealed at 95°C to form a hairpin structure. This structure was then attached to gold nanoparticles via thiol groups and incubated overnight at 4°C. After centrifugation at 10,000 rpm, the DNA strand was redispersed in TE buffer. A Pax-5a gene solution (0.00001–10 pmol / L) was then added to open the hairpin structure. Subsequently, 50 U / 100 µL of ExoIII enzyme was added at 37°C to cleave the DNA walker. The resulting solution was stored at 4°C until further use. (3) Preparation of DNA tracks. First, DNA strand L2 was treated with TCEP for one hour. Then, DNA single strands L1, L2, and L3 were rapidly annealed. L1, L2, and L3 were then mixed and hybridized at 37°C for two hours. In the hybridized track, L3 with -COOH modification was activated with carbodiimide (0.2 M) and N-hydroxysuccinimide (0.05 M) for one hour. 200 mmol / L dopamine was added and shaken at 4°C overnight. The resulting solution was stored at 4°C until use.

[0020] Example 7 The detection of the Pax-5a gene, a marker of acute B-lymphocytic leukemia, comprises the following steps: (1) The MPI-E electrochemiluminescence analysis system was used for the test in a three-electrode system. The Ag / AgCl electrode was used as the reference electrode, the Pt electrode was used as the counter electrode, and the prepared electrochemical biosensor based on the high-efficiency Ce / Tb metal organic gel and the rigid ordered chain DNA track was used as the working electrode. The voltage of the photomultiplier tube was set to 600 V. The cyclic voltammetry test voltage was -2.0 ~ 0.0 V, and the scan rate was 200 mV / s. (2) In 10 mL of phosphate buffer solution containing 0.1 mol / L potassium persulfate at pH = 7.38, detect the electrochemiluminescence signal generated by the Pax-5a gene at different concentrations and draw a working curve; (3) The sample solution to be tested is used instead of the Pax-5a gene solution, a marker of acute B-lymphocytic leukemia, for testing.

Claims

1. A method for preparing an electrochemical biosensor based on a high-efficiency Ce / Tb metal organic gel and a rigid ordered chain DNA track, comprising the following steps: (1) The GCE electrode surface was polished with 0.3 and 0.05 μm Al2O3 powders, ultrasonically treated with ethanol and ultrapure water, and dried with nitrogen to obtain a clean and smooth electrode surface; (2) Take 10 μL of 0.8 ~ 2.4 mg / mL Ce / Tb metal organic gel dispersion and drop it onto the electrode surface and let it dry at room temperature; (3) Add 10 μL of gold nanoparticles to the electrode surface to attach DNA and let it dry at room temperature; (4) Take 10 μL of DNA track solution with 100-300 mmol / L dopamine and drop it onto the electrode surface. Rinse the electrode surface with deionized water and incubate in a refrigerator at 4°C overnight. (5) Continue to take 10 μL of 50 ~ 150 mmol / L 1-hexanethiol and add it to the electrode surface to block the nonspecific active sites on the electrode surface. Rinse the electrode surface with phosphate buffer (pH = 7.38) and dry it in a refrigerator at 4°C. (6) Finally, 10 μL of DNA walker solution prepared from the acute B lymphoblastic leukemia marker Pax-5a gene at different concentrations of 0.00001 ~ 10 pmol / L was added to the electrode surface, and 10 μL of 10 ~ 50 U / 100 μL of ExoIII enzyme was added for shearing. The solution was incubated at room temperature for 1 ~ 5 hours, and the electrode surface was rinsed with phosphate buffer (pH = 7.38) to obtain an electrochemical biosensor based on high-efficiency Ce / Tb metal organic gel and rigid ordered chain DNA track. The solution was stored in a refrigerator at 4 °C for future use.

2. The method for preparing an electrochemical biosensor based on a high-efficiency Ce / Tb metal organic gel and a rigid ordered chain DNA track according to claim 1, wherein the three-dimensional network structured Ce / Tb metal organic gel dispersion is characterized in that: Here are the steps: (1) Preparation of Ce / Tb metal organic gel Dissolve 40.92 mg of 2,2':6',2''-terpyridine-4,4',4''-tricarboxylic acid in 1 mL of ultrapure water. Add 15 µL of triethylamine to deprotonate and promote dissolution during ultrasonic treatment. At the same time, weigh 24.32 mg of Ce(NO3)3 and 25.37 mg of Tb(NO3)3 and dissolve them in 1 mL of ultrapure water to obtain a central ion solution with the same concentration as the ligand. Next, quickly add the ligand solution to the mixed Ce solution. 3+ 、Tb 3+ The initial product was obtained in the solution. The glass bottle was inverted to ensure successful gel formation. The product was then washed and centrifuged three times with ultrapure water. After freeze-drying, 0.8 to 2.4 mg of the dried powder was added to 1 mL of ultrapure water and sonicated for 5 minutes to produce a three-dimensional network-structured Ce / Tb metal organic gel dispersion. (2) Preparation of DNA walker First, DNA strand H1 was treated with TCEP for one hour, then annealed at 95°C to form a hairpin structure, which was then attached to gold nanoparticles via thiol groups. The strands were incubated overnight at 4°C, centrifuged at 10,000 rpm, and redispersed in TE buffer. A Pax-5a gene solution (0.00001–10 pmol / L) was then added to open the hairpin structure. Subsequently, 10–50 U / 100 µL of ExoIII enzyme was added at 37°C to cleave the DNA walker. The resulting solution was stored at 4°C until further use. (3) Preparation of DNA tracks First, DNA strand L2 was treated with TCEP for one hour. Then, single-stranded DNA solutions L1, L2, and L3 were rapidly annealed. L1, L2, and L3 were then mixed and hybridized at 37°C for two hours. The hybridized track was activated for one hour with carbodiimide (0.2 M) and N-hydroxysuccinimide (0.05 M). 10–200 mmol / L dopamine was added, and the mixture was shaken overnight at 4°C. The resulting solution was stored at 4°C until use.

3. The electrochemical biosensor based on high-efficiency Ce / Tb metal organic gel and rigid ordered chain DNA track prepared by the preparation method according to claim 1 is used for detecting the Pax-5a gene, a marker of acute B-lymphocytic leukemia, characterized in that: Here are the steps: (1) The MPI-E electrochemiluminescence analysis system was used for the test in a three-electrode system. The Ag / AgCl electrode was used as the reference electrode, the Pt electrode was used as the counter electrode, and the prepared three-dimensional network structure Ce / Tb metal organic gel electrochemical biosensor was used as the working electrode. The voltage of the photomultiplier tube was set to 600 V. The cyclic voltammetry test voltage was -2.0 ~ 0.0 V, and the scan rate was 200 mV / s. (2) In 10 mL of phosphate buffer solution containing 0.1 mol / L potassium persulfate at pH = 7.38, detect the electrochemiluminescence signal generated by the Pax-5a gene at different concentrations and draw a working curve; (3) The sample solution to be tested is used instead of the Pax-5a gene solution, a marker of acute B-lymphocytic leukemia, for testing.