Development method of terminal nucleotidyl transferase electrochemical luminescence sensor based on porous ZnCo-MOF material

Abstract

The invention designs a development method of a terminal nucleotidyl transferase electrochemical luminescence sensor based on a porous ZnCo-MOF material, the porous ZnCo-MOF material is formed in situ by electrochemically applying constant voltage, and the porous ZnCo-MOF material shows excellent electrochemical luminescence (ECL) intensity due to the excellent electron transport capability of Ru (bpy) 3 < 2 + >. The electrochemical luminescence intensity of the porous ZnCo-MOF material is gradually reduced along with the addition of Hg (II), a DNA chain rich in thymine T is synthesized after TdT is introduced, and Hg (II) is combined with thymine in the DNA double chain to form a T-Hg-T structure, so that the concentration of free Hg (II) is reduced, the electrochemical luminescence intensity is recovered, and fluorescence detection of Hg (II) and TdT is realized on the basis. At present, an electrochemical luminescence sensor based on a porous ZnCo-MOF photoelectric material is not found yet, and the electrochemical luminescence sensor is expanded to be used for analysis and detection of Hg (II), terminal transferase and an inhibitor Na4P2O7 (PP) of the terminal transferase, especially for analysis and monitoring of activity of intracellular TdT.

Description

Technical Field

[0001] The present invention relates to an electrochemiluminescence sensor and its application, in particular to a terminal deoxynucleotidyl transferase electrochemiluminescence sensor based on porous ZnCo-MOF material and its application, and it is used for monitoring the activity of TdT in cervical cancer cells, belonging to the fields of functional biomaterials and chemosensing analysis. Background Art

[0002] The development of porous materials is changing with each passing day, and related research has gradually penetrated into various fields of cutting-edge technology. Highly ordered metal-organic frameworks (MOFs) are crystalline porous materials formed by the self-assembly of metal ions / clusters and organic ligands through coordination bonds, and they have been a research hotspot in the fields of chemistry and materials science in recent decades. Compared with traditional inorganic materials, MOFs have characteristics such as a large specific surface area, a high porosity, diverse structural types, and adjustable functions, as well as a rich variety of organic ligands and metal nodes, making MOFs highly plastic in terms of charge, polarity, chirality, redox potential, photoactivity, hydrophobicity / hydrophilicity, aromaticity / lipophilicity, stereochemistry, etc. However, it is still extremely difficult to construct MOF materials with a large specific surface area or a hierarchically ordered pore structure to improve the sensitivity of sensing methods. Therefore, the research and development of new optoelectronic MOF materials are still extremely challenging. Thanks to the diversity of MOFs' organic ligands and metal nodes, MOFs exhibit a rich variety of structural types and can endow MOFs with special functions through methods such as adjusting the pore environment and post-synthetic modification. Among many MOF materials, electrochemically synthesized porous MOFs stand out due to their excellent specific surface area and porous structure stability, and are very suitable for the design of analytical sensing methods.

[0003] Terminal deoxynucleotidyl transferase (TdT) is a template-free isothermal nucleic acid amplification enzyme that can repeatedly add single nucleotides to the 3'-OH end of an oligonucleotide chain, and DNA extension can be achieved at room temperature. This property makes TdT a widely used tool enzyme in many fields. Clinical studies have shown that abnormal expression of TdT plays a crucial role in cancer development. For example, it may reduce the response of cancer to anticancer chemotherapy, and the expression of TdT has been observed in cases of acute lymphoblastic leukemia. Traditional TdT activity detection methods include gel electrophoresis, immunosorbent assay, biochemical analysis, etc. However, due to difficulties in operation procedures, the need for multiple steps of immobilization and separation, certain environmental pollution, time-consuming and laborious, and high cost, more and more researchers are attracted to develop label-free and non-radioactive TdT detection methods. Therefore, the accurate evaluation of TdT activity is of great significance for biochemical research such as medical diagnosis and tumor drug development.

[0004] The present invention designs a method for developing an electrochemical luminescence sensor for terminal deoxynucleotidyl transferase based on porous ZnCo-MOF material. By electrochemically applying a constant voltage, porous ZnCo-MOF material is in-situ formed, and Ru(bpy)3 2+ With excellent electron transport ability, the porous ZnCo-MOF material exhibits excellent electrochemiluminescence (ECL) intensity. With the addition of Hg(II), the ECL intensity of the porous ZnCo-MOF material gradually decreases. After introducing TdT, a DNA strand rich in thymine T is synthesized. Hg(II) binds to thymine in the DNA double strand to form a T-Hg-T structure, thereby reducing the concentration of free Hg(II) and restoring the ECL intensity. Based on this, fluorescence detection of Hg(II) and TdT is achieved, and finally it is applied to the monitoring of TdT activity in cells. Currently, no electrochemical luminescence sensor based on porous ZnCo-MOF optoelectronic material has been found, and it is extended for the analysis and detection of Hg(II), terminal transferase and its inhibitor Na4P2O7 (PP), especially for the analysis and detection of intracellular TdT. Summary of the Invention

[0005] The present invention proposes a method for developing an electrochemical luminescence sensor for terminal deoxynucleotidyl transferase based on porous ZnCo-MOF material. This method first uses porous ZnCo-MOF material for the construction of an electrochemical luminescence sensor and is used for rapid and ultrasensitive analysis and detection of TdT activity.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows: A method for developing an electrochemical luminescence sensor for terminal deoxynucleotidyl transferase based on porous ZnCo-MOF material, and the specific steps are as follows:

[0007] (1) Preparation of ZnCo-MOF precursor solution

[0008] Precursor solution 1: Dissolve ZnAc2 (0.3 - 0.45 mM, 0.1647 - 0.2470 g), CoAc2 (0.3 - 0.45 mM, 0.1593 g), and KAc (0.15 - 0.3 mM, 0.0662 - 0.1323 g) in 2 - 3 mL of water.

[0009] Precursor solution 2: Dissolve trimesic acid (H3btc) (0.3 - 0.5 mM, 0.063 - 0.1050 g) in 2 - 3 mL of dimethyl sulfoxide (DMSO).

[0010] The above precursor solution 1 and precursor solution 2 are uniformly mixed at room temperature, and Ru(bpy)3Cl2·6H2O (2 - 2.5 mM, 0.0090 - 0.0112 g) is added under stirring. Stir at room temperature in the dark for 1.5 - 2 h to obtain the ZnCo-MOF solution.

[0011] (2) Preparation of Electrochemiluminescence Biosensor

[0012] a. Preparation of Electrode 1: The glassy carbon electrode (GCE, with a diameter of 3 mm) was polished successively with 1, 0.3, and 0.05 μm alumina powder on suede for 1 - 5 min, then ultrasonically washed with water and ethanol successively for 1 - 5 min, and then dried with nitrogen, marked as Electrode 1.

[0013] b. Preparation of Electrode 2: The three - electrode system (reference electrode, glassy carbon electrode, counter electrode) was immersed in the porous ZnCo - MOF material solution in the above step 1, and a voltage of - 1.3 - - 1.4 V was applied using the i - t method for 500 - 600 s. The obtained electrode was marked as Electrode 2.

[0014] c. Preparation of Electrode 3: 8 - 10 μL of 5 μM Hg(II) was dropped onto Electrode 2, incubated in the dark at room temperature for 25 - 30 min, and then placed in the electrolyte solution for electrochemiluminescence detection, marked as Electrode 3.

[0015] d. Preparation of Electrode 4: 800 - 1000 U / mL, 1 μL of TdT (final concentration in 100 μL solution: 8000 - 10000 U / L), 8 - 10 μM, 1 μL of ssDNA (final concentration in 100 μL solution: 0.08 - 0.1 μM), 8 - 10 mM, 1 μL of dTTP (final concentration in 100 μL solution: 0.08 - 0.1 mM), 10×TdT buffer (1 - 2 μL) and distilled water were uniformly mixed to make the volume 8 - 10 μL. It was reacted in a water bath at 35 - 37 °C for 2 - 2.5 h, then the temperature of the constant - temperature water bath was raised to 70 - 75 °C, and reacted for 10 - 15 min to inactivate the enzyme to terminate the polymerization reaction. Subsequently, 40 - 50 μM, 10 μL of Hg(II) (final concentration: 4 - 5 μM) and 3 - 5 μL of HEPES buffer solution (10 mM pH = 7.0) were added to the above 5 μL of TdT reaction solution. After incubating at room temperature for 25 - 30 min, 8 - 10 μL was taken and dropped onto the surface of Electrode 2, incubated in the dark at room temperature for 25 - 30 min, and then placed in the electrolyte solution for electrochemiluminescence detection, marked as Electrode 4.

[0016] Subsequently, the BPCL T15 analysis system was used to measure the electrochemiluminescence (ECL) intensity. The high voltage was set to 600 - 700 V, the scanning speed was 50 - 100 mV / s, and the scanning voltage was 0 - 2 V. The electrochemiluminescence responses of the prepared electrochemical biosensor to Hg(II) and TdT in PBS (0.1 M, pH 7.0, containing 0.1 M KCl and 100 mM triethanolamine) were detected to obtain the electrochemiluminescence magnitudes corresponding to Hg(II) and TdT. The ECL quenching intensity (QE%) was expressed as (I0 - I) / I0; the ECL recovery intensity was expressed as (I - I0) / I0, where I0 and I represent the ECL intensities in the absence and presence of Hg(II), respectively. Based on this, a quantitative relationship between the luminescence intensity and the concentrations of Hg(II) and TdT was established to achieve the detection of Hg(II) and TdT and the screening of their inhibitors.

[0017] Principle of the invention: The present invention is a method for developing an electrochemiluminescence sensor for terminal deoxynucleotidyl transferase based on a porous ZnCo-MOF material, Ru(bpy)3 2+ A MOF complex (porous ZnCo-MOF material) with excellent electrochemiluminescence properties was synthesized by electrodeposition. On the one hand, Hg(II) can quench the ECL intensity of ZnCo-MOF; on the other hand, it can selectively bind to the T base. The T-T base pair has a high selectivity for Hg(II), and the two combine to form a T-Hg-T structure. Therefore, the concentration of Hg(II) decreases, that is, the ECL intensity recovers. When the inhibitor Na4P2O7 (PP) is added, the activity of TdT is inhibited, and the formation of the T-Hg-T structure is inhibited, so the ECL intensity decreases, thus realizing the screening of TdT inhibitors. Through the relationship between the logarithmic concentrations of Hg(II), TdT, and their inhibitor PP and the electrochemiluminescence quenching and recovery intensities, the quantitative analysis of Hg(II), TdT, and their inhibitor PP in the sample to be measured is achieved. Based on this, a simple, rapid, stable, and highly sensitive electrochemiluminescence sensor was constructed.

[0018] Advantages of the present invention: The present invention constructs a method for developing an electrochemiluminescence sensor for terminal deoxynucleotidyl transferase based on a porous ZnCo-MOF material. First, a porous ZnCo-MOF material thin film is formed on the electrode surface by applying a constant voltage to obtain a closely arranged and multi-folded electrochemiluminescence sensor. Within a certain range, the greater the concentration of Hg(II), the more obvious the electrochemiluminescence quenching effect on the porous ZnCo-MOF material; the greater the concentration of TdT, the more T-Hg-T structures are formed, and the more obvious the electrochemiluminescence recovery intensity. The experimental results show that the electrochemiluminescence quenching and recovery intensities are linearly related to the logarithms of the concentrations of Hg(II) and TdT within a certain range, and the analysis and detection of Hg(II) and TdT are preferably achieved. The advantages are as follows:

[0019] (1) The synthesis method is simple and the material has good stability. The synthesis method of the porous ZnCo-MOF material does not require harsh chemical reactions and extreme experimental conditions. It only needs to set the scanning technique on an electrochemical workstation to complete the synthesis simply and quickly. Since this material has a porous structure and a stable pore environment, this optoelectronic MOF material has weak photobleaching and stable fluorescence intensity, making it very suitable as an optoelectronic probe.

[0020] (2) High sensitivity. The linear range of the electrochemiluminescence intensity of the experimentally obtained sensor with respect to the logarithm of the Hg(II) concentration is 0.0005 - 5000 nM, and its linear correlation equation is y = 0.1234lgC Hg(II)+ + 0.4181, R 2 = 0.9980, and the detection limit is 0.031 pM; the linear range of the logarithm of the TdT concentration is 0.03 - 1000 U / L, and the linear correlation equation is y = 0.6618lgC TdT + 0.9054, R 2 = 0.9964, and the detection limit is 0.0025 U / L, enabling highly sensitive detection of Hg(II) and TdT.

[0021] (3) High specificity. For the detection of Hg(II): calcium ions (Ca 2+ ), magnesium ions (Mg 2+ ), ferrous ions (Fe 2+ ), chromium ions (Cr 2+ ), titanium ions (Ti 2+ ), and cadmium ions (Cd 2+ ) have no interference with this system; for the detection of TdT: other common enzymes, such as Papain, Thrombin, Lysozyme (LZM), and Acetylcholinesterase (AChE), have no interference with this detection system.

[0022] (4) The reagents used in the detection method are in small amounts, the detection speed is fast, and the cost is low; it is finally applied to the monitoring of intracellular TdT activity.

[0023] (5) The results are accurate. The average deviation is less than 5%, and the results are accurate and reliable.

[0024] In summary, the method for developing a terminal deoxynucleotidyl transferase electrochemiluminescence sensor based on porous ZnCo-MOF material of the present invention has the advantages of simple synthesis method, high specificity, high sensitivity, and low cost. It can detect low concentrations of Hg(II) and TdT and has good application prospects. Description of the Drawings

[0025] Figure 1This is the feasibility analysis diagram of the electrochemiluminescence biosensor of the present invention;

[0026] Figure 2 This is the linear relationship diagram of the quenching intensity of the electrochemiluminescence biosensor of the present invention for Hg(II) versus the logarithm of concentration;

[0027] Figure 3 This is the linear relationship diagram of the recovery intensity of the electrochemiluminescence biosensor of the present invention for TdT versus the logarithm of concentration;

[0028] Figure 4 This is the linear relationship diagram of the quenching intensity of the electrochemiluminescence biosensor of the present invention for PP versus the logarithm of concentration;

[0029] Figure 5 This is the selectivity experiment diagram of the electrochemiluminescence biosensor of the present invention for TdT.

[0030] Figure 6 This is the anti-interference experiment diagram of the electrochemiluminescence biosensor of the present invention for Hg(II);

[0031] Figure 7 This is the intracellular TdT monitoring experiment diagram of the electrochemiluminescence biosensor of the present invention; Detailed implementation manners

[0032] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0033] Example 1 Preparation of the precursor solution for synthesizing ZnCo-MOF

[0034] Precursor solution 1: Dissolve ZnAc2 (0.45 mM, 0.2 four seven zero g), CoAc2 (0.45 mM, 0.2 three nine zero g), KAc (0.3 mM, 0.1 three two three g) in 3 mL of water.

[0035] Precursor solution 2: Dissolve trimesic acid (H3btc) (0.5 mM, 0.1 zero five zero g) in 3 mL of dimethyl sulfoxide (DMSO).

[0036] The above precursor solution 1 and precursor solution 2 are uniformly mixed at room temperature, and Ru(bpy)3Cl two · 6H2O (2.5 mM, 0.0 one one two g) is added under stirring, and stirred at room temperature in the dark for 1.5 h to obtain the ZnCo-MOF solution.

[0037] Example 2 Preparation of the electrochemiluminescence biosensor

[0038] a. Preparation of Electrode 1: Polish the glassy carbon electrode (GCE, with a diameter of 3 mm) on suede successively with 1, 0.3, and 0.05 μm alumina powder for 1 - 5 min, then ultrasonically wash it with water and ethanol successively for 1 - 5 min, and then dry it with nitrogen, marked as Electrode 1.

[0039] b. Preparation of Electrode 2: Immerse the three - electrode system (reference electrode, glassy carbon electrode, counter electrode) into the porous ZnCo - MOF material solution in the above step 1, apply a voltage of - 1.4 V for 500 s using the i - t method, and mark the obtained electrode as Electrode 2.

[0040] c. Preparation of Electrode 3: Drop 10 μL of 5 μM Hg(II) on Electrode 2, incubate it in the dark at room temperature for 30 min, and place it in the electrolyte solution for electrochemiluminescence detection, marked as Electrode 3.

[0041] d. Preparation of Electrode 4: Uniformly mix 1000 U / mL, 1 μL TdT (final concentration in 100 μL solution: 10000 U / L), 10 μM, 1 μL ssDNA (final concentration in 100 μL solution: 0.1 μM), 10 mM, 1 μL dTTP (final concentration in 100 μL solution: 0.1 mM), 10×TdT buffer (1 μL), and 6 μL distilled water, place it in a water bath at 37 °C for 2 h, then raise the temperature of the constant - temperature water bath to 75 °C and react for 10 min to inactivate the enzyme to terminate the polymerization reaction. Subsequently, add 50 μM, 10 μL Hg(II) (final concentration: 5 μM) and 5 μL HEPES buffer solution (10 mM, pH = 7.0) to the above 5 μL TdT reaction solution, incubate it at room temperature for 30 min, then take 10 μL and drop it on the surface of Electrode 2, incubate it in the dark at room temperature for 30 min, and place it in the electrolyte solution for electrochemiluminescence detection, marked as Electrode 4.

[0042] Subsequently, use the BPCL T15 analysis system to measure the electrochemiluminescence (ECL) intensity. Set the high voltage to 700 V, the scanning speed to 100 mV / s, and the scanning voltage to 0 - 2 V. Detect the electrochemiluminescence responses of the prepared electrochemical biosensor to Hg(II) and TdT in PBS (0.1 M, pH 7.0, containing 0.1 M KCl and 100 mM triethanolamine) to obtain the electrochemiluminescence magnitudes corresponding to Hg(II) and TdT. The ECL quenching intensity is expressed as (I0 - I) / I0; the ECL recovery intensity is expressed as (I - I0) / I0, where I0 and I represent the ECL intensities in the absence and presence of Hg(II), respectively. Based on this, establish the quantitative relationship between the luminescence intensity and the concentrations of Hg(II) and TdT to achieve the detection of Hg(II) and TdT and the screening of their inhibitors.

[0043] To prove that the electrochemiluminescence sensor of the present invention can achieve the detection of Hg(II), an electrochemiluminescence sensor was prepared based on Examples 1 and 2. By comparing the electrochemiluminescence intensity responses of various sensors, as Figure 1 shown, it can be seen that compared with Electrode 2, the electrochemiluminescence intensity of Electrode 3 decreased after adding Hg(II), indicating that Hg(II) effectively quenched the electrochemiluminescence intensity of ZnCo-MOF; when TdT was introduced, a DNA rich in T bound to Hg(II) to form a stable T-Hg-T structure, restoring the electrochemiluminescence intensity of Electrode 4. This shows that the electrochemiluminescence sensor prepared in the present invention has good responses to Hg(II) and TdT and can achieve the analytical detection of Hg(II) and TdT.

[0044] Example 3 Analytical Detection of Hg(II) and TdT

[0045] Analysis and Detection of Hg(II):

[0046] Based on the steps of Examples 1 and 2, by changing the concentration of 10 μL Hg(II) in Example 2c (final concentrations: 0, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000, 5000, 10000, 50000 nM) while keeping other steps unchanged, the detection of Hg(II) can be achieved. The results are as Figure 2 shown. As the concentration of Hg(II) increases, the ECL quenching intensity continuously increases. The logarithm of the Hg(II) concentration in the range of 0.0005 - 5000 nM has a good linear relationship with the ECL quenching intensity, and its linear correlation equation is: y = 0.1234lgC Hg(II) + 0.4181, R 2 = 0.9980, and the detection limit is 0.031 pM.

[0047] Analysis and Detection of TdT:

[0048] Based on the steps of Example 1 and Example 2, control the reaction system to be 100 μL. By changing the concentration of 1 μL TdT in Example 2d (the final concentration in the 100 μL system is: 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 20, 50, 80, 100, 300, 500, 800, 1000, 2000, 3000 U / L), add 10 μM, 1 μL ssDNA (the final concentration in the 10 μL solution is: 1 μM), 10 mM, 1 μL dTTP (the final concentration in the 10 μL solution is: 1 mM), 10×TdT buffer (1 μL) and 6 μL distilled water and mix evenly. Place it in a water bath at 37 °C and react for 2 h. Then raise the temperature of the constant temperature water bath to 75 °C and react for 10 min to inactivate the enzyme to terminate the polymerization reaction. Then add 50 μM, 10 μL Hg(II) (final concentration: 5 μM) and 5 μL HEPES buffer solution (10 mM pH = 7.0) to the above 5 μL TdT reaction solution. After incubating at room temperature for 30 min, add 80 μL porous ZnCo-MOF material solution and continue to incubate for 30 min, and then measure its ECL intensity. Based on this, the detection of TdT can be realized. The results are as Figure 3 shown. As the concentration of TdT increases, the recovery intensity of ECL continuously increases. The logarithm of the TdT concentration in the range of 0.03 - 1000 U / L has a good linear relationship with the recovery intensity of ECL. The linear correlation equation is: y = 0.6618lgC TdT + 0.9054, R 2 = 0.9964, and the detection limit is 0.0025 U / L.

[0049] Detection of TdT inhibitor PP in Example 4

[0050] Based on Example 1 and Example 2, Example 2d was changed. In 1 μL of TdT solution with a final concentration of 1000 U / L, 2 μL of different concentrations of PP (final concentrations in a 100 μL system: 0, 0.0001, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 0.8, 1, 3, 8, 10, 20, 40, 60, 80, 100, 150 mM) were added respectively. Then, 1 μL of 10 μM ssDNA (final concentration in a 100 μL solution: 0.1 μM), 1 μL of 10 mM dTTP (final concentration in a 100 μL solution: 0.1 mM), 1 μL of 10×TdT buffer, and 4 μL of distilled water were added and mixed evenly. It was placed in a water bath at 37 °C for reaction for 2 h. Subsequently, the temperature of the constant-temperature water bath was raised to 75 °C, and the reaction was carried out for 10 min to inactivate the enzyme to terminate the polymerization reaction. Then, 10 μL of 50 μM Hg(II) (final concentration: 5 μM) and 5 μL of HEPES buffer solution (10 mM pH = 7.0) were added to the above 5 μL of TdT reaction solution. After incubation at room temperature for 30 min, 80 μL of distilled water was added and mixed evenly, and then incubated for another 30 min. The ECL light intensity was measured, and based on this, the detection of PP could be realized. The results are as Figure 4 shown. As the concentration of PP increased, the ECL quenching intensity of the porous ZnCo-MOF material increased again, indicating that the inhibitory effect of PP on TdT was stronger. For PP, the half-inhibitory concentration was 0.26 mM, and the screening of TdT inhibitors was achieved.

[0051] Example 5 Selective Detection of TdT

[0052] To verify the selectivity of the fluorescence probe for TdT, according to the preparation steps of Example 1 and Example 2 above, papain, thrombin, lysozyme, acetylcholinesterase (AChE), horseradish peroxidase (HRP), glucose oxidase (GOx), and alkaline phosphatase (ALP) with the same concentration as TdT were added respectively to participate in the reaction instead of TdT. The results are as Figure 5 shown. It can be seen that the ECL quenching intensity of other enzymes hardly changed, indicating that the ECL sensor had good selectivity for TdT.

[0053] Example 6 Interference Resistance Detection of Hg(II)

[0054] To verify the interference resistance of the ECL sensor to Hg(II), according to the preparation steps of Example 1 and Example 2 above, Hg(II) and calcium ions (Ca 2+ ), magnesium ions (Mg 2+ ), and ferrous ions (Fe 2+ ), chromium ions (Cr 2+ ), titanium ions (Ti 2+ ), cadmium ions (Cd 2+ ) participated in the reaction. The results are as Figure 6 shown. It can be seen that other metal ions have little effect on the quenching intensity of Hg(II), indicating that this electrochemiluminescence sensor has good anti-interference performance for Hg(II).

[0055] Example 7 Practical Application Analysis

[0056] The inventive method was used to monitor the TdT activity in cells. The TdT activities in normal cells and cervical cancer cells were compared. First, the cells were lysed, and nuclear proteins were extracted from the lysate and applied to the experiment. The results are as Figure 7 shown. The signal influence in normal cells (3) is greater than that in cervical cancer cells (5), and the addition of inhibitor PP can reversely affect the signal output (4, 6), indicating the presence of TdT. Moreover, compared with normal cells, the expression level of TdT in cervical cancer cells is reduced, which provides a reference value for the clinical analysis and diagnosis of cervical cancer and drug development.

[0057] It should also be noted that the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Those skilled in the relevant art of this technical field who make changes, modifications, additions, or substitutions within the essence of the present invention should also fall within the protection scope of the claims of the present invention.

Claims

1. A method for developing an electrochemical luminescence sensor for terminal deoxynucleotidyl transferase based on porous ZnCo-MOF material, and its mechanism is as follows: The metal-organic framework porous ZnCo-MOF material is electrochemically synthesized in the present invention, and Ru(bpy)3 2+ undergoes a redox reaction with the electron donor tripropylamine. During the reaction process, the product of Ru(bpy)3 2+ emits photons with a wavelength of 620 nm. Therefore, by adding Ru, the optical signal is enhanced. Hg(II) can effectively quench its electrochemical signal, and under the action of Hg(II), the single-stranded DNA rich in T extended by TdT is converted into hairpin DNA, thereby reducing the free Hg(II) and restoring the ECL signal value. Based on this, the activity of TdT is detected, and finally this method is applied to monitor the activity of TdT in cells, which has great scientific significance.

2. The method for developing an electrochemical luminescence sensor for terminal nucleotidyl transferase based on a porous ZnCo-MOF material according to claim 1, characterized in that: The synthesis method of the porous ZnCo-MOF material does not require harsh chemical reactions and extreme experimental conditions. Only by setting the scanning technique on an electrochemical workstation can the synthesis be completed simply and quickly. Since the material has a porous structure and a stable pore environment, the MOF material has weak photobleaching and stable fluorescence intensity, making it an excellent optoelectronic probe.

3. According to claims 1-2, a method for developing an electrochemical luminescence sensor for terminal nucleotide transferase based on porous ZnCo-MOF material, characterized in that: For the first time, the porous ZnCo-MOF material was used in the development of an electrochemiluminescence sensor and applied to the analysis and detection of TdT and Hg(II), which has good scientific significance.

4. According to claims 1 to 2, a method for developing an electrochemical luminescence sensor for terminal nucleotide transferase based on a porous ZnCo-MOF material, characterized in that: Set the voltage to a high voltage of 700 V, the scanning speed to 100 mV / s, and the scanning voltage to 0 - 2 V. Analyze and detect different concentrations of Hg(II) and TdT using electrochemiluminescence method. The logarithm of the target substance concentration shows a good linear relationship with the electrochemiluminescence quenching and recovery intensity. The detection limit of Hg(II) is 0.031 pM, and the detection limit of TdT is 0.0025 U / L. The inhibitor IC 50 = 0.26 mM.

5. According to claims 1 to 2, a method for developing an electrochemical luminescence sensor for terminal nucleotide transferase based on porous ZnCo-MOF material, characterized in that: This method was applied to the monitoring of TdT activity in normal cells and cervical cancer cells. Compared with normal cells, the expression level of TdT in cervical cancer cells decreased, which provides a reference value for the analysis and diagnosis of cervical cancer and drug development in clinical practice.

Images