Fluorescence sensing method based on on-off-on state change and application of fluorescence sensing method in intracellular terminal transferase detection

Abstract

The invention relates to a fluorescence sensing method based on on-off-on state change and application of the fluorescence sensing method in intracellular terminal transferase detection, electrically synthesized porous Zr-BDC-MOF has fluorescence characteristics and shows an on state, Hg (II) can quench the fluorescence intensity of Zr-BDC-MOF, at the moment, an off state is presented, and on the basis of the specific binding effect of a T-rich aptamer and Hg (II), the detection sensitivity of the T-rich aptamer is improved. According to the present invention, the Zr-BDC-MOF fluorescence recovery is caused, the Zr-BDC-MOF fluorescence is recovered, the Zr-BDC-MOF re-presents the on state, the fluorescence response aiming at Hg (II) and TdT is achieved along with the on-off-on state change, and when the inhibitor Na4P2O7 (PP) is added, the TdT activity is inhibited, and the combined Hg (II) is reduced, such that the fluorescence intensity is reduced so as to achieve the TdT inhibitor screening; through the relationship between the molar concentration of Hg (II), TdT and the inhibitor PP of TdT and fluorescence quenching and recovery strength, the activity monitoring analysis of TdT in Jurket cells is realized. On the basis, an on-off-on fluorescence sensing method which is simple, convenient, rapid, reliable and high in sensitivity is constructed.

Description

Technical Field

[0001] The present invention relates to a fluorescence sensing method, in particular to an on-off-on fluorescence sensing method based on Zr-BDC-MOF and its application research in the analysis and detection of mercury ions and terminal transferase, especially the analysis of intracellular terminal transferase activity, belonging to the fields of functional synthetic materials and chemical biosensing. Background Art

[0002] Terminal deoxynucleotidyl transferase (TdT) is a special type of DNA polymerase composed of two polypeptide chains. Its main function is to bind single deoxynucleotides to the 3'-OH end of the substrate DNA molecule without a template. TdT is a strong specific intracellular marker for immature lymphocytes in the hematopoietic system and is often used as one of the important indicators for diagnosing lymphoblastic lymphoma / acute lymphoblastic leukemia (LBL / ALL) in clinical practice. However, there are occasionally cases of TdT-negative LBL / ALL found in clinical practice, but they are quite different from TdT-positive LBL / ALL patients in clinical characteristics and prognosis. The clinical method for detecting TdT activity is based on the binding of TdT antigen and antibody, including gel electrophoresis, biochemical analysis, and immunological assays. These methods are not intuitive enough and require the cooperation of biological instruments. Most importantly, there is still great room for the development of intracellular TdT activity monitoring. Therefore, developing a simple, rapid, low-cost, and highly selective analytical sensing method for TdT activity detection has good application value in clinical practice.

[0003] Metal-organic frameworks (MOFs) are porous nanomaterials composed of metal nodes and organic ligands through coordination bonds. They can be designed and optimized for the analyte during the preparation stage, thereby improving the sensitivity of detection, and can be used in the research of fluorescence probes, nonlinear optics, photocatalysis, electroluminescent devices, and biomedical imaging. Traditional MOF synthesis methods have disadvantages such as long synthesis time, cumbersome and complex process procedures, high energy consumption, and strict requirements for the reaction equipment environment to varying degrees. The electrochemical synthesis process has advantages such as simple process, low pollution, environmental friendliness, simple operation, and can be operated at normal temperature and pressure, which has attracted extensive research by many scientists. However, synthesizing MOFs using electrochemical methods without heating and pressurization is still an urgent problem to be solved.

[0004] The present invention designs a fluorescence sensing method based on the on-off-on state change and its application in the detection of intracellular terminal transferase. This method constructs a porous Zr-BDC-MOF material synthesized by an electrochemical direct current method, which exhibits excellent fluorescence intensity and can be used as a fluorescence (FL) probe. With the addition of Hg(II), the fluorescence intensity of Zr-BDC-MOF gradually decreases. Then, TdT is introduced to synthesize a DNA strand rich in thymine T, which can compete with Hg(II) with quenching ability in the solution to form a thymine-Hg(II)-thymine (T-Hg(II)-T) structure, resulting in the recovery of the FL fluorescence intensity. The FL quenching intensity (QE%) is expressed as (F0 - F) / F0; the FL recovery intensity is expressed as (F - F0) / F0, where F0 and F represent the FL intensities in the absence and presence of Hg(II), respectively. Finally, this patent applies this fluorescence method to the monitoring of intracellular TdT activity. Currently, there is no publicly reported case of a fluorescence sensing method based on porous Zr-BDC-MOF with an on-off-on state change at home and abroad, nor any relevant reports on the use of Zr-BDC-MOF materials for the analysis and detection of intracellular terminal transferase. Summary of the Invention

[0005] The present invention proposes a fluorescence sensing method based on the on-off-on state change and its application in the detection of intracellular terminal transferase. This method is the first to use Zr-BDC-MOF for the development of a fluorescence sensing method with an on-off-on state change and applies it to the detection of Hg(II), TdT, and its inhibitors.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows: A fluorescence sensing method based on the on-off-on state change and its application in the detection of intracellular terminal transferase, the specific steps are as follows:

[0007] (1) Electrochemical synthesis of Zr-BDC-MOF

[0008] Weigh 0.4 g of tetrabutylammonium bromide and 0.1 g of 2-aminoterephthalic acid (NH2-BDC), add them to a mixture of 5 mL of N,N-dimethylformamide (DMF) and acetic acid (volume ratio 1:1), and ultrasonicate for 10 min to fully dissolve them to obtain a clear light yellow solution.

[0009] Put the polished zircon wire (Zr) into the above mixed solution, pass direct current for reaction, and continuously stir magnetically for about 2 h. At this time, the solution becomes turbid. Centrifuge at a high speed (8000 rpm / h), wash three times with ethanol, then place it in an oven to dry thoroughly and grind it into uniform fine powder. Weigh 5 mg of MOF material and put it into 50 mL of deionized water, and ultrasonically disperse it for 20 min to make Zr-BDC-MOF evenly dispersed in water with a concentration of 0.1 mg / mL for standby.

[0010] (2) Preparation of fluorescent probe

[0011] a. Preparation of solution 1: Take 10 - 20 μL of 0.1 mg / mL Zr-BDC-MOF solution and place it in 80 - 90 μL of PBS (10 mM, pH = 7.0) solution, and label it as solution 1.

[0012] b. Preparation of solution 2: Add 40 - 50 μM, 10 μL of Hg(II) (final concentration: 4 - 5 μM) to solution 1, and incubate in the dark at room temperature for 25 - 30 min, and label it as solution 2.

[0013] c. Preparation of solution 3: Mix 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 evenly to make the volume 8 - 10 μL. Place it in a water bath at 35 - 37 °C for reaction for 2 - 2.5 h. Then raise the constant temperature water bath temperature to 70 - 75 °C and react for 10 - 15 min to inactivate the enzyme to terminate the polymerization reaction. Subsequently, add 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) to the above 5 μL of TdT reaction solution. After incubating at room temperature for 25 - 30 min, add 70 - 80 μL of Zr-BDC-MOF solution and continue to incubate for 25 - 30 min, and label it as solution 3.

[0014] Based on the above experimental steps, then use a fluorescence detector to measure the fluorescence (FL) intensity. Set the voltage to 300 - 400 V, the scanning speed to 1000 - 1200 nm / min, the slit width to 5 - 7 nm, the starting wavelength of the emission spectrum to 500 - 520 nm, and the ending wavelength to 720 - 750 nm to realize the detection of Hg(II), TdT and the screening of their inhibitors.

[0015] Principle of the invention: The present invention is a fluorescence sensing method based on the on-off-on state change and its application in the detection of intracellular terminal transferase. The electro-synthesized porous Zr-BDC-MOF has fluorescence characteristics and shows an "on" state, so it is very suitable for the development of fluorescence sensing methods. This patent finds that Hg(II) can quench the fluorescence intensity of Zr-BDC-MOF. At this time, the method presents an "off" state. Based on the specific binding of the T-rich aptamer to Hg(II), we use TdT to generate a long T-rich DNA chain, which can capture a lot of Hg(II) through the T-Hg-T structure, resulting in the recovery of the fluorescence of Zr-BDC-MOF and presenting an "on" state again. With the on-off-on state change, a fluorescence response method for Hg(II) and TdT is realized. When the inhibitor Na4P2O7 (PP) is added, since the activity of TdT is inhibited, the generated T-rich DNA chain is reduced, and the bound Hg(II) is reduced, so the fluorescence intensity will also decrease, thus realizing the screening of TdT inhibitors. Through the relationship between the logarithmic concentrations of Hg(II), TdT and its inhibitor PP and the fluorescence quenching and recovery intensities, the quantitative analysis of Hg(II), TdT and its inhibitor PP in the sample to be tested is realized. Based on this, a simple, convenient, fast, reliable and highly sensitive on-off-on fluorescence sensing method is constructed and applied to the analysis of TdT activity in Jurket cells.

[0016] Advantages of the present invention: The present invention constructs a fluorescence sensing method based on the on-off-on state change and its application in the detection of intracellular terminal transferase. First, a porous Zr-BDC-MOF material with strong fluorescence is formed by applying a constant voltage. Within a certain range, the higher the concentration of Hg(II), the more obvious the quenching effect on Zr-BDC-MOF and the smaller the FL fluorescence intensity; the higher the concentration of TdT, the more T-rich DNA is generated, competing for a lot of Hg(II), and the greater the FL fluorescence intensity. The test results show that the fluorescence intensity has a linear relationship with the logarithmic concentrations of Hg(II) and TdT within a certain range, and the analysis and detection of Hg(II) and TdT are better realized. Its advantages are as follows:

[0017] (1) High reliability of results. Zr-BDC-MOF overcomes the drawback of the instability of traditional fluorescent molecules as FL emitters in aqueous solutions, and this material presents a porous structure with stable fluorescence signals and accurate and reliable results.

[0018] (2) High sensitivity. For the first time, an on-off-on state change fluorescence sensing method is constructed based on Zr-BDC-MOF. This method has good sensitivity, the detection limit of Hg(II) is 0.33 pM, and the detection limit of TdT is 0.029 U / L.

[0019] (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+ ), cadmium ions (Cd 2+ ) have no interference on this system; for the detection of TdT: other common enzymes, such as Papain, Thrombin, Lysozyme (LZM) and Acetylcholinesterase (AChE), have no interference on this detection system.

[0020] (4) Simple method. This fluorescence method is set as a homogeneous experiment, with good repeatability and reproducibility, simple operation, and can realize the rapid and sensitive analysis and detection of the target substance, and the recovery rate is between 96% - 102.5%.

[0021] (5) The reaction conditions of the present invention are mild, environmentally friendly, and have stable performance, and are suitable for practical applications in biosensing and ecological environment, such as the analysis of TdT activity in Jurket cells.

[0022] In summary, the present invention is a fluorescence sensing method based on the on-off-on state change and its application in the detection of intracellular terminal transferase, which has the advantages of convenient operation, high specificity, high sensitivity, etc., can realize the detection of lower concentration TdT in Jurket cells, and has good application prospects. Brief Description of the Drawings

[0023] Figure 1 It is the feasibility analysis diagram of the fluorescence probe of the present invention;

[0024] Figure 2 It is the linear relationship diagram of the quenching intensity of the fluorescence probe of the present invention for Hg(II) versus the logarithm of the concentration;

[0025] Figure 3 It is the linear relationship diagram of the recovery intensity of the fluorescence probe of the present invention for TdT versus the logarithm of the concentration;

[0026] Figure 4 It is the linear relationship diagram of the quenching intensity of the fluorescence probe of the present invention for PP versus the logarithm of the concentration;

[0027] Figure 5 It is the selectivity experiment diagram of the fluorescence probe of the present invention for Hg(II);

[0028] Figure 6 It is the selectivity experiment diagram of the fluorescence probe of the present invention for TdT.

[0029] Figure 7This is the experimental graph of the fluorescence probe of the present invention for monitoring the activity of TdT in Jurket cells. Specific Embodiments

[0030] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0031] Example 1 Electrochemical Synthesis of Zr-BDC-MOF

[0032] Weigh 0.4 g of tetrabutylammonium bromide and 0.1 g of 2-aminoterephthalic acid (NH2-BDC), add them to a mixture of 5 mL of N,N-dimethylformamide (DMF) and acetic acid (volume ratio 1:1), and ultrasonically dissolve for 10 min to obtain a clear light yellow solution.

[0033] Put the polished zirconium wire (Zr) into the above mixed solution, pass a direct current for reaction, continuously stir magnetically for about 2 h. At this time, the solution becomes turbid. After centrifuging at a high speed (8000 rpm / h), wash it three times with ethanol, then dry it thoroughly in an oven and grind it into a uniform fine powder. Weigh 5 mg of the MOF material and put it into 50 mL of deionized water, ultrasonically disperse for 20 min to uniformly disperse Zr-BDC-MOF in water with a concentration of 0.1 mg / mL, and set it aside for use.

[0034] Example 2 Preparation of Fluorescence Probe

[0035] a. Preparation of Solution 1: Take 10 μL of 0.1 mg / mL Zr-BDC-MOF solution and place it in 80 μL of PBS (10 mM pH = 7.0) solution, and label it as Solution 1.

[0036] b. Preparation of Solution 2: Add 50 μM, 10 μL of Hg(II) (final concentration: 5 μM) to the above-prepared 90 μL of Solution 1, incubate in the dark at room temperature for 30 min, and label it as Solution 2.

[0037] c. Preparation of Solution 3: Uniformly mix 1000 U / mL, 1 μL of TdT (final concentration in 100 μL solution: 10 U / mL), 10 μM, 1 μL of ssDNA (final concentration in 100 μL solution: 0.1 μM), 10 mM, 1 μL of dTTP (final concentration in 100 μL solution: 0.1 mM), 10×TdT buffer (1 μL), and 6 μL of distilled water. 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 and terminate the polymerization reaction. Subsequently, add 50 μM, 10 μL of Hg(II) (final concentration: 5 μM) and 5 μL of HEPES buffer solution (10 mM pH = 7.0) to the above 5 μL of TdT reaction solution. Incubate at room temperature for 30 min, and then add 80 μL of Zr-BDC-MOF solution and continue to incubate for 30 min, labeled as Solution 3.

[0038] Based on the above solutions, use a fluorescence detector to measure the fluorescence (FL) intensity. Set the voltage to 350 V, the scanning speed to 1200 nm / min, the slit width to 5 nm, the starting wavelength of the emission spectrum to 520 nm, and the ending wavelength to 720 nm to achieve the detection of Hg(II), TdT, and the screening of their inhibitors.

[0039] To prove that the fluorescence probe of the present invention can detect Hg(II), prepare the fluorescence probe based on Example 1 and Example 2. Comparing the fluorescence intensity responses of various probes, as shown in Figure 1 It can be seen that when Hg(II) is present, the fluorescence intensity of Solution 2 decreases relative to that of Solution 1, indicating that Hg(II) has a good fluorescence quenching effect on Zr-BDC-MOF. When TdT is present, the DNA rich in T binds to Hg(II), causing the fluorescence intensity of Solution 3 to recover, indicating that the fluorescence probe has a good response to Hg(II) and TdT and can be used for the analysis and detection of Hg(II) and TdT.

[0040] Example 3 Analysis and Detection of Hg(II) and TdT

[0041] Analysis and Detection of Hg(II):

[0042] Based on the steps of Example 1 and Example 2a, 2b, control the reaction system to be 100 μL. By changing the concentration of 10 μL of Hg(II) in Example 2b (final concentration: 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 300, 800, 2500, 5000, 100000, 20000, 30000 nM) and keeping other steps unchanged, the detection of Hg(II) can be achieved. The results are as shown in Figure 2As shown, with the increase of Hg(II) concentration, the fluorescence quenching intensity continuously increases. The logarithm of Hg(II) concentration in the range of 0.005 - 10000 nM has a good linear relationship with the fluorescence quenching intensity. The linear correlation equation is y = 0.1461lgC Hg(II) + 0.3167, R 2 = 0.9909, and the detection limit is 0.33 pM.

[0043] TdT analysis and detection:

[0044] Based on the steps of Example 1 and Example 2, control the reaction system to be 100 μL. By changing the 1 μL TdT concentration in Example 2c (the final concentration in the 100 μL system is: 0, 0.1, 0.5, 1, 2, 5, 10, 20, 40, 80, 100, 150, 300, 400, 650, 900, 1200, 1800, 2400, 3000 U / L), and 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. 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. After incubating at room temperature for 30 min, add 80 μL Zr-BDC-MOF solution and continue to incubate for 30 min, and then measure its fluorescence intensity. Based on this, the detection of TdT can be realized. The results are as Figure 3 shown. With the increase of TdT concentration, the fluorescence recovery intensity continuously increases. The logarithm of TdT concentration in the range of 0.5 - 1800 U / L has a good linear relationship with the fluorescence recovery intensity. The linear correlation equation is: y = 1.116lgC TdT + 0.3147, R 2 = 0.9978, and the detection limit is 0.029 U / L. It shows that this fluorescence probe realizes the highly sensitive detection of Hg(II) and TdT.

[0045] Detection of TdT inhibitor PP in Example 4

[0046] Based on Example 1 and Example 2, in a 1 μL solution with a final concentration of 1200 U / L of TdT, 2 μL of different concentrations of PP were added respectively (the final concentrations in a 100 μL system were: 0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 2, 5, 8, 10, 20, 40, 60, 80, 100, 150 mM). Then, 10 μM, 1 μL of ssDNA (the final concentration in a 10 μL solution was: 1 μM), 10 mM, 1 μL of dTTP (the final concentration in a 10 μL solution was: 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, 50 μM, 10 μL of Hg(II) (the final concentration was: 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 Zr-BDC-MOF solution was added and incubated for another 30 min, and then its fluorescence intensity was measured. Based on this, the detection of PP can be realized. The results are as Figure 4 shown. As the concentration of PP increased, the fluorescence quenching intensity of Zr-BDC-MOF increased again, indicating that the inhibitory effect of PP on TdT was stronger. For PP, the half-inhibitory concentration was 0.79 mM, and the screening of TdT inhibitors was achieved.

[0047] Example 5 Selective Detection

[0048] To verify the selectivity of this fluorescent probe for Hg(II), according to the preparation steps of Example 1 and Example 2 above, calcium ions (Ca 2+ ), magnesium ions (Mg 2+ ), ferrous ions (Fe 2+ ), chromium ions (Cr 2+ ), titanium ions (Ti 2+ ), cadmium ions (Cd 2+ ) with the same concentration as Hg(II) were added respectively to participate in the reaction. The results are as Figure 5 shown. It can be seen that compared with Hg(II), the fluorescence quenching intensity (QE%) of other metal ions was stronger, indicating that this fluorescent probe had good selectivity for Hg(II).

[0049] To verify the selectivity of this fluorescent probe for TdT, according to the preparation steps of Example 1 and Example 2 above, papain, thrombin, lysozyme (LZM), and acetylcholinesterase (AChE) with the same concentration as TdT were added respectively to participate in the reaction. The results are as Figure 6As shown, it can be seen that the fluorescence quenching intensity (QE%) of other enzymes remains basically unchanged, indicating that this fluorescence probe has good selectivity for TdT.

[0050] Example 6 Monitoring of TdT Activity in Jurket Cells

[0051] To verify the economic and social benefits of this sensing method, this patent uses Jurket cells as an experimental carrier to complete the intracellular TdT activity monitoring experiment. Jurkat cells are a type of suspension cells related to acute T cell leukemia, and it has currently been found that TdT activity is closely related to this disease. First, Jurkat cells are prepared into suspension solutions of 5000 cells / mL, 10000 cells / mL, 50000 cells / mL, and 100000 cells / mL, and then nuclear proteins are extracted from the cells and used as samples in the experiment. From Figure 7 Figure A, we found that as the number of cells increased, the QE% value decreased, indicating that Jurkat cells contain TdT and the TdT content is continuously increasing. To verify that this change is caused by TdT, this patent selected the 100000 cells / mL suspension solution as the object and added the inhibitor PP, and found that the QE% value increased ( Figure 7 Figure B), indicating that the decrease in the value in Figure A is caused by TdT. In addition, this patent preliminarily explored the TdT activity in normal cells and Jurkat cells. The results of this experiment are the average of 30 parallel test results. It was found that the content in Jurkat cells is higher than that in normal cells, P < 0.05, which is statistically significant. Our results provide a new perspective for TdT-related clinical diagnosis and the discovery of cancer tumor markers.

[0052] 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 scope of the essence of the present invention should also fall within the protection scope of the claims of the present invention.

Claims

1. A fluorescence sensing method based on the on-off-on state change and its application in the detection of intracellular terminal transferase. The mechanism is as follows: In this method, a porous Zr-BDC-MOF material is synthesized under electrochemical direct current conditions. Zr-BDC-MOF exhibits excellent fluorescence intensity and can be used as a fluorescence (FL) probe. With the addition of Hg(II), the fluorescence intensity of Zr-BDC-MOF gradually decreases. Then, TdT is introduced to synthesize a DNA strand rich in thymine T. This DNA can compete with Hg(II) with quenching ability in the solution to form a thymine-Hg(II)-thymine (T-Hg(II)-T) structure, resulting in the recovery of the FL fluorescence value. The FL quenching intensity (QE%) is expressed as (F0 - F) / F0; the FL recovery intensity is expressed as (F - F0) / F0, where F0 and F represent the FL intensities in the absence and presence of Hg(II), respectively. Currently, there is no publicly reported case of a fluorescence sensing method based on porous Zr-BDC-MOF with an on-off-on state change at home and abroad, nor any relevant reports on the use of Zr-BDC-MOF materials for the analysis and detection of terminal transferase.

2. The fluorescence sensing method based on on-off-on state change according to claim 1 and its application in intracellular terminal transferase detection, characterized in that: A porous Zr-BDC-MOF material was synthesized for the first time, which has strong fluorescence. However, Hg(II) can quench its fluorescence, and the T-rich DNA generated by TdT can compete with Hg(II). Based on this, a simple, rapid, and environmentally friendly fluorescence sensing method with an "on-off-on" state change was constructed.

3. A fluorescence sensing method based on the change of on-off-on state and its application in intracellular terminal transferase detection according to claims 1 to 3, characterized in that: The analysis and detection of different concentrations of Hg(II) and TdT were achieved through a porous Zr-BDC-MOF material. A good linear relationship was presented between the logarithm value of the target substance concentration and the fluorescence quenching intensity and fluorescence recovery intensity. The detection limit of Hg(II) was 0.33 pM, and the detection limit of TdT was 0.029 U / L. The inhibitor IC 50 = 0.79 mM.

4. The fluorescence sensing method based on the on-off-on state change and its application in the detection of intracellular terminal transferase according to claims 1 to 3, characterized in that: This method can be applied to monitor the activity of TdT in Jurkat cells. Through statistical methods, it is found that the content of TdT in Jurkat cells is highly expressed, with P < 0.05, which is statistically significant, providing a new perspective for TdT-related clinical diagnosis and the discovery of cancer tumor markers.

Images