阅读说明：本技术 一种酶集成稀土配位聚合物的制备方法及应用 (Preparation method and application of enzyme-integrated rare earth coordination polymer ) 是由 谭宏亮 翁宇豪 朱巧玉 于 2021-08-06 设计创作，主要内容包括：本发明涉及荧光探针的制备及应用领域,具体涉及一种酶集成稀土配位聚合物的制备方法及应用。本发明将酶与具有荧光特性的稀土配位聚合物结合,构建了集成酶的稀土配位聚合物荧光探针,并应用于超氧阴离子的时间分辨荧光检测,解决了现有荧光探针在实际检测中不溶于水、光稳定性差、背景干扰严重的问题。稀土配位聚合物可以保护酶结构免遭环境因素的破坏,极大提高酶的催化活性和稳定性。在超氧阴离子检测过程中,酶能够特异性识别超氧阴离子,并通过分子内电荷转移过程猝灭稀土配位聚合物的荧光。稀土荧光长的荧光寿命可以消除背景荧光的干扰,进而实现时间分辨荧光检测。本发明的方法具有操作简便、成本低和效率高等优点。(The invention relates to the field of preparation and application of fluorescent probes, in particular to a preparation method and application of an enzyme-integrated rare earth coordination polymer. The invention combines enzyme and rare earth coordination polymer with fluorescence characteristic, constructs the rare earth coordination polymer fluorescent probe integrated with enzyme, is applied to time-resolved fluorescence detection of superoxide anion, and solves the problems of water insolubility, poor light stability and serious background interference of the existing fluorescent probe in actual detection. The rare earth coordination polymer can protect an enzyme structure from being damaged by environmental factors, and the catalytic activity and the stability of the enzyme are greatly improved. In the superoxide anion detection process, the enzyme can specifically recognize superoxide anions and quench fluorescence of the rare earth coordination polymer through an intramolecular charge transfer process. The long fluorescence lifetime of rare earth fluorescence can eliminate the interference of background fluorescence, thereby realizing time-resolved fluorescence detection. The method has the advantages of simple and convenient operation, low cost, high efficiency and the like.)

1. A preparation method of an enzyme-integrated rare earth coordination polymer is characterized in that an enzyme is combined with a rare earth coordination polymer with a fluorescent characteristic, and specifically comprises the following steps:

(1) pre-stirring the metal salt aqueous solution and a certain amount of superoxide dismutase for 30-60 min;

(2) adding an aqueous solution of ligand adenosine triphosphate, reacting for 3-5 h, and separating to obtain a solid product;

(3) and (3) mixing the solid product obtained by separation in the step (2) with carboxyphenylboronic acid, reacting for 12-24 h, and separating to obtain a final solid product, namely the enzyme-integrated rare earth coordination polymer.

2. The method for preparing an enzyme-integrated rare earth coordination polymer according to claim 1, wherein the metal salt in step (1) is terbium nitrate.

3. The method of claim 1, wherein the molar ratio of metal salt to ligand is 8: 3.

4. The method of claim 1, wherein the stirring time in step (1) is 30min, the reaction time in step (2) is 3h, and the reaction time in step (3) is 12 h.

5. The method for preparing an enzyme-integrated rare earth coordination polymer according to claim 1, wherein a solid product is obtained by centrifugation in step (2) at 13000rpm for 10 min.

6. The method of claim 1, further comprising a washing step of washing the solid product 3 times with water in step (3) and reacting the solid product with the antenna molecule carboxyphenylboronic acid in a mixed manner.

7. An enzyme-integrated rare earth coordination polymer, characterized by being prepared by the preparation method of any one of claims 1 to 6.

8. The use of the enzyme-integrated rare earth coordination polymer of claim 7 in time-resolved fluorescence detection of superoxide anions.

Technical Field

The invention relates to the field of preparation and application of fluorescent probes, in particular to a method for forming a composite probe by combining a rare earth coordination polymer and an enzyme.

Background

Superoxide anion (O)₂ ^·-) Is one of Reactive Oxygen Species (ROS) and plays an important role in cell growth and metabolism. However,O₂ ^·-can cause oxidative stress in the organism, leading to serious diseases such as rheumatoid arthritis, cardiovascular diseases and cancer. Thus, to O₂ ^·-The high sensitivity and selectivity of detection is of great importance for assessing its role in physiological and pathological processes. At present, various kinds of O have been established₂ ^·-Techniques for detection such as electron spin resonance, electrochemistry, chemiluminescence, and fluorescence. Among them, the fluorescence analysis method is often considered to be an ideal analysis method because of its advantages of simplicity, high sensitivity, easy operation, and the like. Although there is currently O₂ ^·-Fluorescent probes have been reported, but most of them are complex organic molecules constructed based on ethidium hydride, nitrobenzene, caffeic acid, naphthalimide and the like. The preparation of these fluorescent probes requires a time-consuming and laborious multistep synthesis and surface modification process. In addition, they have disadvantages such as poor water solubility, low photostability, and serious background interference. Therefore, it is urgently needed to construct O with simple preparation, stable luminescence property and high sensitivity₂ ^·-A fluorescent probe.

The enzyme has the catalytic advantages of ultrahigh conversion number and unique substrate selectivity, so that the enzyme is widely applied to the fields of food processing, wastewater treatment, pharmaceutical industry and the like. However, the catalytic activity of natural enzymes is highly susceptible to external environments (e.g., pH, temperature, and organic solvents). In recent years, coordination polymers have been considered as ideal carriers for immobilized enzymes. Different from traditional solid carriers such as porous silica, hydrogel and the like, the coordination polymer is a hybrid material consisting of metal ions and organic bridge ligands, and has adjustable structure and performance. In addition, as an enzyme immobilization carrier, the coordination polymer not only has excellent mechanical and thermal stability, but also can enhance the affinity to the enzyme through hydrophobic and/or coordination interaction, thereby obtaining higher enzyme loading rate and negligible enzyme leakage. More attractive is that the enzyme can be well maintained in its conformation in the coordination polymer due to the self-adaptive encapsulation properties of the coordination polymer. Therefore, in the limited domain environment of the coordination polymer, the catalytic activity and the stability of the enzyme can be obviously improved. Nevertheless, most of the current research focuses on the use of coordination polymers as carriers to improve the catalytic performance of enzymes, and the research using enzyme-integrated coordination polymers as nanoprobes is still in the first stage.

Disclosure of Invention

Aiming at the technical current situations of complicated preparation of a fluorescent probe, serious background interference, low sensitivity, poor selectivity and the like in the traditional superoxide anion detection method, the invention prepares the enzyme-integrated rare earth coordination polymer and can detect superoxide anions by combining a time-resolved fluorescence technology.

The technical scheme adopted by the invention is as follows:

a preparation method of an enzyme-integrated rare earth coordination polymer combines an enzyme with a rare earth coordination polymer with a fluorescent characteristic, and specifically comprises the following steps:

(1) pre-stirring the metal salt water solution and 20-25 mug/mL of superoxide dismutase (SOD) for 30-60 min;

(2) adding an aqueous solution of ligand Adenosine Triphosphate (ATP), reacting for 3-5 h, and separating to obtain a solid product;

(3) and (3) mixing the solid product obtained by separation in the step (2) with 400 mu L of carboxyphenylboronic acid (CPBA) with the concentration of 200mM for reaction for 12-24 h, and separating to obtain a final solid product, namely the enzyme-integrated rare earth coordination polymer.

Preferably, the metal salt in step (1) is terbium nitrate (Tb (NO)₃)₃)。

Preferably, the molar ratio of metal salt to ligand is 8: 3.

Preferably, the stirring time in the step (1) is 30min, the reaction time in the step (2) is 3h, and the reaction time in the step (3) is 12 h.

Preferably, the solid product is obtained by centrifugal separation in the step (2), and the centrifugal condition is 13000rpm for 10 min.

Preferably, the preparation method of the enzyme-integrated rare earth coordination polymer further comprises a washing step, wherein in the step (3), the solid product is washed 3 times by water and then mixed with the carboxyphenylboronic acid for reaction.

The invention also provides the enzyme-integrated rare earth coordination polymer prepared by the preparation method.

The invention also comprises the application of the enzyme-integrated rare earth coordination polymer in time-resolved fluorescence detection of superoxide anions.

The invention has the beneficial effects that:

(1) the preparation method of the invention does not contain toxic elements, has mild conditions, and the rare earth coordination polymer can protect the enzyme from the influence of external environment and can effectively improve the catalytic activity and stability of the enzyme.

(2) The enzyme-integrated rare earth coordination polymer prepared by the preparation method can be used for time-resolved fluorescence detection of superoxide anions. In the process of detecting the super anions, the superoxide dismutase can specifically recognize the super anions to perform transduction, so that the detection sensitivity and selectivity are improved. In addition, the rare earth coordination polymer has unique rare earth fluorescence emission characteristics, and can effectively eliminate the interference of background fluorescence through a time resolution technology.

(3) The method of the invention has simple operation, low price and high efficiency.

Drawings

FIG. 1 is the SEM pictures of ATP/Tb and SOD @ ATP/Tb-CPBA in example 1.

FIG. 2 is an absorption spectrum and a photograph of a water-soluble tetrazolium salt reagent (WST-1) in example 2 under different conditions.

FIG. 3 shows the emission spectra of ATP/Tb-CPBA and SOD @ ATP/Tb-CPBA in example 3 under different conditions.

FIG. 4 is a graph comparing the time-dependent fluorescence response of SOD @ ATP/Tb-CPBA and free SOD + ATP/Tb-CPBA systems to superoxide anions in example 4.

FIG. 5 is a graph comparing the activity of SOD @ ATP/Tb-CPBA and free SOD + ATP/Tb-CPBA systems in example 5 on superoxide anion response after storage for various periods of time.

Detailed Description

In order that the above objects, features and advantages of the present invention can be more clearly understood, a more particular description of the invention will be rendered by reference to the appended drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments disclosed below.

A preparation method of an enzyme-integrated rare earth coordination polymer combines an enzyme with a rare earth coordination polymer with a fluorescent characteristic, and specifically comprises the following steps:

(1) the metal salt terbium nitrate (Tb (NO)₃)₃) Pre-stirring the aqueous solution and 20-25 mug/mL of superoxide dismutase (SOD) for 30 min;

(2) adding aqueous solution of ligand Adenosine Triphosphate (ATP), reacting for 3h, centrifuging at 13000rpm for 10min, and separating to obtain solid product, wherein the metal salt terbium nitrate (Tb (NO)₃)₃) The molar ratio of the Adenosine Triphosphate (ATP) to the ligand is 8: 3;

(3) washing the solid product obtained by separation in the step (2) with water for 3 times, mixing the solid product with antenna molecule carboxyphenylboronic acid (CPBA) for reaction for 12 hours, and separating the solid product to obtain the final solid product, namely the enzyme-integrated rare earth coordination polymer.

In the following examples, superoxide dismutase was labeled as SOD, adenosine triphosphate was labeled as ATP, carboxyphenylboronic acid was labeled as CPBA, samples without CPBA were labeled as SOD @ ATP/Tb, samples without SOD were labeled as ATP/Tb-CPBA, and the final samples were labeled as SOD @ ATP/Tb-CPBA.

Example 1

SOD @ ATP/Tb-CPBA was prepared according to the following procedure:

(1) dissolving 5.5mg of terbium nitrate in 4mL of water to obtain a terbium nitrate aqueous solution;

(2) dissolving 0.1mg of SOD in 0.1mL of water to obtain an SOD aqueous solution;

(3) dissolving 6.1mg ATP in 4mL water to obtain ATP aqueous solution;

(4) mixing 4mL of terbium nitrate aqueous solution with 0.1mL of SOD aqueous solution, and stirring for 30 min; then adding ATP aqueous solution, and continuing stirring for 3 h; washing with water for 3 times to obtain SOD @ ATP/Tb;

(5) dissolving 13.3mg of CPBA in 0.4mL of water to obtain a CPBA aqueous solution;

(6) and (3.6 mL of SOD @ ATP/Tb (0.1mg/mL) aqueous solution prepared in the step (4) and the step (5) is mixed with 0.4mL of CPBA aqueous solution, the mixture is stirred for 12 hours and then centrifuged (13000rpm for 10min), and the mixture is washed for 3 times by water to obtain SOD @ ATP/Tb-CPBA.

Example 2

Detecting superoxide anion by combining a water-soluble tetrazolium salt reagent (WST-1), and recording the absorbance corresponding to the maximum absorption peak of the superoxide anion by using an ultraviolet spectrophotometer to detect the catalytic activity of the superoxide anion of the SOD. The method comprises the following steps:

(1) 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) with pH 7.4 is used as a buffer solution, the total volume is 200 μ L, and the reaction temperature is 37 ℃;

(2) superoxide anion is generated from KO dissolved in dimethyl sulfoxide (DMSO) solution₂Obtaining;

(3) numbering the reaction wells as a, b, c and d respectively by using a 96-well plate; wherein the content of the first and second substances,

the preparation method of the solution in the reaction hole a comprises the following steps: HEPBS buffer solution with pH 7.4 containing 10 μ M WST-1 reagent, and water bath reaction at 37 ℃ for 20 min;

the preparation method of the solution in the reaction hole b comprises the following steps: HEPBS buffer solution with pH 7.4 containing 10 μ M WST-1 reagent and 50 μ M superoxide anion solution, and reacting in water bath at 37 deg.C for 20 min;

the preparation method of the solution in the reaction hole c comprises the following steps: HEPBS buffer solution with pH 7.4 containing 10 μ M WST-1 reagent, 50 μ g SOD and 50 μ M superoxide anion, and reacting in water bath at 37 deg.C for 20 min;

the preparation method of the solution in the reaction hole d comprises the following steps: HEPBS buffer solution with pH 7.4 containing 10 μ M WST-1 reagent, 50 μ g SOD @ ATP/Tb and 50 μ M superoxide anion, and reacting in water bath at 37 deg.C for 20 min;

(4) after the reaction holes a, b, c and d react for 20min at the same time, observing that the reaction hole d is colorless and is similar to the reaction hole a, and the rest reaction holes are yellow;

(5) the absorbance corresponding to the maximum absorption peak of each of the samples in the 4 reaction wells was measured at 440nm using an ultraviolet spectrophotometer.

As shown in FIG. 2, the absorbance of the reaction product of free SOD and superoxide anion was much higher than that of SOD @ ATP/Tb, indicating that the catalytic activity of SOD embedded in ATP/Tb was greatly enhanced.

Example 3

And recording the fluorescence intensity corresponding to the maximum emission peak by using a fluorescence spectrometer to detect the capacity of identifying superoxide anions of SOD @ ATP/Tb-CPBA. The specific method comprises the following steps:

(1) 4 centrifuge tubes with the number of a, b, c and d are respectively used for 0.5 mL;

(2) the preparation method of the solution in the centrifuge tube a comprises the following steps: adding 5 mu L of 1mg/mL ATP/Tb-CPBA into 95 mu L HEPES buffer solution with pH value of 7.4, and reacting in water bath at 37 ℃ for 3 min;

(3) the preparation method of the solution in the centrifuge tube b comprises the following steps: adding 5 μ L of 1mg/mL ATP/Tb-CPBA into 95 μ L HEPES buffer solution with pH 7.4 containing 50 μ g SOD and 50 μ M superoxide anion, and reacting in water bath at 37 deg.C for 3 min;

(5) the preparation method of the solution in the centrifuge tube c comprises the following steps: adding 5 mu L of 1mg/mL SOD @ ATP/Tb-CPBA into 95 mu L of HEPES buffer solution with pH value of 7.4, and carrying out water bath reaction at 37 ℃ for 3 min;

(6) the preparation method of the solution in the centrifuge tube d comprises the following steps: adding 5 μ L of 1mg/mL SOD @ ATP/Tb-CPBA into 95 μ L of pH 7.4HEPES buffer solution containing 50 μ M superoxide anion, and reacting in water bath at 37 deg.C for 3 min;

(7) after the reaction is finished, the fluorescence intensity corresponding to the maximum emission peak of the samples in the 4 centrifugal tubes is measured by using a fluorescence spectrometer under the excitation wavelength of 300nm in a time resolution fluorescence mode.

The detection result is shown in figure 3, the fluorescence of the SOD @ ATP/Tb-CPBA after the superoxide anion is added is obviously quenched, and the quenching degree is far greater than that of a mixed system of the SOD and the ATP/Tb-CPBA, so that the SOD @ ATP/Tb-CPBA has sensitive superoxide anion recognition capability.

Example 4

Comparing superoxide anion responsiveness of SOD @ ATP/Tb-CPBA and ATP/Tb-CPBA + SOD systems, and recording fluorescence intensity corresponding to a maximum emission peak by using a fluorescence spectrometer; the method comprises the following specific steps:

referring to the experimental procedure in the test tube when the fluorescence response experiment was performed in example 3, the final concentrations of SOD and superoxide anion were set to 50. mu.M, 0.05mg/mL ATP/Tb-CPBA and SOD @ ATP/Tb-CPBA, and 100. mu.L of the total final reaction volume, and the reaction was carried out in a 37 ℃ water bath for 3min, and after the completion of the reaction, the fluorescence intensities were measured at 300nm excitation wavelengths in a time-resolved fluorescence mode using a fluorescence spectrometer.

The detection result is shown in FIG. 4, the fluorescence response speed of SOD @ ATP/Tb-CPBA to superoxide anion is faster than that of a mixed system of SOD and ATP/Tb-CPBA, and therefore the SOD @ ATP/Tb-CPBA has higher fluorescence quenching efficiency and is used for detecting superoxide anion.

Example 5

Comparing the storage stability of the SOD @ ATP/Tb and the ATP/Tb + SOD system, and recording the fluorescence intensity corresponding to the maximum emission peak by using a fluorescence spectrometer. The method comprises the following specific steps:

(1) respectively placing the SOD @ ATP/Tb system and the SOD system at room temperature for 1-7 days, and monitoring the concentration of superoxide anions every day;

(2) referring to the experimental operation method in the test tube when the fluorescence response experiment was performed in example 3, the final concentration of SOD was set to 50. mu.M, the final concentration of superoxide anion was set to 50. mu.M, the final concentration of SOD @ ATP/Tb was set to 0.05mg/mL, and the total final reaction volume was set to 100. mu.L, and the reaction was carried out in a water bath at 37 ℃ for 3min, and after the reaction was completed, the fluorescence intensities of the SOD mixed system and the free SOD @ ATP/Tb system were measured by a fluorescence spectrometer under the excitation wavelength of 300nm in a time-resolved fluorescence mode, respectively.

As shown in FIG. 5, the catalytic stability of superoxide anion of SOD @ ATP/Tb is much higher than that of free SOD within 7 days, so that the encapsulation of SOD in ATP/Tb can greatly enhance the catalytic stability of SOD.

In conclusion, the enzyme-integrated rare earth coordination polymer provided by the invention is formed by combining the enzyme and the rare earth coordination polymer. In the preparation process of the enzyme-integrated rare earth coordination polymer, the enzyme still has higher catalytic activity and stability because of no toxic elements and mild conditions. The enzyme-integrated rare earth coordination polymer prepared by the preparation method disclosed by the invention can be used for detecting superoxide anions by combining a time-resolved fluorescence technology, and has the fluorescence characteristics of rare earth metals and the specific recognition function of enzyme, so that the detection sensitivity and selectivity are improved; compared with the common superoxide anion detection, the method is simpler and more efficient.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.