Protein and rare earth complex co-crystallized Cu < 2 + > fluorescence sensor, method and application
The co-crystallization of rare earth complexes and proteins to form hybrid luminescent crystals, which solves the problem of insufficient stability of rare earth complexes in biological samples, and achieves high sensitivity and high selectivity Cu2+ detection, which has good biocompatibility.
Patent Information
- Application Number
- CN202510451539.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-08-15
AI Technical Summary
The application of existing rare earth complexes in biological samples has insufficient stability and environmental interference, resulting in insufficient sensitivity and selectivity for metal ion detection.
By co-crystallizing the rare earth complex with proteins to form hybrid luminescent crystals, and enhancing their stability through chemical cross-linking, a Cu2+ fluorescence sensor co-crystallized by proteins and rare earth complexes was prepared.
It realizes Cu2+ detection with high sensitivity and selectivity, has good biocompatibility, can effectively avoid environmental interference, and provides efficient and accurate metal ion detection methods.
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Figure CN120485326A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of nano functional materials, and in particular to a fluorescent sensor based on protein and rare earth complex cocrystallization and a preparation method thereof and a method for detecting Cu 2+ Application in. Background Art
[0002] Metal ions (such as Cu 2+ 、Zn 2+ 、Fe 2+ / Fe 3+ , Ca 2+ Mg 2+ ) plays a key physiological function in organisms, and abnormal changes in their concentration are closely related to the occurrence and development of various diseases. Therefore, the development of a highly sensitive and selective method for detecting metal ions has important scientific significance and application value. In recent years, fluorescent sensors have attracted widespread attention due to their rapid response, high sensitivity and good biocompatibility. Rare earth complexes are considered to be ideal fluorescent probe materials due to their unique optical properties (such as long fluorescence lifetime and large Stokes shift). However, the application of rare earth complexes in biological samples still faces problems such as insufficient stability and environmental interference.
[0003] Therefore, the existing technology has defects and needs to be improved. Summary of the Invention
[0004] The purpose of the present invention is to provide a Cu based on protein and rare earth complex cocrystallization 2+ A fluorescence sensor, its preparation method, and its application. The sensor has high sensitivity, high selectivity, and good biocompatibility, can effectively avoid environmental interference, and provides a new, efficient and accurate method for the detection of metal ions.
[0005] The technical solution of the present invention comprises the following steps:
[0006] A method for preparing a fluorescent sensor of protein and rare earth complex co-crystallization comprises the following steps: blending a rare earth complex solution and a protein solution, precipitating the product by a co-crystallization method to obtain a rare earth complex-doped luminescent protein crystal, and annealing the crystal, supplemented by chemical cross-linking, to obtain a crystalline structure with enhanced stability.
[0007] In the preparation method, the protein is hen egg white lysozyme.
[0008] In the preparation method, the rare earth complex solution is Eu(TTA)3phen or Tb(ACAC)3phen.
[0009] The preparation method comprises the following steps:
[0010] A1. Dissolve hen egg white lysozyme in ultrapure water to prepare a solution with a concentration of 0.001-0.1 M.
[0011] A2. Preparation of rare earth complex solution: 0.01M EuCl3 ethanol solution or 0.01M TbCl3 ethanol solution, 0.03M 2-thenoyltrifluoroacetone (TTA) ethanol solution and 0.01M o-phenanthroline (phen) ethanol solution were prepared respectively, and the three solutions were mixed in a ratio of 1:1:1 to prepare a rare earth complex solution;
[0012] A3, co-crystallization process: the above rare earth complex solution is mixed with hen egg white lysozyme (HEWL) solution to form hybrid luminescent crystals by anti-solvent crystallization method;
[0013] A4, annealing treatment of crystal;
[0014] A5. Cross-linking treatment of crystals.
[0015] A6. The cross-linked crystals were washed with ethanol.
[0016] The preparation method, the specific method of step A3 is: mixing the HEWL solution and the Eu(TTA)3phen solution in a centrifuge tube at a volume ratio of 1:3, and standing at -18-4°C for 40-80 minutes to generate crystals.
[0017] The preparation method, the specific method of step A4 is: placing the crystal on a glass slide and using a constant temperature drying oven for annealing. The equipment needs to be calibrated in advance to ensure the accuracy of temperature and time. During the annealing process, slowly increase the temperature to the target temperature of 25-80°C at a rate of 5-10°C / min and maintain it at the selected temperature for 12-48 hours; after annealing is completed, naturally cool to room temperature.
[0018] The specific method of the preparation method, step A5 is: transferring the generated crystals into a 4 vol% glutaraldehyde solution for cross-linking for 24 hours.
[0019] The protein and rare earth complex co-crystallized Cu obtained by any of the preparation methods 2+ Fluorescence sensor.
[0020] According to the co-crystallization of protein and rare earth complex Cu 2+ Fluorescence sensor in detecting Cu 2+ Application in.
[0021] The beneficial effects of the present invention are: protein crystals can be used as carriers with good biocompatibility, and hybrid luminescent crystals can be obtained by co-crystallization with rare earth complexes. The hybrid luminescent crystals can effectively detect Cu in blood or solution. 2+Concentration: As the ion concentration changes, the luminescence intensity of the crystal changes. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 Optical microscope images of crystals formed between protein solution and Eu(TTA)3phen at different temperatures and crystallization times;
[0023] Figure 2 Microscopic spectrum of protein crystals co-crystallized with Eu(TTA)3phen;
[0024] Figure 3 Confocal images of protein crystals co-crystallized with Eu(TTA)3phen;
[0025] Figure 4 The effect of different concentrations of copper ions on the luminescence intensity of the crystal;
[0026] Figure 5 Fitting curves of crystal fluorescence quenching at different concentrations of different metal ions; DETAILED DESCRIPTION
[0027] The present invention is described in detail below with reference to specific embodiments.
[0028] Example 1
[0029] Egg white lysozyme was dissolved in ultrapure water, and the rare earth complex was dissolved in ethanol, and crystals were cultivated by anti-solvent co-crystallization method.
[0030] The hen egg white lysozyme was dissolved in ultrapure water to prepare a solution with a concentration of 0.01 M, and an ultrasonic machine was used to accelerate the dissolution and disperse it evenly.
[0031] Prepare a europium complex solution: Prepare 0.01M EuCl3 ethanol solution, 0.03M 2-thenoyltrifluoroacetone (TTA) ethanol solution, and 0.01M phenanthroline (phen) ethanol solution. Mix the three solutions in a 1:1:1 ratio to create a 0.01M Eu(TTA)3phen solution.
[0032] The europium complex and protein solution were placed in the same 4°C environment for 40 min.
[0033] Preparation of co-crystallization crystals of europium complex and protein: keeping the ambient temperature constant, the egg white lysozyme solution and the europium complex solution are mixed in a centrifuge tube at a solution volume ratio of 1:3, and crystals are generated in the centrifuge tube.
[0034] Annealing of the crystal: The obtained rare earth complex hybrid luminescent crystal is washed and dried to remove impurities. Then, the crystal is placed on a glass slide to ensure that it is heated evenly during the heating process. Next, annealing is performed using equipment such as a constant temperature drying oven. The equipment needs to be calibrated in advance to ensure the accuracy of temperature and time. During the annealing process, the temperature is slowly raised to the target temperature of 25-80°C at a rate of 5-10°C / min and maintained at the selected temperature for 12-48 hours. After annealing is completed, it is naturally cooled to room temperature to avoid damage to the crystal structure caused by rapid cooling;
[0035] The glutaraldehyde solution was diluted with ultrapure water to prepare a glutaraldehyde crosslinking agent with a concentration of 4 vol %. The crystals were transferred to the 4 vol % glutaraldehyde solution and immersed in the crosslinking solution for 24 hours.
[0036] The cross-linked crystals were transferred to ethanol and washed 2-3 times for a period of time to remove excess rare earth complexes, protein chains, and glutaraldehyde.
[0037] The fluorescence of the rare earth complex in the eutectic crystals was altered using metal ions. Ultrapure water was used as the solvent to prepare metal ions at concentrations ranging from 0 to 0.1 mg / ml. The metal ions used were copper, zinc, iron, calcium, and magnesium. Crystals were added to the metal ion solutions (0 to 0.1 mg / ml) and the fluorescence intensity was measured after 10 to 60 minutes. The crystals were then immersed in the solutions (from low to high concentrations) using an immersion method. Microscopic observation and fluorescence detection of the microscopic area were performed at the same location.
[0038] Anticoagulant Performance Test: The crystalloid's anticoagulant properties were evaluated by mixing it with blood of varying concentrations and measuring the hemolysis rate. The results showed that the crystalloid exhibited low hemolysis rates at all volume concentrations, confirming its good blood compatibility.
[0039] Figure 2 and Figure 3 The following are microscopic and confocal spectra of protein crystals co-crystallized with Eu(TTA)3phen. The effectiveness of the composite was further confirmed by microscopic spectrophotometry, which enabled us to accurately measure the fluorescence intensity of the crystals. The emission spectrum revealed a prominent peak at approximately 610 nanometers, which is attributed to the rare earth ions. The confocal image shows a uniform distribution of fluorescence within the crystals, indicating that the rare earth dopant is uniformly dispersed within the crystal lattice. This uniformity is crucial for the reliability and reproducibility of the sensor's response to metal ions.
[0040] Figure 4The effect of different concentrations of copper ions on the luminescence intensity of crystals is demonstrated. In the experiment, co-crystallized crystals formed by protein and europium complex prepared by antisolvent co-crystallization method were immersed in a series of copper ion solutions with different concentrations. The concentration range of these solutions covers physiologically relevant levels from low to high. The fluorescence intensity of the same position of each sample was detected using a microscopic spectrophotometer to ensure the consistency and comparability of the data. Figure 4 It can be observed that as the copper ion concentration increases, the fluorescence intensity of the crystal shows a significant downward trend. This phenomenon indicates that copper ions have a significant quenching effect on the crystal, and the quenching effect is positively correlated with the copper ion concentration. Specifically, the fluorescence intensity begins to decrease at low copper ion concentrations, and this quenching effect becomes increasingly significant as the copper ion concentration gradually increases.
[0041] Figure 5 The graph shows the fluorescence quenching effect of different metal ion concentrations on the crystal. It can be observed that copper ions have the most significant fluorescence quenching effect on the crystal, followed by iron and zinc ions, while calcium and magnesium ions have relatively weaker quenching effects. Specifically, as the concentration of copper, iron, and zinc ions in the solution increases, the luminescence intensity of the crystal shows a clear downward trend. This phenomenon indicates that there is a strong interaction between these metal ions and the crystal, resulting in a decrease in fluorescence intensity.
[0042] Example 2
[0043] Egg white lysozyme was dissolved in ultrapure water, and the rare earth complex was dissolved in ethanol, and crystals were cultivated by anti-solvent co-crystallization method.
[0044] Egg white lysozyme was dissolved in ultrapure water to prepare a solution with a concentration of 0.01 M.
[0045] Prepare a europium complex solution: Prepare 0.01M EuCl3 ethanol solution, 0.03M 2-thenoyltrifluoroacetone (TTA) ethanol solution, and 0.01M phenanthroline (phen) ethanol solution. Mix the three solutions in a 1:1:1 ratio to create a 0.01M Eu(TTA)3phen solution.
[0046] The europium complex and protein solution were placed in the same 4°C environment for 60 min.
[0047] Preparation of co-crystallization crystals of europium complex and protein: keeping the ambient temperature constant, the egg white lysozyme solution and the europium complex solution are mixed in a centrifuge tube at a solution volume ratio of 1:3, and crystals are generated in the centrifuge tube.
[0048] The annealing treatment of the crystal is the same as in Example 1;
[0049] The glutaraldehyde solution was diluted with ultrapure water to prepare a glutaraldehyde crosslinking agent with a concentration of 4 vol %. The crystals were transferred to the 4 vol % glutaraldehyde solution and immersed in the crosslinking solution for 24 hours.
[0050] The cross-linked crystals were transferred to ethanol and washed 2-3 times for a period of time to remove excess rare earth complexes, protein chains, and glutaraldehyde.
[0051] Example 3
[0052] Egg white lysozyme was dissolved in ultrapure water, and the rare earth complex was dissolved in ethanol, and crystals were cultivated by anti-solvent co-crystallization method.
[0053] Egg white lysozyme was dissolved in ultrapure water to prepare a solution with a concentration of 0.01 M.
[0054] Prepare a europium complex solution: Prepare 0.01M EuCl3 ethanol solution, 0.03M 2-thenoyltrifluoroacetone (TTA) ethanol solution, and 0.01M phenanthroline (phen) ethanol solution. Mix the three solutions in a 1:1:1 ratio to create a 0.01M Eu(TTA)3phen solution.
[0055] The europium complex and protein solution were placed in the same 4°C environment for 80 min.
[0056] Preparation of co-crystallization crystals of europium complex and protein: keeping the ambient temperature constant, the egg white lysozyme solution and the europium complex solution are mixed in a centrifuge tube at a solution volume ratio of 1:3, and crystals are generated in the centrifuge tube.
[0057] The annealing treatment of the crystal is the same as in Example 1;
[0058] The glutaraldehyde solution was diluted with ultrapure water to prepare a glutaraldehyde crosslinking agent with a concentration of 4 vol %. The crystals were transferred to the 4 vol % glutaraldehyde solution and immersed in the crosslinking solution for 24 hours.
[0059] The cross-linked crystals were transferred to ethanol and washed 2-3 times for a period of time to remove excess rare earth complexes, protein chains, and glutaraldehyde.
[0060] Example 4
[0061] Egg white lysozyme was dissolved in ultrapure water, and the rare earth complex was dissolved in ethanol, and crystals were cultivated by anti-solvent co-crystallization method.
[0062] Egg white lysozyme was dissolved in ultrapure water to prepare a solution with a concentration of 0.01 M.
[0063] Prepare a europium complex solution: Prepare 0.01M EuCl3 ethanol solution, 0.03M 2-thenoyltrifluoroacetone (TTA) ethanol solution, and 0.01M phenanthroline (phen) ethanol solution. Mix the three solutions in a 1:1:1 ratio to create a 0.01M Eu(TTA)3phen solution.
[0064] Place the europium complex and protein solution in the same -18°C environment for 40 minutes.
[0065] Preparation of co-crystallization crystals of europium complex and protein: keeping the ambient temperature constant, the egg white lysozyme solution and the europium complex solution are mixed in a centrifuge tube at a solution volume ratio of 1:3, and crystals are generated in the centrifuge tube.
[0066] The annealing treatment of the crystal is the same as in Example 1;
[0067] The glutaraldehyde solution was diluted with ultrapure water to prepare a glutaraldehyde crosslinking agent with a concentration of 4 vol %. The crystals were transferred to the 4 vol % glutaraldehyde solution and immersed in the crosslinking solution for 24 hours.
[0068] The cross-linked crystals were transferred to ethanol and washed 2-3 times for a period of time to remove excess rare earth complexes, protein chains, and glutaraldehyde.
[0069] Example 5
[0070] Egg white lysozyme was dissolved in ultrapure water, and the rare earth complex was dissolved in ethanol, and crystals were cultivated by anti-solvent co-crystallization method.
[0071] Egg white lysozyme was dissolved in ultrapure water to prepare a solution with a concentration of 0.01 M.
[0072] Prepare a europium complex solution: Prepare 0.01M EuCl3 ethanol solution, 0.03M 2-thenoyltrifluoroacetone (TTA) ethanol solution, and 0.01M phenanthroline (phen) ethanol solution. Mix the three solutions in a 1:1:1 ratio to create a 0.01M Eu(TTA)3phen solution.
[0073] The europium complex and protein solution were placed in the same -18°C environment for 60 minutes.
[0074] Preparation of co-crystallization crystals of europium complex and protein: keeping the ambient temperature constant, the egg white lysozyme solution and the europium complex solution are mixed in a centrifuge tube at a solution volume ratio of 1:3, and crystals are generated in the centrifuge tube.
[0075] The annealing treatment of the crystal is the same as in Example 1;
[0076] The glutaraldehyde solution was diluted with ultrapure water to prepare a glutaraldehyde crosslinking agent with a concentration of 4 vol %. The crystals were transferred to the 4 vol % glutaraldehyde solution and immersed in the crosslinking solution for 24 hours.
[0077] The cross-linked crystals were transferred to ethanol and washed 2-3 times for a period of time to remove excess rare earth complexes, protein chains, and glutaraldehyde.
[0078] Example 6
[0079] Egg white lysozyme was dissolved in ultrapure water, and the rare earth complex was dissolved in ethanol, and crystals were cultivated by anti-solvent co-crystallization method.
[0080] Egg white lysozyme was dissolved in ultrapure water to prepare a solution with a concentration of 0.01 M.
[0081] Prepare a europium complex solution: Prepare 0.01M EuCl3 ethanol solution, 0.03M 2-thenoyltrifluoroacetone (TTA) ethanol solution, and 0.01M phenanthroline (phen) ethanol solution. Mix the three solutions in a 1:1:1 ratio to create a 0.01M Eu(TTA)3phen solution.
[0082] The europium complex and protein solution were placed in the same -18°C environment for 80 min.
[0083] Preparation of co-crystallization crystals of europium complex and protein: keeping the ambient temperature constant, the egg white lysozyme solution and the europium complex solution are mixed in a centrifuge tube at a solution volume ratio of 1:3, and crystals are generated in the centrifuge tube.
[0084] The annealing treatment of the crystal is the same as in Example 1;
[0085] The glutaraldehyde solution was diluted with ultrapure water to prepare a glutaraldehyde crosslinking agent with a concentration of 4 vol %. The crystals were transferred to the 4 vol % glutaraldehyde solution and immersed in the crosslinking solution for 24 hours.
[0086] The cross-linked crystals were transferred to ethanol and washed 2-3 times for a period of time to remove excess rare earth complexes, protein chains, and glutaraldehyde.
[0087] Example 7
[0088] Egg white lysozyme was dissolved in ultrapure water, and the rare earth complex was dissolved in ethanol, and crystals were cultivated by anti-solvent co-crystallization method.
[0089] Egg white lysozyme was dissolved in ultrapure water to prepare a solution with a concentration of 0.01 M.
[0090] Prepare a europium complex solution: Prepare 0.01M EuCl3 ethanol solution, 0.03M 2-thenoyltrifluoroacetone (TTA) ethanol solution, and 0.01M phenanthroline (phen) ethanol solution. Mix the three solutions in a 1:1:1 ratio to create a 0.01M Eu(TTA)3phen solution.
[0091] The europium complex and protein solution were placed in the same -70°C environment for 40 minutes.
[0092] Preparation of co-crystallization crystals of europium complex and protein: keeping the ambient temperature constant, the egg white lysozyme solution and the europium complex solution are mixed in a centrifuge tube at a solution volume ratio of 1:3, and crystals are generated in the centrifuge tube.
[0093] The annealing treatment of the crystal is the same as in Example 1;
[0094] The glutaraldehyde solution was diluted with ultrapure water to prepare a glutaraldehyde crosslinking agent with a concentration of 4 vol %. The crystals were transferred to the 4 vol % glutaraldehyde solution and immersed in the crosslinking solution for 24 hours.
[0095] The cross-linked crystals were transferred to ethanol and washed 2-3 times for a period of time to remove excess rare earth complexes, protein chains, and glutaraldehyde.
[0096] Example 8
[0097] Egg white lysozyme was dissolved in ultrapure water, and the rare earth complex was dissolved in ethanol, and crystals were cultivated by anti-solvent co-crystallization method.
[0098] Egg white lysozyme was dissolved in ultrapure water to prepare a solution with a concentration of 0.01 M.
[0099] Prepare a europium complex solution: Prepare 0.01M EuCl3 ethanol solution, 0.03M 2-thenoyltrifluoroacetone (TTA) ethanol solution, and 0.01M phenanthroline (phen) ethanol solution. Mix the three solutions in a 1:1:1 ratio to create a 0.01M Eu(TTA)3phen solution.
[0100] The europium complex and protein solution were placed in the same -70°C environment for 60 min.
[0101] Preparation of co-crystallization crystals of europium complex and protein: keeping the ambient temperature constant, the egg white lysozyme solution and the europium complex solution are mixed in a centrifuge tube at a solution volume ratio of 1:3, and crystals are generated in the centrifuge tube.
[0102] The annealing treatment of the crystal is the same as in Example 1;
[0103] The glutaraldehyde solution was diluted with ultrapure water to prepare a glutaraldehyde crosslinking agent with a concentration of 4 vol %. The crystals were transferred to the 4 vol % glutaraldehyde solution and immersed in the crosslinking solution for 24 hours.
[0104] The cross-linked crystals were transferred to ethanol and washed 2-3 times for a period of time to remove excess rare earth complexes, protein chains, and glutaraldehyde.
[0105] Example 9
[0106] Egg white lysozyme was dissolved in ultrapure water, and the rare earth complex was dissolved in ethanol, and crystals were cultivated by anti-solvent co-crystallization method.
[0107] Egg white lysozyme was dissolved in ultrapure water to prepare a solution with a concentration of 0.01 M.
[0108] Prepare a europium complex solution: Prepare 0.01M EuCl3 ethanol solution, 0.03M 2-thenoyltrifluoroacetone (TTA) ethanol solution, and 0.01M phenanthroline (phen) ethanol solution. Mix the three solutions in a 1:1:1 ratio to create a 0.01M Eu(TTA)3phen solution.
[0109] The europium complex and protein solution were placed in the same -70°C environment for 80 min.
[0110] Preparation of co-crystallization crystals of europium complex and protein: keeping the ambient temperature constant, the egg white lysozyme solution and the europium complex solution are mixed in a centrifuge tube at a solution volume ratio of 1:3, and crystals are generated in the centrifuge tube.
[0111] The annealing treatment of the crystal is the same as in Example 1;
[0112] The glutaraldehyde solution was diluted with ultrapure water to prepare a glutaraldehyde crosslinking agent with a concentration of 4 vol %. The crystals were transferred to the 4 vol % glutaraldehyde solution and immersed in the crosslinking solution for 24 hours.
[0113] The cross-linked crystals were transferred to ethanol and washed 2-3 times for a period of time to remove excess rare earth complexes, protein chains, and glutaraldehyde.
[0114] Figure 1Figures a, b, and c show optical microscopic images of crystals formed from a protein solution and Eu(TTA)3phen at 4°C for 40, 60, and 80 minutes, respectively. Figures d, e, and f show optical microscopic images of crystals formed from a protein solution and Eu(TTA)3phen at -18°C for 40, 60, and 80 minutes, respectively. Figures g, h, and i show optical microscopic images of crystals formed from a protein solution and Eu(TTA)3phen at -70°C for 40, 60, and 80 minutes, respectively. Controlling crystallization conditions is crucial to ensuring high-quality crystals. Crystals co-crystallized with a protein and europium complex at 4°C exhibited good morphology but were of moderate quantity. Crystals co-crystallized at -18°C and for 60 minutes exhibited the greatest quantity and the best morphology. Crystals co-crystallized at -70°C exhibited poor morphology and were fewer in number. Therefore, when the crystallization temperature is -18°C, crystals with excellent properties in all aspects are obtained.
[0115] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all such improvements and changes should fall within the scope of protection of the appended claims of the present invention.
Claims
1. A method for preparing a fluorescent sensor of protein and rare earth complex co-crystallization, characterized in that: The following steps are involved: The rare earth complex solution and the protein solution are mixed, and the product is precipitated by a co-crystallization method to obtain rare earth complex-doped luminescent protein crystals. The crystals are annealed and supplemented with chemical cross-linking to obtain a crystalline structure with enhanced stability.
2. The preparation method according to claim 1, characterized in that The protein is hen egg white lysozyme.
3. The preparation method according to claim 2, characterized in that The rare earth complex solution is Eu(TTA)3phen or Tb(ACAC)3phen.
4. The preparation method according to claim 3, characterized in that The following steps are involved: A1. Dissolve hen egg white lysozyme in ultrapure water to prepare a solution with a concentration of 0.001-0.1 M. A2. Preparation of rare earth complex solution: 0.01M EuCl3 ethanol solution or 0.01M TbCl3 ethanol solution, 0.03M 2-thenoyltrifluoroacetone (TTA) ethanol solution and 0.01M o-phenanthroline (phen) ethanol solution were prepared respectively, and the three solutions were mixed in a ratio of 1:1:1 to prepare a rare earth complex solution; A3, co-crystallization process: the above rare earth complex solution is mixed with hen egg white lysozyme (HEWL) solution to form hybrid luminescent crystals by anti-solvent crystallization method; A4, annealing treatment of crystal; A5. Cross-linking treatment of crystals. A6. The cross-linked crystals were washed with ethanol.
5. The preparation method according to claim 4, characterized in that The specific method of step A3 is: mixing the HEWL solution and the Eu(TTA)3phen solution in a centrifuge tube at a volume ratio of 1:3, and standing at -18-4°C for 40-80 minutes to generate crystals.
6. The preparation method according to claim 4, characterized in that The specific method of step A4 is as follows: placing the crystal on a glass slide and performing annealing treatment in a constant temperature drying oven. The equipment needs to be calibrated in advance to ensure the accuracy of temperature and time. During the annealing process, the temperature is slowly increased to a target temperature of 25-80°C at a rate of 5-10°C / min and maintained at the selected temperature for 12-48 hours. After annealing is completed, the crystal is naturally cooled to room temperature.
7. The preparation method according to claim 4, characterized in that The specific method of step A5 is: transferring the generated crystals into a 4 vol% glutaraldehyde solution for cross-linking for 24 hours.
8. The protein and rare earth complex co-crystallized Cu obtained by the preparation method according to any one of claims 1 to 7 2+ Fluorescence sensor.
9. The protein and rare earth complex co-crystallized Cu according to claim 8 2+ Fluorescence sensor in detecting Cu 2+ Application in.