A virtual screening method of a protein optical switch and a probe screened by the method

Abstract

The present application belongs to the field of protein fluorescence analysis and biosensing technology, and particularly relates to a virtual screening method of protein optical switch and a probe screened by the method. The method comprises the following steps: firstly, a virtual compound library of metal polypyridyl complexes containing different auxiliary ligand structures, dppz ligand numbers and substituent types is constructed; then, a crystal structure of a target protein is selected as a receptor, and a molecular docking method is used to calculate the binding energy of each candidate complex in the virtual compound library and the target protein, and the candidate complexes are sorted according to the predicted binding affinity; finally, the candidate complexes with high ranking are selected for synthesis, and the luminescence performance of the candidate complexes in an aqueous solution and after being combined with the target protein is tested, so that a protein optical switch probe which emits weak luminescence in an aqueous solution and emits significantly enhanced luminescence after being combined with the target protein is screened. The target probe screened by the present application has high sensitivity, fast response and excellent selectivity.

Description

Technical Field

[0001] This invention belongs to the field of protein fluorescence analysis and biosensing technology, specifically relating to a method for designing, synthesizing, virtually screening, and analyzing protein photoswitches based on ruthenium polypyridine complexes, particularly relating to the screening of human serum albumin and β-lactoglobulin photoswitching probes and their detection applications in biological samples. Background Technology

[0002] Ruthenium(II)-dipyrido[3,2-a:2′,3′-c]phenazine (dppz) complexes are a typical type of environmentally sensitive "molecular photoswitches." In aqueous solution, they exhibit weak luminescence due to non-radiative decay, but their luminescence is significantly enhanced upon entering a hydrophobic microenvironment. Currently, these complexes are mainly used for DNA recognition, but their application in protein detection faces challenges.

[0003] In existing technologies, the discovery of high-performance Ru-dppz complexes mainly relies on empirical design and repeated trial and error, resulting in long development cycles and difficulties in revealing structure-activity relationships. Furthermore, the complexity of protein structures and the diversity of binding sites make it difficult to predict the binding behavior and luminescent response of complexes to different proteins. While existing protein quantification methods (such as HPLC and immunoassay) are accurate, they typically rely on large instruments, complex pretreatment processes, and long detection times, hindering rapid on-site screening. Therefore, there is an urgent need to establish an efficient method for the rational design and screening of protein photoswitches. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the shortcomings of existing protein detection technologies, such as probe discovery relying on experience, low screening efficiency, expensive detection equipment, and cumbersome pretreatment. This invention provides a virtual screening method for protein photoswitches and a high-performance probe obtained based on this method, enabling rapid, sensitive, and selective detection of human serum albumin and β-lactoglobulin.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A virtual screening method for protein photoswitches includes the following steps:

[0007] S1: Construct a virtual compound library of metal polypyridine complexes containing different auxiliary ligand structures, dppz ligand numbers, and substituent types;

[0008] S2: Select the crystal structure of the target protein as the acceptor, calculate the binding energy of each candidate complex in the virtual compound library to the target protein using the molecular docking method, and sort them according to the predicted binding affinity.

[0009] S3: Select the top-ranked candidate complexes for synthesis and test their luminescence properties in aqueous solution and after binding with the target protein. Screen out protein photoswitching probes that exhibit weak luminescence in aqueous phase and significantly enhanced luminescence after binding with the target protein.

[0010] Furthermore, the metal polypyridine complex is a ruthenium(II) polypyridine complex, with the following general structural formula:

[0011]

[0012] R is H or CH3; X - To counter ion Cl - or Br - x and y are integers between 0 and 3, and x + y ≤ 3; M is Ru, Fe, or Os.

[0013] Furthermore, the target protein is human serum albumin or β-lactoglobulin.

[0014] A human serum albumin light-switching probe, obtained by the above method, is a ruthenium complex Ru4, and its structure is as follows:

[0015]

[0016] [Ru(dppz)2dip] 2+ (Ru4)

[0017] A β-lactoglobulin photoswitching probe, obtained by the above method, is a ruthenium complex Ru6, and its structure is as follows:

[0018]

[0019] [Ru(dip)2dppx] 2+ (Ru6)

[0020] Compared with the prior art, the advantages and effects of the present invention are as follows:

[0021] 1. This invention establishes for the first time a technical route that combines computational virtual screening and experimental verification for protein photoswitches, overcoming the problem that traditional probe development relies on empirical design and repeated trial and error. It can quickly discover high-performance probes suitable for the detection of target proteins without having to synthesize all candidate molecules one by one.

[0022] 2. The target probes screened by this invention have low background and significant luminescence activation in aqueous phase. They achieve 280.0-fold and 346.1-fold luminescence enhancement for human serum albumin and β-lactoglobulin, respectively, with detection limits of 0.0093 mg / L and 0.0057 mg / L, respectively. Moreover, the response time is less than 1 s, demonstrating high sensitivity, fast response and excellent selectivity.

[0023] 3. This invention can realize quantitative analysis of proteins in complex samples. The detection of human serum albumin can be used for serum and urine samples, and the detection of β-lactoglobulin can be used for raw milk samples. The results are in good agreement with the standard method, indicating that this invention has good accuracy, reproducibility and practical application value.

[0024] 4. This invention combines luminescence detection with smartphone image analysis, and uses blue LED excitation and simplified optical devices to build a portable POC detection platform, which is suitable for rapid on-site screening and low-cost applications. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the protein photo-switching computational virtual screening strategy and detection principle of the present invention.

[0026] Figure 2 The results of computational virtual screening and experimental verification of HSA photoswitching probes for human serum albumin are shown in the figure. In the figure, a) is a heatmap of the predicted binding ability of different metal polypyridine complexes in the virtual compound library to HSA molecules; b) is a comparison of the luminescence intensity of candidate complexes under conditions without HSA and with HSA; c) is a figure showing the predicted binding energy and corresponding luminescence enhancement factor of some candidate complexes.

[0027] Figure 3 The graphs show the detection performance of the HSA photoswitching probe, where a) is the change in the emission spectrum of the target probe under different concentrations of HSA and the color change under blue light irradiation; b) is the result of the change in emission intensity with HSA concentration; c) is the linear relationship between I / I0 and HSA concentration; d) is the response time curve; e) is the emission stability under different pH conditions; and f) is the evaluation graph of selectivity and anti-interference performance.

[0028] Figure 4 The diagram shows the detection mechanism of the HSA photoswitching probe, where a) is the UV-Vis absorption spectrum of the target probe under different concentrations of HSA; b) is the luminescence lifetime decay curve before and after the target probe interacts with HSA; c) is a three-dimensional molecular docking diagram of the binding mode between the target probe and HSA; d) is a two-dimensional interaction diagram of the binding mode between the target probe and HSA; and e) is a diagram showing the change in luminescence intensity after the addition of site-competing molecules such as ibuprofen, warfarin, and ferric chloride heme.

[0029] Figure 5 The figures show the results of real-time detection and actual sample analysis based on the HSA photoswitching probe, where: a) is a schematic diagram of the portable POC detection device; b) are images of HSA at different concentrations and actual serum samples under blue light irradiation; c) is a quantitative relationship graph between R / (R+G+B) value and HSA concentration; d) is a comparison graph of the HSA determination results in serum by the method of the present invention and the standard bromocresol green method; e) is a luminescence spectrum of HSA at different concentrations in an artificial urine system; and f) is a linear relationship graph between I / I0 and HSA concentration in the artificial urine system.

[0030] Figure 6 The results of computational virtual screening and experimental verification of β-LG photoswitching probes targeting β-lactoglobulin are shown in the figure. In the figure, a) is a heatmap of the predicted binding ability of different metal polypyridine complexes in the virtual compound library to β-LG molecules; b) is a comparison of the luminescence intensity of candidate complexes under conditions without and with β-LG; c) is a figure showing the predicted binding energy and corresponding luminescence enhancement factor of some candidate complexes.

[0031] Figure 7 The graphs show the detection performance of the β-LG photoswitching probe, where a) is the change in the emission spectrum of the target probe under different concentrations of β-LG and the color change under blue light irradiation; b) is the result of the change in emission intensity with β-LG concentration and its linear fitting result; c) is the response time curve; and d) is the evaluation graph of selectivity and anti-interference performance. Detailed Implementation

[0032] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the invention.

[0033] Figure 1 This is a schematic diagram illustrating the protein photoswitching computational virtual screening strategy and detection principle of the present invention. For the specific complex structures, synthesis methods, and characterization methods of the candidate metal polypyridine complex library involved in the examples, please refer to the invention patent "A Metal Polypyridine Complex for Detecting and Removing Perfluorinated Compounds and Its Application," which will not be elaborated upon in this specification.

[0034] Example 1: Human serum albumin photoswitching probe screening method based on computational virtual screening

[0035] 1. Preparation of candidate coordination library and target protein

[0036] HSA was selected as the target protein, and the HSA crystal structure from a protein database was used as the molecular docking acceptor. A virtual compound library of metal polypyridine complexes composed of different auxiliary ligands and dppz-type ligands was constructed for subsequent computational screening and experimental verification. The overall screening principle is as follows: Figure 1 As shown, the process includes four steps: target protein selection, virtual library construction, molecular docking and sequencing, and experimental verification. Figure 1 a is a schematic diagram illustrating the calculation of the virtual screening strategy. Figure 1 b is a schematic diagram of the ligand types related to the virtual compound library. Figure 1 c is a schematic diagram of the detection mechanism of the designed protein photoswitch.

[0037] 2. Molecular docking screening

[0038] The binding affinity between each candidate complex and HSA was calculated using molecular docking, and the complexes were ranked according to their predicted binding energies. Preferably, molecular docking was performed using AutoDock 4 and AutoDock Tools in a semi-flexible docking mode. The PDB number of the HSA protein structure was 4K2C, the docking grid size was set to 126 × 98 × 120 Å, and the lowest energy conformation was selected as the optimal binding conformation. Figure 2 The figure shows a heatmap of the predicted binding ability of different candidate complexes to HSA. It can be seen that different auxiliary ligand structures, the number of dppz ligands and the type of substituents will affect the binding ability of candidate complexes to HSA.

[0039] 3. Experimental verification

[0040] Candidate metal polypyridine complexes were prepared into ultrapure water stock solutions of appropriate concentrations and stored in the dark. HSA was prepared in 5 mM HEPES buffer at pH 7.4. During testing, the candidate complex solution was mixed with the HEPES buffer, and then the HSA solution was added, adjusting the total volume to 2 mL. The emission spectra were recorded at room temperature. Preferably, the excitation wavelength was 450 nm, and the emission wavelength range was 550-800 nm. Figure 2 b presents the comparison results of the luminescence intensity of the candidate complexes with and without HSA. Figure 2 c gives the structure number, predicted binding energy, and corresponding luminescence enhancement factor of each candidate coordination compound.

[0041] 4. Screening Results

[0042] like Figure 2 As shown, different candidate complexes exhibited varying degrees of luminescence enhancement after interaction with HSA, with the enhancement factor following the order: Ru4 > Ru3 > Ru5 > Ru1 > Ru2. Among them, Figure 2C showed that Ru4 exhibited a luminescence enhancement factor of 280.0-fold, significantly superior to other candidate complexes and two orders of magnitude higher than the control probe Ru2 without dppz ligands. Table 1 further lists the luminescence properties of each candidate complex under HSA conditions, including luminescence intensity, enhancement factor, quantum yield, and detection limit. Ru4 showed a higher quantum yield and lowest detection limit in the bound state. Therefore, Ru4 can be identified as the preferred photoswitching probe for HSA detection. The results indicate that the auxiliary ligand structure, the number of dppz ligands, and their ratio all affect the binding affinity of candidate complexes to HSA and their luminescence-on performance.

[0043] Table 1. Luminescence properties of different Ru complexes with and without HSA.

[0044]

[0045] Note: a The complex concentration was 1 μM, and the solvent system was HEPES buffer (5 mM, pH = 7.4). b The concentration of the complex was 1 μM, and the concentration of HSA was 15 μM. c Quantum yield (Φ) was determined using [Ru(bpy)3]Cl2 as a standard.

[0046] Example 2: Human serum albumin detection method and sample analysis based on human serum albumin photoswitching probe

[0047] 1. Preparation of HSA and probe solutions

[0048] The HSA photoswitching probes screened in Example 1 were prepared in 5 mM HEPES buffer at pH 7.4, with a preferred probe concentration of 1 μM, and stored protected from light. HSA standards were dissolved in the same buffer to prepare standard solutions of different concentrations, which were further diluted as needed for detection.

[0049] 2. Construction of the luminescence detection system

[0050] Two mL of probe solution was added to a quartz cuvette, followed by HSA standard solutions of different concentrations. After thorough mixing, the emission spectrum was measured. Preferably, the excitation wavelength was 450 nm, and the emission wavelength range was 550-800 nm. As the HSA concentration increased, the probe luminescence gradually increased, exhibiting a visible change from colorless to red under blue light illumination. Figure 3 a presents the emission spectra and color changes at different HSA concentrations. Figure 3 b shows the corresponding changes in luminous intensity.

[0051] 3. Test performance results

[0052] The results show that the HSA photo-switching probe has a significant concentration-dependent response to HSA, with continuously enhanced luminescence in the 0-15 μM range, such as... Figure 3 a and Figure 3 As shown in b; its I / I0 value has a good linear relationship with the HSA concentration in the range of 0-0.7 μM, and the linear equation is I / I0 = 151.81 × [HSA] + 0.98, R 2 = 0.990, such as Figure 3 As shown in c. The detection limit, calculated using the 3σ / k method, is 0.14 nM, or 0.0093 mg / L. The probe's response time to HSA is less than 1 s, as... Figure 3 As shown in d; and it maintains good luminescence stability within the pH range of 5.0-8.0, such as Figure 3 As shown in e.

[0053] 4. Selectivity and anti-interference performance

[0054] Glucose, lactic acid, lysozyme, glutathione, cysteine, citric acid, creatinine, urea, and HCO3 were added. - H2PO4 - SO4 2- Cl - Mg 2+ NH4 + and Na + Common small molecules and ions were added to the detection system to investigate their effects on the probe luminescence signal. The results showed that none of the aforementioned interfering substances induced a significant luminescence response. Further interference experiments were conducted on major coexisting proteins in serum. Under serum-related ratios (HSA / human immunoglobulin G (hIgG) / transferrin (Tf) / fibrinogen (Fib) / α1-antitrypsin (AAT) = 70 / 14 / 5.7 / 2.8 / 0.7 (w / w), their effect on the probe signal was negligible. Figure 3 f presents the selectivity and anti-interference results of the probe for representative small molecules, ions and major serum proteins, indicating that the method has good selectivity and anti-interference ability.

[0055] 5. Mechanism of Action Analysis

[0056] like Figure 4 As shown, the absorption spectrum, luminescence lifetime, and binding mode of the probe before and after interaction with HSA were systematically analyzed. Figure 4 The results showed that as the HSA concentration increased, the probe exhibited a significant hypocoloration effect at 370 nm, indicating that the complex bound to HSA and caused perturbation of the dppz ligand-related π→π* transition. Figure 4b shows that the excited-state lifetime of the probe increased from 85.5 ns to 121.2 ns, indicating that after binding to HSA, the contact between the dppz site and water molecules was suppressed, and the non-radiative decay was weakened. Figure 4 c presents the three-dimensional molecular docking results between the probe and HSA, indicating that the probe tends to bind to the IB subregion of HSA; Figure 4 Figure d shows a two-dimensional interaction diagram, which shows that residues such as LEU115, PRO118, MET123, ALA126, and LYS137 interact hydrophobically with the dppz ligand. Among them, LYS137 also participates in π-cation interaction, and residues such as VAL116, PRO118, VAL122, and ALA126 further interact with the auxiliary ligand, thus forming a hydrophobic microenvironment that protects the dppz site. Figure 4 The results of the site competition experiment (e) show that the luminescence of the Ru4-HSA system significantly decreased after the addition of hemin chloride, which binds to the IB subregion of HSA. Warfarin, which binds to the IIA subregion, and ibuprofen, which binds to the IIIA subregion, caused only minor changes, indicating that the probe mainly binds to the IB subregion of HSA. In summary, the hydrophobic and spatially confined microenvironment provided by the HSA binding pocket effectively shields the interaction of water molecules with the nitrogen atom of the phenazine ligand in the dppz ligand, causing the dark state to become unstable and the bright state to dominate, thus producing a significant light-switching effect.

[0057] 6. POC Detection and Actual Sample Analysis

[0058] like Figure 5 As shown, a portable detection device consisting of a blue LED, a long-pass filter, an eight-tube sample cell, and a smartphone imaging module was constructed. Figure 5 a is a schematic diagram of the POC detection device. During detection, a 1 μM probe is added to an eight-tube array, followed by HSA standard solution or the sample to be tested, and images are acquired under blue light irradiation. Figure 5 b shows that as the HSA concentration increased from 0 to 10 mg / L, the sample color gradually changed from colorless to red. Further RGB values ​​were extracted and the R / (R+G+B) value was calculated. Figure 5 c shows that the R / (R+G+B) value has a good linear relationship with the HSA concentration in the range of 0-10 mg / L. The linear equation is R / (R+G+B) = 0.009×[HSA] + 0.268, R² = 0.987, and the detection limit is 0.59 mg / L.

[0059] In actual sample analysis, when testing commercial human serum samples, the HSA concentrations measured by luminescence immunoassay and smartphone imaging were 48.2 ± 0.3 mg / mL and 49.7 ± 1.3 mg / mL, respectively, consistent with the result of 52.1 ± 0.6 mg / mL obtained by the standard bromocresol green method. Figure 5 Section d provides a comparison of the results from three methods, demonstrating that the method of this invention has good accuracy. For urine samples, an artificial urine system can be used for evaluation. Figure 5 The results showed that as the HSA concentration in artificial urine increased, the probe luminescence gradually increased. Figure 5 f shows a good linear relationship between the I / I0 value and the HSA concentration in the range of 0-60 mg / L, with the correlation equation being I / I0 = 0.0122×[HSA] + 1, R 2 = 0.997, and the limit of detection was 2.22 mg / L. Further recovery experiments were conducted at spiking levels of 10, 30, and 40 mg / L, with recoveries ranging from 90.3% to 98.5% and relative standard deviations from 1.7% to 4.9% (Table 2), indicating that this method is suitable for rapid quantitative analysis of HSA in complex samples.

[0060] Table 2. Results of HSA content determination in artificial urine samples using Ru4.

[0061]

[0062] Example 3: A β-lactoglobulin photoswitching probe screening method based on computational virtual screening

[0063] 1. Preparation of candidate coordination library and target protein

[0064] β-LG was selected as the target protein, and the β-LG crystal structure from a protein database was used as the molecular docking acceptor. A virtual compound library of metal polypyridine complexes of the same type as in Example 1 was constructed for subsequent computational screening and experimental verification.

[0065] 2. Molecular docking screening

[0066] The binding affinity between each candidate complex and β-LG was calculated using molecular docking methods, and the complexes were sorted according to their predicted binding energies. Preferably, the PDB number of the β-LG protein structure was 1BSO, the docking grid size was set to 124 × 124 × 124 Å, and the lowest energy conformation was selected as the optimal binding conformation. Figure 6 The figure shows a heatmap of the predicted binding ability of different candidate complexes to β-LG. It can be seen that different auxiliary ligand structures, the number of dppz ligands and their ratios also significantly affect the binding ability of candidate complexes to β-LG.

[0067] 3. Experimental verification

[0068] Candidate metallopolypyridine complexes were prepared into ultrapure water stock solutions of appropriate concentrations and stored in the dark. β-LG was prepared in 5 mM HEPES buffer at pH 7.4. For testing, the candidate complex solution was mixed with the HEPES buffer, and then the β-LG solution was added, adjusting the total volume to 2 mL. The emission spectra were recorded at room temperature. Preferably, the excitation wavelength was 450 nm, and the emission wavelength range was 550-800 nm. Figure 6 b presents the comparison results of the luminescence intensity of the candidate complexes with and without β-LG. Figure 6 c gives the predicted binding energy and corresponding luminescence enhancement factor for each candidate coordination compound.

[0069] 4. Screening Results

[0070] like Figure 6 As shown, different candidate complexes exhibited varying degrees of luminescence enhancement after interaction with β-LG, with the enhancement factor following the order: Ru6 > Ru4 > Ru5 > Ru3 > Ru2. Ru6 showed the highest enhancement factor at 346.1 times, while Ru4, Ru5, Ru3, and Ru2 showed enhancement factors of 174.1 times, 167.5 times, 41.8 times, and 2.6 times, respectively. Therefore, Ru6 can be identified as the preferred optical switching probe for β-LG detection. The results demonstrate that despite the structural differences between β-LG and HSA, the strategy combining computational virtual screening and experimental verification can effectively screen for high-performance optical switching probes targeting β-LG, indicating the good versatility of this strategy.

[0071] Example 4: β-lactoglobulin detection method and sample analysis based on β-lactoglobulin photoswitching probe

[0072] 1. Preparation of β-LG and probe solutions

[0073] The β-LG photoswitching probe screened in Example 3 was prepared in 5 mM HEPES buffer at pH 7.4, with a preferred probe concentration of 1 μM, and stored protected from light. β-LG standards were dissolved in the same buffer to prepare standard solutions of different concentrations, which were further diluted as needed for detection.

[0074] 2. Construction of the luminescence detection system

[0075] Two mL of probe solution was added to a quartz cuvette, followed by the addition of β-LG standard solutions of different concentrations. After thorough mixing, the emission spectrum was measured. Preferably, the excitation wavelength was 450 nm, and the emission wavelength range was 550-800 nm. As the β-LG concentration increased, the probe luminescence gradually increased, exhibiting a visible change from colorless to red under blue light illumination. Figure 7 a presents the emission spectra and color changes at different β-LG concentrations. Figure 7 b gives the corresponding changes in luminescence intensity and the results of its local linear fitting.

[0076] 3. Test performance results

[0077] The results show that the β-LG photoswitching probe exhibits a significant concentration-dependent response to β-LG, with continuously enhanced luminescence in the 0-60 μM range, such as... Figure 7 a and Figure 7 As shown in b, the I / I0 value and the β-LG concentration exhibit a good linear relationship in the range of 0-10 nM, with the linear equation being I / I0 = 0.850 × [β-LG] + 0.745, R0 2 = 0.980, such as Figure 7 As shown in Figure b. The detection limit, calculated using the 3σ / k method, is 0.31 nM, or 0.0057 mg / L. The response time of this probe to β-LG is less than 1 s, as... Figure 7 As shown in Figure c, the method of the present invention has the characteristics of high sensitivity and fast response.

[0078] 4. Selectivity and anti-interference performance

[0079] Cysteine, glycine, glucose, lactose, and potassium + Ca 2+ Mg 2+ Na + H2PO4 - and Cl - Small molecules and ions were added to the detection system separately to investigate their effects on the probe luminescence signal. The results showed that the aforementioned interfering substances caused only negligible changes in luminescence. Further interference experiments were conducted on major coexisting proteins in whey, including α-LA, bIgG, BSA, and LF. Under whey-related ratio conditions, i.e., β-LG / α-LA / bIgG / BSA / LF = 50 / 20 / 10 / 5 / 1 (w / w), the coexisting proteins had minimal impact on the probe signal, and the interfering substances did not significantly affect the β-LG detection signal. Figure 7 The results of the probe’s selectivity and anti-interference against the above-mentioned small molecules, ions and major coexisting proteins in whey are given in d, indicating that the method has good selectivity.

[0080] 5. Analysis of actual samples

[0081] The raw milk sample was centrifuged at 4 ℃ and 14000 rpm for 10 min to remove the fat layer. The pH of the skim milk was then adjusted to 4.6 with acetic acid to precipitate casein. It was then centrifuged again at 4 ℃ and 14000 rpm for 10 min. The supernatant containing whey protein was collected and filtered through a 0.22 μm filter before analysis. The β-LG content in the raw milk was determined to be 3.76 ± 0.21 mg / mL using the luminescence method described in this invention. A high-performance liquid chromatography (HPLC) method was used for verification, yielding a result of 3.60 ± 0.15 mg / mL. The results from the two methods showed good agreement, and the relevant results are listed in Table 3. The HPLC verification was performed using an Athena C4 column, 210 nm UV detection, and a trifluoroacetic acid-acetonitrile gradient elution system. The above results indicate that the method of this invention is suitable for rapid quantitative analysis of β-LG in milk samples.

[0082] Table 3 shows the results of determining the β-LG content of raw milk using the luminescence method and high performance liquid chromatography method of this invention.

[0083]

[0084] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A virtual screening method for protein photoswitches, characterized in that, Includes the following steps: S1: Construct a virtual compound library of metal polypyridine complexes containing different auxiliary ligand structures, dppz ligand numbers, and substituent types; S2: Select the crystal structure of the target protein as the acceptor, calculate the binding energy of each candidate complex in the virtual compound library to the target protein using the molecular docking method, and sort them according to the predicted binding affinity. S3: Select the top-ranked candidate complexes for synthesis and test their luminescence properties in aqueous solution and after binding with the target protein. Screen out protein photoswitching probes that exhibit weak luminescence in aqueous phase and significantly enhanced luminescence after binding with the target protein.

2. The method according to claim 1, characterized in that, The metal polypyridine complex is a ruthenium(II) polypyridine complex, and its general structural formula is: R is H or CH3; X - To counter ion Cl - or Br - x and y are integers between 0 and 3, and x + y ≤ 3; M is Ru, Fe, or Os.

3. The method according to claim 1, characterized in that, The target protein is human serum albumin or β-lactoglobulin.

4. A human serum albumin light-switching probe, characterized in that, The probe, obtained by screening using the method described in any one of claims 1-3, is a ruthenium complex Ru4, with the following structure: [Ru(dppz)2dip] 2+ (Ru4) 。 5. A β-lactoglobulin light-switching probe, characterized in that, The probe, obtained by screening using the method described in any one of claims 1-3, is a ruthenium complex Ru6, with the following structure: [Ru(dip)2dppx] 2+ (Ru6) 。

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