Method for detecting interaction between platinum nano enzyme and serum protein

Abstract

The invention discloses a method for detecting interaction of platinum nano enzyme and serum protein, and belongs to the field of medicine in nano materials. The method comprises the following steps: S1, synthesizing platinum nano-enzyme; s2, performing ultraviolet spectrum detection on interaction of the platinum nano enzyme and serum protein; s3, performing steady-state fluorescence spectrum detection on the interaction of the platinum nano-enzyme and the serum protein; s4, performing synchronous fluorescence spectrum detection on interaction of the platinum nano enzyme and serum protein; and S5, analyzing the detection results in the steps S2-S4. According to the invention, an interaction mechanism between SA and PtNZs is successfully disclosed through a spectrum technology, and thermodynamic data of interaction is obtained. The thermodynamic data obtained by the invention proves that the binding process is carried out spontaneously and the hydrophobic action is the main driving force, which indicates that the stability of the PtNZs in the blood is higher, the PtNZs is firmly bound with the protein, and a reference basis is provided for optimizing the biocompatibility and targeted medication of the nano-drug.

Description

Technical Field

[0001] The present invention relates to the field of medicine in nanomaterials, and more particularly to a method for detecting the interaction between platinum nanozymes and serum proteins. Background Art

[0002] Inflammation is a complex process in the body's response to pathogen infection or metabolic disorders, involving the recruitment and activation of components of the immune system. Repeated noxious stimuli or uncontrolled immune effector mechanisms can lead to tissue damage. Reactive oxygen species (ROS) play a key role in physiological cell signaling and the destruction of internalized pathogens. However, abnormal production and release of ROS have harmful effects on the surrounding environment, making ROS regulation a priority for reducing inflammation. Most current anti-inflammatory therapies rely on drugs that impair the release of pro-inflammatory mediators and increase enzyme activity to reduce ROS levels.

[0003] As a novel type of mimetic enzyme, nanozymes have the dual identity of both enzymes and nanomaterials. Compared with natural enzymes, nanozymes have higher stability, lower cost, and adjustable catalytic activity, and thus are widely used in the treatment of diseases. For example, they can be important substitutes for natural enzymes to treat pathological conditions characterized by ROS-mediated inflammation.

[0004] However, compared with natural enzymes, nanozymes have poor biosafety. Nanozymes have great potential in disease treatment. However, different from traditional enzymes, nanozymes are mostly inorganic nanoparticles that can enter the lysosomes, mitochondria, or nuclei of cells and may cause damage to cells through irreversible interactions with intracellular components. Taking metal-based nanozymes as an example, although many studies have demonstrated the cytoprotective effect and biocompatibility of nanozymes, metal ion release is still considered a factor that acts on normal tissues due to metal overload.

[0005] The mechanism of action of nanozymes in the inflammatory process remains unknown. Currently, the research on nanozymes mostly focuses on the optimization of catalytic activity, while less attention is paid to the analysis of catalytic mechanisms. Summary of the Invention

[0006] Based on the above technical problems, the present invention proposes a method for detecting the interaction between platinum nanozymes and serum proteins.

[0007] The technical solution adopted by the present invention is as follows:

[0008] A method for detecting the interaction between platinum nanozymes and serum proteins, comprising the following steps:

[0009] S1. Synthesize platinum nanozymes;

[0010] S2. Perform ultraviolet spectrum detection on the interaction between platinum nanozymes and serum proteins;

[0011] The UV-visible absorption spectrum was measured using a spectrophotometer; ultrapure water was used as the reference solution, the optical path of the quartz cuvette was 1.0 cm, and the scanning range was 200 - 900 nm; 1 ml of 8×10 -6 mol·L -1 SA was added to the experimental group, and the PtNZs solution was gradually added in the concentration range of 0 - 3.20×10 - 8 mol·L -1 , and the insufficient part was made up with ultrapure water; in the blank control group, the PtNZs solution in the above steps was replaced with ultrapure water;

[0012] S3. Steady-state fluorescence spectroscopy was used to detect the interaction between platinum nanozyme and serum protein;

[0013] The fluorescence spectrum was measured using an F-4600 fluorescence spectrophotometer and a 1.0 cm optical path quartz cell; the concentration of HSA / BSA was fixed at 1.6×10 -6 mol·L -1 , and the concentration of PtNZs was gradually increased in the concentration range of 0 - 3.2×10 -9 mol·L -1 for the steady-state fluorescence experiment; the excitation wavelength was set at 280 nm, and the emission spectrum was recorded in the range of 300 nm to 500 nm; both the emission and excitation slits were set at 2.5 nm; the fluorescence intensities of the mixed solutions of PtNZs and HSA / BSA were measured successively at the temperatures of 293.15 K, 298.15 K, and 303.15 K;

[0014] S4. Synchronous fluorescence spectroscopy was used to detect the interaction between platinum nanozyme and serum protein;

[0015] The synchronous fluorescence spectrum of the mixed solution of PtNZs and HSA / BSA was measured using an RF-5301PC fluorescence spectrophotometer; the concentration of HSA / BSA was fixed at 6.0×10 -5 mol·L -1 , and the concentration of PtNZs was increased; Δλ = 60 nm, λ ex = 240 nm, λ em = 300 nm - 400 nm; Δλ = 15 nm, λ ex = 265 nm, λ em = 280 nm - 340 nm, and both the excitation and emission slits were set at 10 nm;

[0016] S5. Analyze the detection results in steps S2 - S4.

[0017] Preferably, in step S1: First, 3.6 mL of a 2% chloroplatinic acid hexahydrate solution was added to 496.4 mL of boiling deionized water. After reacting for 1 minute, 11 mL of a mixed solution containing 1% sodium citrate and 0.05% citric acid was added to the system. After 30 seconds, 5.5 mL of a freshly prepared reducing agent solution containing 1% sodium citrate, 0.05% citric acid, and 0.08% sodium borohydride was quickly injected. After the reaction continued for 10 minutes, the reaction system was cooled to room temperature to obtain a platinum nanozyme product.

[0018] Preferably, the detection and analysis results in step S2 are as follows: The absorption intensities of HSA and BSA increase with the increase in the concentration of PtNZs. The absorbance change rates of BSA and HSA are different. At the same concentration, the growth rate of BSA is higher than that of HSA.

[0019] Preferably, the detection and analysis results in step S3 are as follows: With the increase in PtNZs, the maximum fluorescence intensities of HSA and BSA gradually decrease, indicating that PtNZs can cause fluorescence quenching with SA; the affinity of HSA for PtNZs is weaker than that of BSA; and the ΔH value, ΔS value, and ΔG value of the HSA-PtNZs system and the BSA-PtNZs system are calculated; ΔG is negative, indicating that the binding processes of the HSA-PtNZs system and the BSA-PtNZs system are both spontaneous; both ΔH and ΔS are positive, indicating that the binding of SA to PtNZs is driven by hydrophobic bonds.

[0020] Preferably, the detection and analysis results in step S4 are as follows: Synchronous fluorescence experiments are used to study the changes in the molecular microenvironment around chromophore molecules, and the spectra provide characteristic information of tyrosine residues or tryptophan residues; the quenching effect of tyrosine residues on the fluorescence intensity of HSA is weaker than that of tryptophan residues; after adding PtNZs, the maximum fluorescence emission of tyrosine residues undergoes a slight red shift, indicating that the conformations of HSA and BSA have changed.

[0021] The beneficial technical effects of the present invention are as follows:

[0022] (1) The present invention successfully reveals the interaction mechanism between SA and PtNZs through spectroscopic techniques and obtains the thermodynamic data of the interaction. After PtNZs are applied to the human body, they will interact with biological macromolecules and have unknown effects on biological macromolecules. Serum albumin is an important carrier for drug delivery. The thermodynamic data obtained in the present invention prove that the binding process is spontaneous and hydrophobic interaction is the main driving force, indicating that PtNZs have high stability in the blood and bind firmly to proteins, providing a reference basis for optimizing the biocompatibility and targeted drug use of nano-drugs.

[0023] (2) The interaction between PtNZs and serum albumin directly affects their biocompatibility and toxicity. The thermodynamic data obtained in this invention can systematically evaluate potential risks and avoid the destruction of albumin function or immunogenicity caused by the application of PtNZs to the human body.

[0024] (3) In this invention, green reagents citric acid and sodium citrate are used to synthesize PtNZs with high biocompatibility, reducing the toxicity of PtNZs synthesized by thiol chemistry in the past.

[0025] (4) The interaction mechanism between SA and PtNZs has been successfully revealed by spectroscopic techniques in this invention. With the increase in the concentration of PtNZs, the absorption spectrum of SA is significantly enhanced. The fluorescence quenching experiment confirms that the fluorescence quenching of SA by PtNZs is a static quenching. K a and △G values further indicate that the binding process is spontaneous. Both △H and △S values are positive, which means that the binding between SA and PtNZs is mainly driven by hydrophobic bonds. The results of this detection and analysis contribute to a deeper understanding of the unusual interaction between SA and PtNZs. The obtained thermodynamic data provide a basic theoretical reference and a reliable research method for the application of PtNZs to the human body as an anti-inflammatory drug. Description of the Drawings

[0026] Figure 1 It is a characterization diagram of platinum nanozymes (PtNZs), where (A) is the Fourier transform infrared spectrum of PtNZs, (B) is the XRD spectrum of PtNZs, (C) is the TEM image of PtNZs, and (D) is the size distribution of PtNZs;

[0027] Figure 2 It is a visible spectrum diagram, where (A) is the ultraviolet-visible spectrum diagram of the interaction between PtNZs and HAS; (B) is the ultraviolet-visible spectrum diagram of the interaction between PtNZs and BSA;

[0028] Figure 3 It shows the emission spectra under the action of different concentrations of PtNZs, where (A) shows the emission spectra of HSA under the action of different concentrations of PtNZs, and (B) shows the emission spectra of BSA under the action of different concentrations of PtNZs;

[0029] Figure 4 It is a Stern-Volmer diagram of the binding of HSA and BSA with PtNZs at three different temperatures; among them, (A) shows the Stern-Volmer relationship of the binding of HSA with PtNZs, and (B) shows the Stern-Volmer relationship of the binding of BSA with PtNZs;

[0030] Figure 5The Hill plots of HSA and BSA with PtNZs at three different temperatures; among them, (A) is the Hill plot of HSA with PtNZs, and (B) is the Hill plot of BSA with PtNZs;

[0031] Figure 6 The Van't Hoff plots of the SA-PtNZs system and the BSA-PtNZs system; among them, (A) is the Van't Hoff plot of the HSA-PtNZs system, and (B) is the Van't Hoff plot of the BSA-PtNZs system;

[0032] Figure 7 The fluorescence spectra of tyrosine residues (Tyr) and tryptophan residues (Trp) of HSA / BSA at Δλ of 15 nm and 60 nm, respectively. Among them, (A) shows the fluorescence spectrum of HSA-PtNZs at Δλ of 15 nm, (B) shows the fluorescence spectrum of HSA-PtNZs at Δλ of 60 nm, (C) shows the fluorescence spectrum of BSA-PtNZs at Δλ of 15 nm, and (D) shows the fluorescence spectrum of BSA-PtNZs at Δλ of 60 nm. Detailed implementation manners

[0033] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation manners.

[0034] All chemicals used in the present invention are of analytical purity grade and can be used without further purification. Chloroplatinic acid hexahydrate (H2PtCl6·6H2O), sodium citrate dihydrate (Na3C6H5O7·2H2O), and sodium borohydride (NaBH4, purity > 98%) are all purchased from Alfa Aesar (China) Chemical Co., Ltd. Citric acid (C6H8O7.H2O), flufenamic acid (FA, purity > 98%), and phenylbutazone (PB, purity > 98%) are purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. and Shanghai Aladdin Biochemical Technology Co., Ltd., respectively. Bovine serum albumin (BSA, purity > 98%, M w ≈66.0 kDa) and human serum albumin (HSA, purity > 96%, M w ≈66.5 kDa) are purchased from Beijing J&K Scientific Ltd. In the spectroscopic experiments, 10.0 mM Tris-HCl buffer solution (pH 7.40) is used as the solvent for preparation. Ultra-pure water is used throughout the experiment.

[0035] A detection method for the interaction between platinum nanozyme and serum protein, comprising the following steps:

[0036] S1. Synthesize platinum nanozyme;

[0037] First, 3.6 mL of a 2% solution of hexahydrate chloroplatinic acid (H2PtCl6·6H2O) was slowly added to 496.4 mL of boiling deionized water. After reacting for 1 minute, 11 mL of a mixed solution containing 1% sodium citrate and 0.05% citric acid was added to the system. After 30 seconds, 5.5 mL of a freshly prepared reducing agent solution containing 1% sodium citrate, 0.05% citric acid, and 0.08% sodium borohydride was rapidly injected. After the reaction continued for 10 minutes, the reaction system was cooled to room temperature to obtain the platinum nanozyme product.

[0038] Figure 1 Figures for platinum nanozymes (PtNZs), where (A) is the Fourier transform infrared spectrum of PtNZs, (B) is the XRD spectrum of PtNZs, (C) is the TEM image of PtNZs, and (D) is the size distribution of PtNZs. As Figure 1 shown in (A) therein, the Fourier transform infrared spectrum (FT-IR) analyzed the successfully synthesized platinum nanozymes (PtNZs) in the wavelength range of 4000 cm -1 to 500 cm -1 The absorption peaks appearing at 3512 cm -1 and 1629 cm -1 correspond to the O-H group and the C═C bond, respectively. The peaks at 1403 cm -1 and 1384 cm -1 are the C-H plane bending and C-H bending vibrations, respectively. The spectrum also detected other functional groups, including the C-O-H deformation at 1385 cm -1 and the C-O stretching vibration at 1085 cm -1 . Finally, the peak found at 668 cm -1 indicated the presence of the C-Cl (chloroethylene) group. Since the metal nanozyme was successfully synthesized under sodium citrate conditions, all the above functional groups were present. X-ray diffraction is an important research tool for determining the crystal structure of the prepared NPs. Using the X-ray diffraction pattern, as Figure 1 shown in (B) therein, the crystal properties of PtNZs were calculated. The diffraction peaks found at 45.1°, 55.8°, 67.2°, and 76.1° belong to platinum (111), platinum (200), platinum (220), and platinum (311), respectively, confirming the face-centered cubic (fcc) phase of platinum. Therefore, the XRD results indicated that PtNZs were successfully synthesized using a sodium citrate solution. A typical TEM image obtained using a JEM-2100F transmission electron microscope (TEM, JEOL Ltd., Japan) is shown in Figure 1 shown in (C) therein. The average diameter of PtNZs was calculated using particle size analysis software, and the result was approximately 5.1 nm.

[0039] S2. Perform ultraviolet spectroscopy detection on the interaction between platinum nanozyme and serum protein, and analyze the detection results;

[0040] The study used a UV-2501PC spectrophotometer (Shimazdu, Japan) to measure ultraviolet-visible absorption (UV-vis) spectra. Ultra-pure water was used as the reference solution. The optical path of the quartz cuvette was 1.0 cm. The scanning range was 200 - 900 nm. The study was divided into a blank control group and an experimental group. In the experimental group, 1 ml of 8×10 -6 mol·L -1 HAS was added, and PtNZs solution was gradually added within the concentration range of 0 - 3.20×10 -8 mol·L -1 , ensuring that the system was 3 ml. The insufficient part was made up with ultra-pure water. In the blank control group, the PtNZs solution in the above steps was replaced with ultra-pure water, and the concentration and volume of SA remained unchanged, ensuring that the SA-H2O system was 3 ml.

[0041] Ultraviolet-visible spectroscopy is an effective technique for determining the structural changes of protein molecules and the formation of complexes between drugs / nanoparticles and proteins. When proteins are adsorbed onto the surface of nanoparticles, the absorbance of the proteins will change, resulting in hyperchromic and hypochromic effects. With the SA concentration remaining constant, PtNZs was continuously added to SA and the spectra were recorded. Figure 2 It shows that the absorption intensities of HSA and BSA increase with the increase in PtNZs concentration. The absorption intensity increases sharply, and hyperchromism appears at 278 nm, indicating that PtNZs has a strong adsorption ability for SA at a constant concentration under electrostatic attraction and hydrogen bonding. Figure 2 In [ ], [HSA] = 8.0×10 -6 mol·L -1 , [BSA] = 8.0×10 -6 mol·L -1 ; from (a) to (g), the concentrations of PtNZs were 0.00, 5.33×10 -9 , 1.07×10 -8 , 1.60×10 -8 , 2.13×10 -8 , 2.67×10 -8 , 3.20×10 - 8 mol·L -1 . The absorbance change rates of BSA and HSA are different. At the same concentration, the growth rate of BSA is slightly higher than that of HSA. The reason may be that BSA has two tryptophan residues (Try 134 and Try 213), while HSA only contains one conserved tryptophan residue (Try 214) at position 214.

[0042] S3. Perform steady-state fluorescence spectroscopy detection on the interaction between platinum nanozyme and serum protein;

[0043] Use an F-4600 fluorescence spectrophotometer (Hitachi, Japan) and a 1.0 cm optical path quartz cell to measure the fluorescence spectrum. Fix the concentration of HSA / BSA at 1.6×10 -6 mol·L -1 , and gradually increase the concentration of PtNZs in the concentration range of 0 - 3.2×10 -9 mol·L -1 to perform steady-state fluorescence experiments. Set the excitation wavelength at 280 nm and record the emission spectrum in the range of 300 nm to 500 nm. Both the emission and excitation slits are set at 2.5 nm. At temperatures of 293.15 K, 298.15 K, and 303.15 K, the fluorescence intensities of the mixed solutions of PtNZs and HSA / BSA were measured in sequence.

[0044] Figure 3 The emission spectra under the action of different concentrations of PtNZs are shown, where (A) shows the emission spectra of HSA under the action of different concentrations of PtNZs, and (B) shows the emission spectra of BSA under the action of different concentrations of PtNZs. The concentrations of HSA and BSA are both fixed at 1.6×10 -6 mol·L -1 . The concentrations of PtNZs are 0.00, 5.33×10 -10 , 1.07×10 -9 , 1.60×10 -9 , 2.13×10 -9 , 2.67×10 -9 , 3.20×10 -9 mol·L -1 .

[0045] Figure 3 In (A) and (B), the quenching effects of PtNZs on HSA and BSA in 10 mM Tris-HCl buffer are respectively shown. Due to the presence of tryptophan and tyrosine residues, HSA and BSA will emit strong fluorescence at a wavelength of 343 nm when excited at a wavelength of 280 nm. As the concentration of PtNZs increases, the maximum fluorescence intensities of HSA and BSA gradually decrease, indicating that PtNZs can cause fluorescence quenching with SA. Fluorescence quenching is due to the formation of a supramolecular structure after HSA and BSA bind to the surface of PtNZs.

[0046] Fluorescence quenching experiments can be used to observe the changes in fluorescence intensity after proteins are adsorbed onto the surface of NPs. Generally, according to the interaction mode between proteins and quenchers, there are two mechanisms of fluorescence quenching: dynamic and static. These two quenching behaviors can be distinguished by the influence of temperature. The fluorescence spectra of SA were recorded at three different temperatures (298.15, 303.15, and 308.15 K) in the absence and presence of different concentrations of PtNZs as quenchers. As can be seen from the figure, with the increase in the concentration of PtNZs, the fluorescence intensities of HSA and BSA both decreased regularly, and the emission wavelength did not change.

[0047] The fluorescence quenching data at different temperatures were analyzed using the Stern-Volmer equation to study the quenching mechanism of the binding of HSA and BSA to PtNZs.

[0048] F0 / F = 1 + K sv [Q] = 1 + K q τ0[Q] (1)

[0049] where F0 and F represent the fluorescence intensities of SA in the absence and presence of PtNZs, respectively. K sv is the Stern-Volmer quenching constant, [Q] is the concentration of the quencher, K q is the biomolecular quenching constant, and τ0 is the average fluorescence lifetime of the excited biomolecule in the absence of the quencher. τ0 is approximately 5.78×10 -9 s and 1.0×10 -8 s for HSA and BSA, respectively. Figure 4 Figure shows the Stern-Volmer plots for the binding of HSA and BSA to PtNZs at three different temperatures. Among them, (A) and (B) show the Stern-Volmer relationships for the binding of HSA and BSA to PtNZs, respectively. The values of K sv and K q can be obtained from these figures and are listed in Tables 1 and 2. The K q values of HSA and BSA at different temperatures are all higher than the maximum diffusion collision constant (2.0×10 10 L·mol -1 ·s -1 ). The quenching mechanism of the binding of HSA / BSA to PtNZs is static quenching caused by specific interactions between them. That is, fluorescence quenching is due to the formation of a complex rather than dynamic collision. In addition, the Ksv value gradually decreases with the increase in temperature, which also indicates that the quenching mechanism is static quenching.

[0050] The Stern-Volmer quenching constants for the binding of HSA to PtNZs at different temperatures are shown in Table 1.

[0051] Table 1

[0052]

[0053] The Stern-Volmer quenching constants for the binding of BSA to PtNZs at different temperatures are shown in Table 2 below.

[0054] Table 2

[0055]

[0056] The binding mechanism between SA and PtNZs is analyzed as follows:

[0057] When the quenching mechanism is static quenching, the interaction system conforms to the Hill equation (Equation 2), as shown below:

[0058] log[(F0 - F) / F] = logK a + nlog[Q] (2)

[0059] where K a is the binding constant and n is the Hill coefficient, representing the degree of cooperativity of the binding of SA to the surface of PtNZs. The values of K a and n are obtained from the linear fitting plot of log[(F0 - F) / F] vs log[Q] (see Figure 5 ). Tables 5 and 6 summarize these values. At the same temperature, the K a value for the binding of HSA to PtNZs is less than the K a value for the binding of BSA to PtNZs. These results indicate that the affinity of HSA for PtNZs is relatively weaker than that of BSA. This phenomenon may be due to the fact that BSA has two tryptophan molecules (Trp 135 and Trp 214) located in subdomains IA and IIA, while there is only one tryptophan residue (Trp 214) in the structure of HSA located in subdomain IIA [5, 13 - 15].

[0060] In addition, the n value for the binding of BSA to PtNZs is greater than 1, indicating that if other ligands have been adsorbed on the NP surface, the binding force of the ligand will be enhanced. However, the n value for the binding of HSA to PtNZs is less than 1, indicating a weakened binding force of the ligand. This conclusion is consistent with the K a value.

[0061] The binding constant (K a ) and Hill coefficient (n) for the binding of HSA to PtNZs are shown in Table 3 below.

[0062] Table 3

[0063]

[0064] The binding constant (K a ) and Hill coefficient (n) of BSA and PtNZs are shown in Table 4 below.

[0065] Table 4

[0066]

[0067] The driving force analysis of the binding process is as follows:

[0068] The driving forces for the binding of biomolecules to NPs mainly include hydrogen bonding, electrostatic interactions, van der Waals interactions, etc. Thermodynamic parameters, such as enthalpy change (ΔH) and entropy change (ΔS), can determine the type of interaction force. From a thermodynamic perspective, ΔH>0 and ΔS>0 mean that hydrophobic interaction is the main force; ΔH<0 and ΔS<0 indicate that van der Waals force or hydrogen bonding plays a major role.

[0069] ΔH<0 and ΔS>0 indicate that electrostatic force plays a major role. When the temperature does not change significantly, the values of ΔH and ΔS can be obtained according to the van't Hoff equation (Equation 3). ΔH and ΔS are calculated based on the slope and the intercept at the origin of the fitting line ( Figure 6 ).

[0070]

[0071] where K a is the binding constant at the corresponding temperature, R is the gas constant 8.314 J·mol -1 ·K -1 , and T is the absolute temperature. The change in Gibbs free energy (ΔG) is estimated by the following formula (Equation 4):

[0072] ΔG = ΔH - TΔS (4)

[0073] Table 6 and Table 7 summarize the ΔH values, ΔS values, and ΔG values of the HSA-PtNZs system and the BSA-PtNZs system, respectively. The negative value of ΔG indicates that the binding processes of both the HSA-PtNZs system and the BSA-PtNZs system are spontaneous. Positive values of both enthalpy and entropy generally represent hydrophobic interactions.

[0074] The thermodynamic parameters of HSA and PtNZs at different temperatures are shown in Table 5 below.

[0075] Table 5

[0076]

[0077] The thermodynamic parameters of BSA and PtNZs at different temperatures are shown in Table 6 below.

[0078] Table 6

[0079]

[0080] S4. Synchronously fluorescence spectroscopy was used to detect the interaction between platinum nanozyme and serum proteins;

[0081] The synchronous fluorescence spectra of PtNZs and HSA / BSA mixed solutions were measured using an RF-5301PC (Shimazdu, Japan) fluorescence spectrophotometer. The concentration of HSA / BSA was fixed at 6.0×10 -5 mol·L -1 , and the concentration of PtNZs was increased. Δλ = 60 nm, λ ex = 240 nm, λ em = 300 nm - 400 nm; Δλ = 15 nm, λ ex = 265 nm, λ em＝ 280 nm - 340 nm, and both the excitation and emission slits were set to 10 nm.

[0082] Synchronous fluorescence experiments were used to study the changes in the molecular microenvironment around chromophore molecules, and the spectra can provide characteristic information of tyrosine residues or tryptophan residues. Figure 7 The fluorescence spectra of tyrosine residues (Tyr) and tryptophan residues (Trp) of HSA / BSA at Δλ of 15 nm and 60 nm respectively are shown. In the figure, the concentrations of HSA and BSA were both fixed at 2.00×10 -5 mol·L-1. The concentrations of PtNZs in (a) to (h) were 0.00, 2.67×10 -9 , 5.33×10 -9 , 8.00×10 -9 , 1.07×10 -8 , 1.33×10 -8 , 1.60×10 -8 , 1.87×10 -8 mol·L -1 .

[0083] Among them, (A) and (B) show that the quenching effect of Tyr on the fluorescence intensity of HSA is weaker than that of Trp. This indicates that Trp residues contribute greatly to the intrinsic fluorescence of HSA. Similarly, the same result was obtained in BSA molecules. At the same time, the fluorescence intensities of HSA and BSA at Δλ = 15 nm and Δλ = 60 nm decreased regularly in the presence of PtNZs. (A) and (C) show that after adding PtNZs, a slight red shift occurred in the maximum fluorescence emission of Tyr residues, indicating that the conformations of HSA and BSA changed.

[0084] The present invention has successfully revealed the interaction mechanism between SA and PtNZs through spectroscopic techniques and obtained the thermodynamic data of the interaction. With the increase in the concentration of PtNZs, the absorption spectrum of SA is significantly enhanced. The fluorescence quenching experiment confirmed that the fluorescence quenching of SA by PtNZs is a static quenching. K a and △G values further indicate that the binding process is spontaneous. The values of △H and △S are both positive, which means that the binding of SA and PtNZs is mainly driven by hydrophobic bonds. This finding helps to deeply understand the unusual interaction between SA and PtNZs. The obtained thermodynamic data provides a basic theoretical reference and a reliable research method for the application of PtNZs in the human body as an anti-inflammatory drug.

[0085] After PtNZs are applied to the human body, they will interact with biomacromolecules and have unknown effects on biomacromolecules. Serum albumin is an important carrier for drug delivery. The thermodynamic data obtained in the present invention proves that the binding process is spontaneous and the hydrophobic interaction is the main driving force, which indicates that PtNZs have high stability in the blood and bind firmly to proteins, providing a reference for optimizing the biocompatibility and targeted drug use of nanodrugs.

[0086] Moreover, the interaction between PtNZs and serum albumin directly affects their biocompatibility and toxicity. The thermodynamic data can systematically evaluate potential risks and avoid damage to albumin function or immunogenicity caused by the application of PtNZs to the human body.

[0087] The parts not described in the above manner can be achieved by adopting or referring to the existing technologies.

[0088] The above-described embodiments are only used to describe the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A detection method for the interaction between platinum nanozyme and serum protein, characterized in that Including the following steps: S1. Synthesize platinum nanozyme; S2. Conduct ultraviolet spectrum detection on the interaction between platinum nanozyme and serum protein; The ultraviolet-visible absorption spectrum was measured using a spectrophotometer; ultrapure water was used as the reference solution, the optical path of the quartz cuvette was 1.0 cm, and the scanning range was 200 - 900 nm; 1 ml of 8×10 -6 mol·L -1 SA was added to the experimental group, and the PtNZs solution was gradually added within the concentration range of 0 - 3.20×10 -8 mol·L -1 , and the insufficient part was made up with ultrapure water; in the blank control group, the PtNZs solution in the above steps was replaced with ultrapure water; S3. Conduct steady-state fluorescence spectrum detection on the interaction between platinum nanozyme and serum protein; The fluorescence spectra were measured using an F-4600 fluorescence spectrophotometer and a 1.0 cm optical path quartz cell; the concentrations of HSA / BSA were fixed at 1.6×10 -6 mol·L -1 , and steady-state fluorescence experiments were carried out by gradually increasing the concentration of PtNZs in the concentration range of 0 - 3.2×10 -9 mol·L -1 ; the excitation wavelength was set at 280 nm, and the emission spectra were recorded in the range of 300 nm to 500 nm; both the emission and excitation slits were set at 2.5 nm; the fluorescence intensities of the mixed solutions of PtNZs and HSA / BSA were measured successively at the temperatures of 293.15 K, 298.15 K, and 303.15 K; S4. Conduct synchronous fluorescence spectrum detection on the interaction between platinum nanozyme and serum protein; The synchronous fluorescence spectra of PtNZs and HSA / BSA mixed solutions were measured using an RF-5301PC fluorescence spectrophotometer; the concentration of HSA / BSA was fixed at 6.0×10 -5 mol·L -1 , and the concentration of PtNZs was increased; Δλ = 60 nm, λ ex = 240 nm, λ em = 300 nm - 400 nm; Δλ = 15 nm, λ ex = 265 nm, λ em = 280 nm - 340 nm, and both the excitation and emission slits were set to 10 nm; S5. Analyze the detection results in steps S2 - S4.

2. The detection method for the interaction between platinum nanozyme and serum protein according to claim 1, wherein In step S1: First, add 3.6 mL of 2% chloroplatinic acid hexahydrate solution to 496.4 mL of boiling deionized water. After reacting for 1 minute, add 11 mL of a mixed solution containing 1% sodium citrate and 0.05% citric acid to the system. After 30 seconds, quickly inject 5.5 mL of freshly prepared reducing agent solution containing 1% sodium citrate, 0.05% citric acid, and 0.08% sodium borohydride. After the reaction continues for 10 minutes, cool the reaction system to room temperature to obtain the platinum nanozyme product.

3. The detection method for the interaction between platinum nanozyme and serum protein according to claim 1, wherein The detection and analysis results in step S2 are as follows: The absorption intensities of HSA and BSA increase with the increase in the concentration of PtNZs. The absorbance change rates of BSA and HSA are different. At the same concentration, the growth rate of BSA is higher than that of HSA.

4. The detection method for the interaction between platinum nanozyme and serum protein according to claim 1, characterized in that The detection and analysis results in step S3 are as follows: With the increase in PtNZs, the maximum fluorescence intensities of HSA and BSA gradually decrease, indicating that PtNZs can cause fluorescence quenching with SA; the affinity between HSA and PtNZs is weaker than that between BSA and PtNZs; And calculate the ΔH value, ΔS value, and ΔG value of the HSA - PtNZs system and the BSA - PtNZs system; ΔG is negative, indicating that the binding processes of both the HSA - PtNZs system and the BSA - PtNZs system are spontaneous; both ΔH and ΔS are positive, indicating that the binding of SA and PtNZs is driven by hydrophobic bonds.

5. The detection method for the interaction between platinum nanozyme and serum protein according to claim 1, wherein The detection and analysis results in step S4 are as follows: Synchronous fluorescence experiments are used to study the changes in the molecular microenvironment around chromophore molecules, and the spectrum provides characteristic information of tyrosine residues or tryptophan residues; the quenching effect of tyrosine residues on the fluorescence intensity of HSA is weaker than that of tryptophan residues; after adding PtNZs, the maximum fluorescence emission of tyrosine residues shows a slight red shift, indicating that the conformations of HSA and BSA have changed.

Images