Supramolecular fluorescent probe for rapid detection of gossypol as well as preparation method and application of supramolecular fluorescent probe

Abstract

The invention belongs to the field of fluorescent probes, discloses a supramolecular fluorescent probe for rapid detection of gossypol as well as a preparation method and application of the supramolecular fluorescent probe, and solves the technical problems of low gossypol content detection accuracy, long response time and environmental pollution easily caused by use of an organic solvent or strong acid in an existing technology for detecting gossypol by a fluorescence method. The supramolecular fluorescent probe for gossypol detection is formed by combining human serum albumin and a probe P2, the structural formula of the probe P2 is as follows: # imgabs0 # P2 and HSA are combined to form a supramolecular compound, and rapid detection of gossypol concentration in a water phase is realized through self object-object interaction of supramolecules. The fluorescence intensity of P2 at HSA and the gossypol concentration (0-8 [mu] M) have a good linear relationship, R2 is equal to 0.9912, the detection limit is 0.0137 [mu] M, and a precious thought and a new strategy are provided for subsequent development of gossypol response type fluorescent probes.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent probes, and particularly to a supramolecular fluorescent probe. Background Art

[0002] Gossypol is a yellow polyphenolic substance with cumulative toxicity produced by the seeds of Malvaceae plants such as cotton to resist pests and diseases. At present, due to the limitations of the processing and utilization technology of cotton seeds, there will be a small amount of gossypol residue in cotton by-products such as cottonseed meal and cottonseed oil obtained from cottonseed processing. And cottonseed oil is a commonly used oil for making fried foods in industry, and cottonseed meal is a high-quality animal feed, which is widely used in the feeding of livestock such as cattle and sheep. Therefore, people may cause secondary accumulation of gossypol, a toxic substance, in the body by consuming animal products and fried products containing gossypol residue. Although gossypol has extensive potential applications in medicine and agriculture and has received wide attention as a possible anti-tumor drug, it has strong toxic effects on the human body and may cause problems such as swelling, bleeding, and nerve abnormalities. In severe cases, it may even inhibit the production of human sperm, resulting in infertility. Therefore, the problem of gossypol pollution has attracted much attention.

[0003] There are many current methods for detecting gossypol. For example, high performance liquid chromatography, gas chromatography - mass spectrometry, enzyme - linked immunosorbent assay, spectrophotometry or test strip detection method. However, the above - mentioned testing methods all have many disadvantages that limit their application in the detection of gossypol in food. High performance liquid chromatography and gas chromatography - mass spectrometry have high sensitivity and good separation effect, but the instruments are expensive, the operation is complex, and the time consumption is long, which is difficult to meet the needs of on - site rapid detection; enzyme - linked immunosorbent assay, this method is simple to operate and low in cost, but the sensitivity is relatively low and it is easily interfered by the matrix. The test strip detection method, such as the one disclosed in CN101598716A which discloses a rapid detection test strip for gossypol and its preparation method and application, its core reagents include a color developer such as ferric acetylacetonate or ferric chloride or ferric sulfate, a mixed acid solution of hydrochloric acid and glacial acetic acid, a complexing agent o - phenanthroline and a solid - loading agent. Although this method can achieve the rapid detection of gossypol, through the method of comparing with a colorimetric card, the sensitivity is low, it is difficult to detect trace gossypol, and it can only perform semi - quantitative or qualitative detection, and it is difficult to accurately determine the content of gossypol.

[0004] Molecular fluorescence probes have the characteristics of high sensitivity, high selectivity, and easy popularization and use, and are widely used in the development of rapid detection methods for food safety. Based on the special chemical reactions of analytes, by combining fluorophores and active reaction sites, specific molecular fluorescence probes can be constructed. However, due to the lack of effective reaction sites in the gossypol molecule, it is difficult to occur rapid reactions at normal temperature, normal pressure, and in aqueous solutions, so it is difficult to construct rapid and effective fluorescence probes. In addition, fluorescence-enhanced probes have the characteristics of high sensitivity and strong selectivity, and are particularly suitable for the establishment of rapid detection methods for food safety. In contrast, fluorescence quenching probes, that is, methods for detecting analytes based on the quenching of fluorescence signals, have low detection sensitivity and are easily interfered by substances in the matrix. Especially when detecting analytes containing various colored components such as cottonseed oil, cottonseed meal, and meat. So far, there has been no reported rapid detection method for gossypol based on molecular fluorescence probes. Developing a fluorescence-enhanced rapid detection method for gossypol in aqueous solution is beneficial to the monitoring of gossypol, reducing the harm caused by gossypol, and helping to solve problems such as long detection time, use of organic solvents or strong acids, and easy environmental pollution in existing methods. Summary of the Invention

[0005] In view of the above technical problems, the present invention provides a supramolecular fluorescence probe for rapid detection of gossypol, its preparation method and application.

[0006] To achieve the above object, the technical solution of the present invention is realized as follows:

[0007] A supramolecular fluorescence probe (P2@HSA) for rapid detection of gossypol, which is composed of human serum albumin (HSA) and probe P2. The structural formula of probe P2 is as follows:

[0008]

[0009] Furthermore, the preparation method of the above probe P2 is as follows:

[0010] (1) React 1,3-indanedione and malondialdehyde diphenylamine hydrochloride in acetic anhydride, separate the solid product by vacuum filtration, and wash the upper solid with a small amount of acetic anhydride, a large amount of deionized water, and an appropriate amount of isopropanol in sequence. Then place the upper solid in a vacuum drying oven for drying, and finally obtain a bright yellow powdery intermediate 2;

[0011] (2) Intermediate 2 undergoes an addition reaction with 1,2,3,3-tetramethyl-3H-indole iodide in ethanol to obtain the fluorescence probe P2.

[0012] In the above step (1), the molar ratio of 1,3-indanedione to malondialdehyde diphenylamine hydrochloride is 1:1. The reaction means reacting first at 140 - 160 °C for 15 - 30 min, and then reacting at room temperature for 24 h. For example, the reaction can be carried out by reacting at 140 - 150 °C for 20 - 30 min and then reacting at room temperature for 24 h; the reaction can be carried out by reacting at 150 - 160 °C for 15 - 20 min and then reacting at room temperature for 24 h; the reaction can be carried out by reacting at 140 °C for 30 min and then reacting at room temperature for 24 h; the reaction can be carried out by reacting at 160 °C for 15 min and then reacting at room temperature for 24 h; the reaction can be carried out by reacting first at 150 °C for 20 min and then reacting at room temperature for 24 h, and so on.

[0013] In the above step (2), the molar ratio of intermediate 2 to 1,2,3,3-tetramethyl-3H-indole iodide is 1:1. The addition reaction means heating under reflux at 85 - 95 °C for 25 - 40 min. For example, the addition reaction means heating under reflux at 85 - 90 °C for 30 - 40 min; the addition reaction means heating under reflux at 90 - 95 °C for 25 - 30 min; the addition reaction means heating under reflux at 85 °C for 40 min; the addition reaction means heating under reflux at 95 °C for 25 min; the addition reaction means heating under reflux at 90 °C for 35 min, and so on.

[0014] The preparation method of the above supramolecular fluorescent probe for gossypol detection is as follows: At room temperature, it is prepared by uniformly mixing human serum albumin and probe P2 in PBS buffer solution. P2@HSA is constructed using human serum albumin HSA as the host and an environment-sensitive disaggregation-induced fluorescence probe as the guest. Probe P2 aggregates in PBS, resulting in fluorescence quenching, while HSA can effectively relieve the aggregation state of probe P2 due to the presence of its own hydrophobic pocket and bind probe P2 at the fissure site of HSA to form the supramolecular compound P2@HAS.

[0015] The specific preparation method is as follows: Dissolve probe P2 in DMSO solution to prepare probe P2 solution; dissolve human serum albumin in PBS buffer solution to prepare HAS solution; at room temperature, mix the probe P2 solution and the HAS solution into PBS buffer solution and prepare by uniform mixing.

[0016] Furthermore, the molar ratio of the above probe P2 to human serum albumin is 1:3. The final concentration of probe P2 in the solution is 1 - 6 μM. For example, the final concentration of probe P2 in the solution is 1 - 2 μM; the final concentration of probe P2 in the solution is 2 - 4 μM; the final concentration of probe P2 in the solution is 4 - 6 μM; the final concentration of probe P2 in the solution is 3 - 5 μM; the final concentration of probe P2 in the solution is 1 μM, 2 μM, 3 μM, 4 μM, 5 μM or 6 μM.

[0017] Furthermore, the pH of the above PBS buffer solution is 7.3 - 7.5, and the concentration of the salt of phosphate in the PBS buffer solution is 10 mM.

[0018] Use of the above supramolecular fluorescent probe for rapid detection of gossypol in the detection of gossypol content. The principle of detecting gossypol is as follows: Gossypol can specifically bind to the IB site of HSA. When gossypol binds to the IB site of P2@HSA that has already bound to P2, gossypol can significantly enhance the fluorescence intensity of P2@HSA through guest-guest interaction. By the change in the fluorescence intensity of P2@HSA, the purpose of detecting the gossypol content in the analyte can be achieved ( Figure 1 ).

[0019] Furthermore, the above detection method is as follows:

[0020] (a) Add standard gossypol solutions with different concentrations to the PBS buffer solution of the above supramolecular fluorescent probe to form a series of gradient standard solutions, then perform fluorescence emission spectroscopy measurement with an excitation wavelength of 590 nm, and plot the working curve between the fluorescence intensity at 614 nm of the solution and the concentration of the added standard gossypol solution;

[0021] (b) Add the analyte gossypol solution to the PBS buffer solution of the above supramolecular fluorescent probe, perform fluorescence emission spectroscopy measurement with an excitation wavelength of 590 nm to obtain the fluorescence intensity of the solution at 614 nm, and substitute it into the working curve in step (a), then the concentration of gossypol in the analyte can be obtained, achieving the purpose of rapid detection.

[0022] Furthermore, the final concentration of the supramolecular fluorescent probe in the above steps (a) and (b) is 4 μM.

[0023] The beneficial effects of the present invention are:

[0024] (1) The present invention proposes a supramolecular fluorescent probe P2@HSA for gossypol detection, realizing rapid and efficient detection of the gossypol content in foods such as cottonseed meal and cottonseed oil. Among them, P2@HSA can achieve a response to gossypol through supramolecular guest-guest interaction and cause an increase in the fluorescence of P2@HSA. The fluorescence intensity of P2@HSA shows a good linear relationship with the concentration of gossypol when the gossypol concentration in the system is 0 - 8 μM, R 2 = 0.9912, and the detection limit is 0.0137 μM, which can easily achieve precise detection of gossypol.

[0025] (2) The present invention proposes a novel fluorescent probe P2, which can detect the concentration of HSA in physiological environment. Probe P2 has poor solubility in PBS solution and aggregates, resulting in fluorescence quenching. However, due to the existence of its hydrophobic cavity, HSA can effectively relieve the aggregation state of probe P2, enabling probe molecule P2 to bind to its own fissure site to form a supramolecular compound P2@HSA, and leading to fluorescence turn-on. At this time, the fluorescence intensity of probe P2 has an excellent linear relationship with the added HSA concentration, R 2 = 0.9775, and the detection limit is 0.0377 μM; moreover, the disaggregation process of probe P2 by HSA can be completed within 20 s, and the fluorescence of probe P2 can remain stable within 25 min after turn-on.

[0026] (3) The supramolecular fluorescent probe P2@HSA in the present invention is an efficient and reliable detection tool for the gossypol content in food, providing valuable ideas and new strategies for the development of subsequent gossypol-responsive fluorescent probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following-described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0028] Figure 1 It is the flow chart and principle diagram for the detection of gossypol by the supramolecular fluorescent probe P2@HSA.

[0029] Figure 2 It is the UV-Vis absorption spectra (a) and fluorescence spectra (b) of probe P2 with different concentration gradients in PBS, and the linear relationship diagram (c) of absorbance and concentration; the UV-Vis absorption spectra (d) and fluorescence spectra (e) of probe P2 with different concentration gradients in DMF, and the linear relationship diagram (f) of absorbance and concentration. Among them, λex = 590 nm.

[0030] Figure 3 It is the UV-Vis absorption spectra (a) and fluorescence spectra (b) of 4 μM probe in different solvents, and the bar chart (c) of the fluorescence intensity of 4 μM probe in different solvents. Among them, λex = 590 nm.

[0031] Figure 4 It is the fluorescence spectra (a) and line chart (b) of 4 μM probe P2 in different ratios of PBS-DIOX, and the line chart (c) of the change of solution fluorescence intensity with the polarity value △f. Among them, λex = 590 nm.

[0032] Figure 5 Fluorescence spectra of 4 μM probe P2 in Gly-PBS with different ratios (a) and line graph of the fluorescence intensity of the solution varying with the glycerol ratio in the mixed solution system (b). Among them, λex = 590 nm.

[0033] Figure 6 UV-visible absorption spectra and fluorescence spectra of 4 μM probe P2 before and after adding HSA in PBS buffer.

[0034] Figure 7 Graph of the fluorescence emission spectrum of probe P2 (4 μM) varying with the concentration of human serum albumin added (0 - 15.15 μM) (a); Linear relationship graph of the fluorescence emission intensity of probe P2 (4 μM) at 614 nm varying with the concentration of human serum albumin added (2 - 6 μM) (b); Time-dependent fluorescence spectrum of probe P2 (4 μM) after adding human serum albumin (c); (d) Calculation of the binding constant between P2 and HSA by fluorescence titration method, equation 1 / (F - F0) = 1 / (Ka(F max - F0)[HSA]) + 1 / (F max - F0), F0: Fluorescence intensity of probe P2 without HSA, F: Fluorescence intensity of probe P2 after adding different concentrations of HSA, F max : Maximum fluorescence intensity of probe P2 with HSA, Ka: Binding constant, λex = 590 nm.

[0035] Figure 8 Fluorescence emission spectra of 4 μM probe P2 in the presence of 15.15 μM HSA, 15.15 μM BSA and 20 μM other interfering substances (a) and bar chart of the fluorescence emission intensity at 614 nm (b); (c) Statistical graph of the fluorescence intensity change of 4 μM probe P2 with 15.15 μM HSA at 614 nm in the presence of 20 μM interfering substances such as anions, cations or amino acids. λex = 590 nm.

[0036] Figure 9 Line graph of the fluorescence intensity change of 4 μM probe P2 and P2@HSA at 614 nm under different pH conditions. Among them, λex = 590 nm.

[0037] Figure 10 Job’s plot curve of probe P2 and HSA in PBS (pH = 7.4, 10 mM), [P2] + [HSA] = 10 μM, λex = 590 nm.

[0038] Figure 11To study the changes in the fluorescence intensities of the drugs themselves, HSA (15.15 μM), and the P2@HSA supramolecular compound with the drug concentration after adding different drugs, such as ibuprofen (a), warfarin (b), and lidocaine (c), the experiment was carried out in PBS (pH 7.4, 10 mM) with λex = 590 nm.

[0039] Figure 12 It is the molecular docking diagram of the probe molecule P2 and HSA. The enlarged images on the right show the conformation of the P2 molecule in the HSA site and its hydrophobic interactions with the residues around the HSA binding site. Among them, the P2 molecule is represented by a yellow stick structure, and the amino acid residues in HSA that interact with P2 are shown in green.

[0040] Figure 13 It is the fluorescence emission spectrum change diagram of the supramolecular fluorescent probe P2@HSA with the change in gossypol content added (a) and the linear relationship diagram between the fluorescence intensity of P2@HSA at 614 nm and the gossypol concentration (b). Among them, λex = 590 nm.

[0041] Figure 14 It is the selective fluorescence emission spectrum diagram (a) of the supramolecular probe P2@HSA in the face of 20 μM cations, anions, and amino acids and other substances, and the bar chart (b) of the change in fluorescence emission intensity at 614 nm; (c) the statistical chart of the change in fluorescence intensity of P2@HSA at 614 nm in the presence of 20 μM interfering substances such as cations, anions, or amino acids. Detailed implementation manners

[0042] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0043] The experimental materials used in the present invention are shown in Table 1.

[0044] Table 1

[0045]

[0046] Examples

[0047] The preparation method of the probe P2 in this example is as follows:

[0048]

[0049] Synthesis of Intermediate 2: Weigh accurately 729.1 mg (5 mmol) of 1,3-indanedione (CAS: 606-23-5), 1290.2 mg (5 mmol) of malondialdehyde diphenylamine hydrochloride (CAS: 28140-60-5), and 419.4 mg (5.1 mmol) of anhydrous sodium acetate (CAS: 127-09-3) respectively. Place them in a sealed reaction tube and add 20 mL of acetic anhydride to completely dissolve the above substances. Then heat and stir the reaction at 150 °C for about 15 minutes. Stop heating and let the sealed reaction tube cool to room temperature, then continue stirring for it to react again for 24 hours. After the reaction is completed, separate the solid product by suction filtration and wash the upper solid successively with a small amount of acetic anhydride, a large amount of deionized water, and an appropriate amount of isopropanol. Then place the upper solid in a vacuum drying oven to dry, and finally obtain bright yellow powdery Intermediate 2. This product can be directly used in subsequent reactions without further purification. The product mass is 826 mg and the yield is 52%.

[0050] Synthesis of Probe P2: Dissolve Intermediate 2 (317.2 mg, 1 mmol), 1,2,3,3-tetramethyl-3H-indole iodide (301.5 mg, 1 mmol, CAS: 5418-63-3), and anhydrous sodium acetate (164.6 mg, 2 mmol) in 6 mL of absolute ethanol. Start stirring and heat under reflux at 85 °C for 25 minutes. After the reaction system cools to room temperature, remove the solvent in the reaction by suction filtration, and wash the upper solid crude product with boiling ethanol. The crude product is purified by silica gel column chromatography (eluent: dichloromethane / petroleum ether = 100:1, V / V), and finally obtain black-purple powdery solid Probe P2. The product mass is 105.7 mg and the yield is 30%. The 1H NMR and 13C NMR data further confirm the chemical structure of the target compound, while the high-resolution mass spectrometry provides accurate molecular weight information, which is highly consistent with the theoretical value, thus ensuring the purity and structural accuracy of the probe.

[0051] 1 H NMR (600 MHz, DMSO-d6) δ 8.12 (t, J = 13.2 Hz, 1H), 7.75 (d, J = 13.0 Hz, 1H), 7.70 (s, 4H), 7.57–7.48 (m, 2H), 7.33 (t, J = 8.3 Hz, 1H), 7.23 (d, J = 7.9 Hz, 1H), 7.13 (t, J = 7.4 Hz, 1H), 6.15 (d, J = 13.4 Hz, 1H), 3.49 (s, 3H), 1.63 (s, 6H).

[0052] 1313C NMR (151 MHz, DMSO) δ 190.91, 190.61, 170.95, 155.39, 146.88, 141.64, 140.32, 134.21, 134.01, 128.59, 123.79, 122.56, 121.64, 121.42, 119.93, 117.40, 110.23, 101.11, 48.37, 40.04, 27.80, 0.57.

[0053] HR-MS: Molecular formula C 24 H 21 NO2, [M + H + Theoretical value: 356.1646, Measured value: 356.1659.

[0054] The optical properties of probe P2 were investigated, and the specific steps and results are as follows:

[0055] Test conditions for spectral measurements: All spectral measurements were carried out at room temperature (25 °C). The absorption spectrum and fluorescence emission spectrum were both measured using the three-dimensional fluorescence and absorption spectrometer Buetta produced by Horiba (Japan). All spectral experiments were performed using a quartz cuvette with a 10 mm optical path length. When measuring the fluorescence spectrum, the slit widths of the excitation light and the emission light were both set to 5 nm.

[0056] (I). Optical properties of probe P2 in organic solvents

[0057] The UV-visible absorption spectra and fluorescence spectra of the probe molecule P2 in the organic solvent DMF and PBS (pH = 7.4, 10 mM) buffer solution are as Figure 2 shown. In the PBS solution, obvious and relatively broad absorption peaks appeared at 590 nm for different concentration gradients of probe P2, and there was a typical tail-end absorption, indicating that the solubility of probe P2 in PBS was poor and aggregation occurred ( Figure 2 a). As the concentration of the probe increased from 2 μM to 10 μM, the absorbance of probe P2 showed a good linear relationship with its concentration (R 2 = 0.9737) ( Figure 2 c). As can be seen from Figure 2 b, probe P2 had almost no fluorescence in PBS, and due to the existence of the aggregation phenomenon, as the concentration of P2 continued to increase, the fluorescence intensity at 614 nm did not change. In DMF, the UV absorption spectra of different concentrations of probe P2 Figure 2As shown in 2 , when the concentration of the probe increased from 2 μM to 10 μM, a good linear relationship was also presented between the absorbance of the solution and the probe concentration (R Figure 2 = 0.9997)( Figure 2 f). With the increase of the concentration of P2, the fluorescence intensity of the solution also increased. However, when the concentration of P2 reached a certain level, aggregation occurred, resulting in the fluorescence intensity no longer changing(

[0058] e). To ensure that the probe had good solubility in organic solvents and PBS and its absorbance was still within the linear range of the Lambert-Beer law, a suitable concentration of 4 μM was selected as the test concentration of P2. The concentration of the P2 probe used in subsequent experiments was 4 μM. Figure 3 The solvation effect of the probe molecule P2 in common solvents, including PBS, methanol, acetonitrile, ethanol, ethylene glycol, N, N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, ethyl acetate, dichloromethane, chloroform and 1,4-dioxane, was further explored, and the results are as Figure 3 shown. It can be seen from Figure 3 a that the absorption peaks of the probe molecule P2 in solvents were all located at 550 nm - 600 nm, and the absorbance of the probe P2 in most organic solvents except chloroform was higher than that in PBS. This was because the P2 molecules aggregated in poor solvents such as PBS and chloroform, resulting in a decrease in the number of effective light-absorbing solute molecules in the solution. It can be clearly seen from Figure 3 b and

[0059] c that the emission wavelength of the probe P2 was located at 610 nm - 625 nm, and the fluorescence intensity of P2 was significantly higher in organic reagents such as DMF, DMSO and ACN. This was because the probe P2 was well dispersed in these reagents. This indicated that the probe had a good solvatochromic effect. Therefore, 590 nm was selected as the best excitation wavelength of P2, and 614 nm was selected as the best emission wavelength of P2 for subsequent research. Figure 4It can be seen from a that as the proportion of PBS increases continuously, the fluorescence intensity of the probe at 614 nm first gradually increases and reaches the maximum when the polarity value is about 0.23. Subsequently, as the proportion of PBS in the mixed solution and the polarity value of the system increase, the fluorescence intensity of probe P2 gradually decreases. Figure 4 b and Figure 4 c). This phenomenon indicates that probe P2 has a high responsiveness to environmental polarity and exhibits the optimal fluorescence emission intensity when the polarity value is 0.23. When the polarity value is too high or too low, the fluorescence intensity is inhibited. In addition, the weak fluorescence intensity of P2 in DIOX is also attributed to the low polarity value of DIOX, resulting in a decrease in the fluorescence quantum yield of the probe.

[0060] The response ability of probe P2 to viscosity was tested by adding the probe to Gly-PBS mixed solutions with different ratios to evaluate its sensing ability to environmental viscosity. The specific method for preparing the test solutions was as follows: In a four-way cuvette, different volume ratios of a mixed solvent of PBS buffer solution and glycerol (Gly) with a total volume of 2 mL were added, and the volume ratio of Gly in the mixed solvent was gradually increased from 0% to 100% in a 10% gradient. Then, probe P2 (final concentration of 4 μM) was added, and the fluorescence intensity of the solution was measured. Figure 5 The results showed that as the proportion of glycerol increased, the fluorescence intensity of the probe at 614 nm gradually increased, indicating that probe P2 has good response performance to viscosity changes. This result indicates that the probe may have a higher fluorescence quantum yield in a high-viscosity environment.

[0061] (II) Response performance of probe P2 to HSA

[0062] The changes in the ultraviolet-visible absorption spectrum and fluorescence spectrum of probe P2 before and after adding HSA in PBS buffer solution were tested. As can be seen from Figure 6 it, before adding HSA, probe P2 showed aggregation in PBS, with an obvious shoulder peak appearing on the left side of the absorption peak and almost no fluorescence. However, after adding HSA, the shoulder peak of the absorption peak of probe P2 disappeared, and both its absorbance and fluorescence intensity increased, especially the fluorescence intensity increased by nearly 400 times. This indicates that the probe underwent disaggregation after binding to HSA. Under the irradiation of a 365 nm ultraviolet lamp, an obvious color change from colorless to pink occurred in the detection system containing probe P2 before and after adding HSA.

[0063] To further explore the response ability of probe P2 to HSA, the following test solutions were prepared for fluorescence performance testing. The preparation of the test solutions is as follows: Add 2 mL of PBS buffer solution (pH = 7.4, 10 mM) and probe P2 with a final concentration of 4 μM to a series of four-way cuvettes. Subsequently, add HSA with increasing gradient concentrations to these cuvettes and then perform fluorescence spectroscopy testing under excitation at 590 nm. In the absence of HSA, the fluorescence emission intensity of probe P2 at 614 nm is very weak. However, as the concentration of HSA in the system continuously increases, the fluorescence emission peak intensity of P2 at 614 nm gradually increases ( Figure 7 a). When the HSA concentration range is between 2 μM and 6 μM, the fluorescence intensity of P2 at 614 nm shows a good linear relationship with the HSA concentration (R 2 = 0.9775). Based on the formula LOD = 3σ / k, the detection limit of probe P2 is calculated to be 0.0377 μM, where σ is the standard deviation of testing the blank solution 10 times, K is the slope of the working curve of probe P2's response to HSA, and LOD is the detection limit ( Figure 7 b). The kinetic response of P2 to HSA was studied. The results show that the initial fluorescence intensity of probe P2 is very weak. In the presence of HSA, the aggregated state of probe P2 is completely removed within 20 s and forms a supramolecular compound P2@HSA with HSA. At this time, the fluorescence intensity reaches the maximum value and remains stable within the next 25 minutes ( Figure 7 c). Compared with P2 aggregated in PBS, the fluorescence intensity of the supramolecular compound P2@HSA formed after P2 binds to HSA is enhanced by more than dozens of times. This indicates that probe P2 has a significant response ability to HSA and the formed supramolecular fluorescent probe P2@HSA has excellent photostability and quantum yield. In addition, a linear relationship graph of 1 / (F - F0) and 1 / [HSA] was made using fluorescence titration ( Figure 7 d), and combined with the Benesi-Hildebrand (B-H) equation: 1 / (F - F0) = 1 / Ka(F max - F0)[HSA]) + 1 / (F max - F0), the binding constant Ka of P2 and HSA was calculated to be 3.6531×10 5 . In the formula, F0 is the fluorescence intensity of probe P2 without HSA, F is the fluorescence intensity of probe P2 after adding different concentrations of HSA, F max is the maximum fluorescence intensity of probe P2 with HSA, and Ka is the binding constant. This calculation result not only shows that P2 and HSA have a strong affinity but also proves the formation of the supramolecular compound P2@HSA.

[0064] Specific selectivity: HSA mainly exists in human body fluids, and actual samples may contain various potential interfering substances such as anions, cations, and amino acids. Therefore, common ions and amino acids in physiological samples were selected as interfering substances to explore the selective recognition ability and anti-interference performance of probe P2 for HSA. Figure 8 a shows the fluorescence emission spectra of probe P2 in the presence of different interfering substances. As can be seen from the results, except for HSA and BSA, which can significantly enhance the fluorescence signal of the system, the remaining interfering substances did not cause obvious fluorescence changes, indicating that probe P2 has excellent selectivity for the detection of HSA. In addition, since HSA and BSA do not appear under the same physiological conditions, and when equal amounts of HSA and BSA are present, the fluorescence enhancement caused by HSA is more significant. Therefore, probe P2 can be used for the rapid detection of HSA concentration under physiological conditions. In addition, Figure 8 The bar chart in b further intuitively shows the change in the fluorescence intensity of probe P2 at 614 nm. Figure 8 c shows that in the presence of 20 μM interfering substances, the detection result of probe P2 for HSA is basically unaffected, indicating that probe P2 has strong anti-interference ability. In summary, the experimental results show that probe P2 can specifically recognize HSA and still maintain good detection performance in a complex physiological environment, with good selectivity and anti-interference ability.

[0065] The applicable pH range largely determines the application scenario of the probe. Therefore, the influence of pH on the fluorescence intensity of probe P2 and the supramolecular compound P2@HSA in the detection system was explored. By mixing equal volumes of 0.04 M H3BO3, H3PO4, and CH3COOH and titrating with 0.2 M NaOH to the target pH (2.0 - 12.0), and finally making up the volume, B-R buffer solutions with different pH values can be obtained. Take 2 mL of B-R buffer solutions with different pH values in a four-way cuvette, then add probe P2 and the supramolecular fluorescent probe P2@HSA and perform fluorescence tests to explore the fluorescence characteristics of probe P2 and the supramolecular fluorescent probe P2@HSA under different pH conditions (adjusted by B-R buffer solutions). Figure 9The results showed that the probe P2 remained stable in the pH range of 2 - 12. However, due to a certain degree of aggregation of P2 in aqueous solution, its fluorescence intensity was generally low in the absence of HSA. After adding HSA to form the supramolecular compound P2@HSA, its fluorescence signal increased significantly, indicating that HSA had a certain depolymerization effect on the P2 aggregates, dispersing the probe and restoring fluorescence. However, under different pH conditions, there were certain fluctuations in the degree of fluorescence enhancement of P2@HSA. This was mainly attributed to the fact that as a biological macromolecule, the structure of HSA was significantly affected by pH changes, thus affecting the binding state of P2@HSA. However, under physiological conditions (pH = 7.4), the structure of HSA was relatively stable, and the fluorescence intensity of P2@HSA was also relatively stable. Therefore, pH = 7.4 was selected as the pH condition of the test solution for subsequent tests. In summary, the probe P2 could stably recognize HSA and form a stable supramolecular compound P2@HSA under physiological pH conditions, showing good biocompatibility and being suitable for the rapid detection and analysis of HSA in physiological environments.

[0066] To explore the binding mechanism between the probe P2 and HSA, the Job Plot method was used to determine the binding ratio of P2 to HSA ( Figure 10 ). Under the condition that the total concentration of P2 and HSA remained unchanged at 10 μM, fluorescence spectroscopy tests were carried out by continuously changing the molar ratio of P2 to HSA. It was observed that the fluorescence signal reached the maximum value when the ratio of [P2] / ([P2]+[HSA]) was 0.2663. This indicated that P2 and HSA formed a supramolecular compound P2@HSA with a ratio of 1:3.

[0067] The preparation method of the supramolecular fluorescent probe (P2@HSA) for the rapid detection of gossypol in this example is as follows:

[0068] (1) Preparation of the probe P2 stock solution: Weigh 3.6 mg of the solid powder of the probe P2 prepared in this example and dissolve it in 5 mL of chromatographically pure DMSO solution to obtain a probe P2 stock solution with a concentration of 2 mM; HSA was dissolved in PBS buffer solution (7.4, 10 mM) to prepare an HSA stock solution with a concentration of 20 mg / mL;

[0069] (2) Add 4 μL of the stock solution of the probe P2 with a concentration of 2 mM and 80 μL of the HSA stock solution with a concentration of 20 mg / mL to 2 mL of PBS buffer solution (pH = 7.4, 10 mM) and mix them evenly to obtain the supramolecular fluorescent probe P2@HSA.

[0070] HSA has multiple known ligand-binding sites, and drugs can be loaded at specific drug-binding sites of HSA through covalent or non-covalent interactions. Four drugs were selected as competitive drugs for common drug-binding sites on HSA: Warfarin - a marker for Sudlow I site, Ibuprofen - a marker for Sudlow II site, Lidocaine - a marker for IB site. The binding site of HSA with P2 was explored using drug competition experiments. The experimental results ( Figure 11 ) showed that after adding Ibuprofen, Warfarin, and Lidocaine, the fluorescence intensity change of the P2@HSA complex was not obvious, indicating that the binding sites of P2 and these labeled drugs on HSA were different. This means that P2 does not bind to the Sudlow I, Sudlow II, or IB sites. The above phenomenon shows that the drugs at the Sudlow site I, Sudlow site II, and IB site did not displace the probe P2 from HSA, which means that there is no direct interaction between P2 and the previous drug molecules, and the binding sites of P2 and HSA are not at the above positions. Therefore, it is speculated that the binding site of P2 and HSA is in the narrow middle pocket of HSA, that is, the fissure site.

[0071] Subsequently, molecular docking simulation was used to verify the binding of P2 and HSA at the fissure site. The results of molecular docking calculations strongly proved that P2 could effectively bind to the fissure site of HSA ( Figure 12 ), and there was a strong binding force between P2 and HSA, and its binding energy was about -5.92 kcal / mol, indicating that P2 could stably bind to HSA at this site and form the supramolecule P2@HSA. The docking results were visualized using PyMOL software to clarify the binding mode of P2 in the protein complex. The analysis showed that the two carbonyl groups in the structure of P2 respectively bound to the amino acid residues LEU-115 and LYS-190 near the fissure binding site of HSA and formed hydrogen bond interactions. Among them: the hydrogen bond distance between LEU-115 and the carbonyl group of P2 was The hydrogen bond distance between LYS-190 and the carbonyl group of P2 was The formation of these hydrogen bonds indicates that P2 highly matches the active pocket of human serum albumin and can stably form a stable complex.

[0072] Application Example

[0073] 1. Detection performance of the supramolecular probe P2@HSA for gossypol

[0074] To achieve the rapid detection of gossypol, this detection method prepares a PBS buffer solution containing a supramolecular fluorescent probe according to the principle of preparing it immediately before use, and obtains a working curve between the fluorescence intensity and the concentration of the added standard gossypol solution by adding a series of gossypol solutions with different concentrations; then the purpose of quantitatively and rapidly detecting the gossypol content can be achieved. The process is as follows:

[0075] (S1) 4 μL of the stock solution of probe P2 with a concentration of 2 mM and 80 μL of the HSA stock solution with a concentration of 20 mg / mL are added to 2 mL of PBS buffer solution (pH = 7.4, 10 mM), and they are mixed evenly to obtain the supramolecular fluorescent probe P2@HSA solution;

[0076] (S2) 0 - 11 μL of the standard gossypol solution with a concentration of 2 mM is added to the solution obtained in step (S1) to form a series of gradient standard solutions, and then the fluorescence emission spectrum is measured at an excitation wavelength of 590 nm, and a working curve between the fluorescence intensity at 614 nm and the concentration of the added standard gossypol solution is plotted. The results are as Figure 13 shown.

[0077] Figure 13 a The results show that gossypol can significantly enhance the fluorescence intensity of P2@HSA at 614 nm, and the fluorescence intensity of P2@HSA shows a good linear relationship with the gossypol concentration (0 - 8 μM)( Figure 13 b), R 2 = 0.9912, and its detection limit is calculated to reach 0.0137 μM according to the formula LOD = 3σ / k. Therefore, the supramolecular compound P2@HSA is a powerful tool for rapidly detecting trace gossypol. The reason why gossypol can enhance the fluorescence of the supramolecular compound P2@HSA is that the binding of gossypol to HSA changes the conformation of HSA itself, and the change in conformation further changes the binding behavior between probe P2 and HSA, thereby leading to the enhancement of the fluorescence of P2@HSA.

[0078] In addition, the selectivity of the supramolecular compound P2@HSA for common cations, anions and amino acids in a complex environment was investigated( Figure 14 a - 14b) and the anti-interference ability of P2@HSA in detecting gossypol in a complex environment( Figure 14 c). The results show that substances such as cations, anions and amino acids other than gossypol will not enhance the fluorescence of P2@HSA, which indicates that the supramolecular fluorescent probe P2@HSA has excellent selectivity for gossypol. The experimental results also prove that when using P2@HSA to detect gossypol, the abundant interfering substances in the environment will not interfere with the detection results. The above results all prove the excellent selectivity and anti-interference ability of P2@HSA in detecting the gossypol concentration in a complex environment.

[0079] 2. Detection of gossypol content in actual food using supramolecular probe P2@HSA

[0080] Cottonseed meal and cottonseed oil are important cotton by-products obtained by pressing cottonseeds. Cottonseed meal is widely used in animal feed, and cottonseed oil is one of the important oils for frying foods in human society. Due to current limitations in cottonseed processing and utilization technologies, it is not yet possible to completely remove the gossypol in cottonseed oil and cottonseed meal obtained by pressing cottonseeds. This toxic substance poses a strong potential hazard to humans and many animals. In animal husbandry, cottonseed meal is often used as animal feed, which is an important protein source for raising livestock. However, the gossypol contained in cottonseed meal not only affects the growth and development of livestock and poultry but may even enter the human body through the food chain and accumulate. Therefore, the gossypol content in cottonseed meal and cottonseed oil needs to be strictly controlled to ensure food safety.

[0081] To further verify the reliability of the detection results of gossypol content in food using P2@HSA, cottonseed meal and cottonseed oil were selected as actual samples to verify this method.

[0082] Extraction of gossypol from cottonseed oil: According to the national standard method GB5009.148-2014, add cottonseed oil (5 g) to 25 mL of ethanol, stir for 10 minutes, then let it stand for stratification. Take the supernatant and perform centrifugation, and then take the supernatant again. Filter the supernatant obtained after centrifugation through a 0.22 μM filter membrane. Take 10 mL of the filtered solution, evaporate it to dryness, dissolve it in DMSO, and then perform subsequent tests.

[0083] Extraction of gossypol from cottonseeds: According to the national standard method GBT13086-2020, take 20 g of cottonseeds, crush them, weigh 1 g of cottonseed meal, add it to 50 mL of solvent A (prepared according to the method in the national standard method GBT13086-2020), oscillate for one hour, filter the reaction solution to obtain the cottonseed extract. Take 10 mL of the cottonseed extract, evaporate it to dryness using a rotary evaporator, add DMSO to obtain the test solution, and then perform subsequent tests. Among them, the preparation of solvent A: Take 500 mL of a mixture of isopropanol and n-hexane (volume ratio 6:4), add 2 mL of 3-amino-1-propanol, 8 mL of glacial acetic acid, and 50 mL of distilled water, transfer it to a 1000 mL volumetric flask, and make up the volume to the mark with a mixture of isopropanol and n-hexane.

[0084] Testing of actual samples: 4 μL of the stock solution of probe P2 with a concentration of 2 mM and 80 μL of the stock solution of HSA with a concentration of 20 mg / mL were added to 2 mL of PBS buffer solution (pH = 7.4, 10 mM) and mixed evenly to obtain the supramolecular fluorescent probe P2@HSA solution. Then, 2 μL of gossypol extract was added for testing. For spike recovery, after adding the extract to the test solution, a standard gossypol sample with a known concentration was added again before testing.

[0085] Regarding how to detect, it is recommended to supplement the specific detection method (including the test solution). The results are shown in Table 2.

[0086] Table 2 Determination results of gossypol content in cottonseed meal and cottonseed oil samples by the supramolecular probe P2@HSA

[0087]

[0088] Table 2 results show that gossypol was not detected in actual samples such as cottonseed oil and detoxified cottonseed cake. In the spike recovery experiment, the recovery rate range of gossypol detection in cottonseed meal and cottonseed oil by this method was 75.50% - 87.75%, and the relative standard deviation (RSD) of the detection results ≤ 3.16%. The above experimental results indicate that the supramolecular probe P2@HSA is an accurate and reliable method for rapid detection of gossypol content in food, with potential and value for practical applications.

[0089] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A supramolecular fluorescent probe for rapid detection of gossypol, characterized in that, The supramolecular fluorescent probe is formed by the combination of human serum albumin and probe P2. The structural formula of probe P2 is as follows: 。 2. The supramolecular fluorescent probe for rapid detection of gossypol according to claim 1, wherein The preparation method of the probe P2 is as follows: 1) React 1,3-indandione and malondialdehyde diphenylamine hydrochloride in acetic anhydride to obtain intermediate 2; 2) Intermediate 2 undergoes an addition reaction with 1,2,3,3-tetramethyl-3H-indole iodide in ethanol to prepare probe P2.

3. The supramolecular fluorescent probe for gossypol detection according to claim 2, characterized in that, In step 1), the molar ratio of 1,3-indandione to malondialdehyde diphenylamine hydrochloride is 1:1; the reaction means reacting at 140-160 °C for 15-30 min first, and then reacting at room temperature for 24 h.

4. The supramolecular fluorescent probe for rapid gossypol detection according to claim 3, characterized in that, In step 2), the molar ratio of intermediate 2 to 1,2,3,3-tetramethyl-3H-indole iodide is 1:1, and the addition reaction means heating under reflux at 85-95 °C for 25-40 min.

5. The preparation method of the supramolecular fluorescent probe for rapid detection of gossypol according to claim 1, characterized in that, The steps are as follows: At room temperature, human serum albumin and the probe P2 described in claim 1 are uniformly mixed in PBS buffer solution to prepare it.

6. The preparation method of the supramolecular fluorescent probe for rapid detection of gossypol according to claim 5, wherein, The molar ratio of the probe P2 to human serum albumin is 1:

3.

7. The preparation method of the supramolecular fluorescent probe for rapid gossypol detection according to claim 6, characterized in that, The final concentration of the probe P2 is 1-6 μM, and the pH of the PBS buffer solution is 7.3-7.

5.

8. Application of the supramolecular fluorescent probe for rapid detection of gossypol described in claim 1 in detecting the gossypol content.

9. The application according to claim 8, wherein The detection method is as follows: a) Add standard gossypol solutions with different concentrations to the PBS buffer solution containing the supramolecular fluorescent probe described in claim 1 to form a series of gradient standard solutions. Then, perform fluorescence emission spectrum measurement with an excitation wavelength of 590 nm, and plot the working curve between the fluorescence intensity at 614 nm of the solution and the concentration of the added standard gossypol solution; b) Add the gossypol solution to be measured to the PBS buffer solution containing the supramolecular fluorescent probe described in claim 1, perform fluorescence emission spectrum measurement with an excitation wavelength of 590 nm to obtain the fluorescence intensity at 614 nm of the solution, and substitute it into the working curve in step a) to obtain the concentration of gossypol in the solution to be measured, achieving the purpose of rapid detection.

10. According to the application described in claim 9, the final concentration of the supramolecular fluorescent probe in steps a) and b) is 4 μM.

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