Preparation and application of fluorescent probe for specifically detecting human serum albumin

Abstract

The invention discloses preparation and application of a fluorescent probe for specifically detecting human serum albumin (HSA). The fluorescent probe can be used as a high-sensitivity and high-selectivity HSA fluorescence analysis means. The self-assembled nano fluorescent probe has the advantages of excellent selectivity, good anti-interference capability, quick response, high sensitivity, good biocompatibility, strong stability, simple preparation, low synthesis cost and the like. The nanoparticles constructed by the quinoline hydrazide Schiff base and the HSA can be used as a visual carrier for carrying platinum drugs. The fluorescent probe has the advantage that the fluorescent probe can emit fluorescence under light excitation, so that a tracing effect on the nano-particles is achieved. After the nanoparticles are dissociated, fluorescence disappears, and release of platinum drugs in a biological system can be monitored.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probe detection and nano drug delivery application, and more specifically, relates to the application of a nano fluorescent probe formed by self-assembly of quinoline hydrazide Schiff base in detecting HSA, and further to the application of fluorescently labeling nanoparticles composed of HSA to achieve tracing of drug molecule distribution and metabolism. Background Art

[0002] Human serum albumin (HSA) is a heart-shaped single-chain protein with a molecular weight of 66.5kDa. It is commonly used clinically to treat symptoms such as bleeding, shock, burns, polycythemia, and hypoalbuminemia. HSA can be used as a biomarker and play an important role in human health monitoring. Its normal concentration in the blood is about 35-55g / L, and the concentration in the urine of healthy individuals should be less than 30mg / L. Accurate tracking of the content and activity of disease-related proteins is essential for clinical diagnosis, drug toxicity monitoring, and disease treatment. Microalbuminuria is an early indicator of diabetes, hypertension, cardiovascular disease, and kidney disease. Low levels of HSA in plasma, i.e., hypoalbuminemia, may mean cirrhosis, liver failure, and chronic hepatitis. Therefore, accurate detection of HSA levels in body fluids is crucial.

[0003] As a soluble carrier protein, HSA is the most abundant in the blood, accounting for 52%-60% of the total plasma protein content. It plays a vital role in maintaining blood osmotic pressure, transporting various substances, and binding hormones, vitamins, fatty acids, calcium ions, and drug molecules. HSA has excellent bioavailability as a drug carrier. Due to its wide distribution in the body, non-toxicity, non-antigenicity, and biodegradability, it does not cause rejection reactions in patients. At the same time, the combination of drugs with HSA can reduce drug load and toxicity and maximize the therapeutic effect. HSA can be combined with drugs with poor water solubility to promote the uniform distribution of drugs in the body, delay drug metabolism, and prolong the circulation time of drug efficacy. Platinum drugs have been widely and effectively used in cancer treatment, and platinum drug delivery systems based on HSA have shown good application prospects in clinical trials. Studying the metabolism and distribution of HSA nanoparticles loaded with platinum drugs in vivo is of great significance for drug development, clinical application, and improving patient treatment effects. By combining fluorescent probe molecules with HSA, HSA nanocarriers can be fluorescently tracked in biological systems. Subsequently, as HSA decomposes, the fluorescent molecules will lose their fluorescent properties, thus enabling the detection of drug delivery and metabolism in vivo.

[0004] Traditional methods for HSA detection and analysis include dye binding, fluorescent immunoassay, LC-MS, electrophoresis, etc. These methods usually require high material and instrument costs, involve complex sample preparation, and have poor specificity and anti-interference ability. Small molecule fluorescent probes have been shown to be a promising option. They have the advantages of easy preparation, fast and convenient detection, non-invasiveness, and non-destructiveness in biomolecule detection. So far, various HSA probes have been developed for the selective and sensitive detection of HSA that may be present in biological samples, but there are still some limitations. For example, some HSA probes show poor water solubility, which hinders their application in biological environments. In addition, some probes covalently bind to the amino or thiol groups of proteins, resulting in HSA denaturation. Most HSA fluorescent probes bind to two classic HSA drug binding sites, site I and site II, so commonly used drug molecules in the body can cause serious interference with fluorescence detection. The development of new small molecule probes for qualitative and quantitative detection of HSA has great practical value and research significance. In recent years, molecular self-assembled HSA probes have become a powerful tool for constructing soft functional materials and have a wide range of applications in materials science and biology. Small molecule organic dyes can be directly assembled into nanoparticles without nanocarriers, showing excellent photostability and biocompatibility, and providing extensive flexibility in molecular design. Quinoline as a fluorophore has the advantages of high fluorescence quantum yield, large Stokes shift, high photostability, good biocompatibility and tunable fluorescence properties; the amide group can increase the binding affinity with HSA; the assembly design makes the different functional groups of the hydrazide Schiff base compound show different hydrophilicity and lipophilicity; all of which are conducive to the application of the synthesized fluorescent probe as an HSA chemical sensor. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a new nano fluorescent probe formed by self-assembly of quinoline hydrazide Schiff base, which can be used as a highly sensitive and highly selective HSA fluorescence analysis method. Compared with the probes (compounds) in the existing literature, the self-assembled nano fluorescent probe of the present invention has the advantages of excellent selectivity, good anti-interference ability, rapid response, high sensitivity, good biocompatibility, strong stability, simple preparation, and low synthesis cost. The nanoparticles constructed by quinoline hydrazide Schiff base and HSA can be used as a visual carrier to carry platinum drugs. Its advantage is that it can emit fluorescence under light excitation, thereby tracing the nanoparticles. The fluorescence disappears after the nanoparticles dissociate, and the release of platinum drugs in the biological system can be monitored.

[0006] To achieve the above purpose, the present invention discloses the following technical contents: A quinoline hydrazide Schiff base fluorescent probe, characterized in that it has the following structural formula:

[0007] The present invention further discloses a method for preparing the quinoline hydrazide Schiff base fluorescent probe, which is characterized by being carried out in the following steps: (1) Dissolve quinoline-2-carboxylic acid in anhydrous ethanol, then add an appropriate amount of concentrated sulfuric acid and heat under reflux for 5-7 h. After the reaction system is cooled, adjust the pH value to 6-9 with saturated NaHCO3 solution, extract with dichloromethane, and remove the solvent by rotary evaporation of the dichloromethane layer to obtain solid quinoline-2-carboxylic acid ethyl ester; (2) dissolving ethyl quinoline-2-carboxylate in methanol, then adding hydrazine hydrate dropwise while stirring, heating and stirring, condensing and refluxing for 1 h, and monitoring the reaction by TLC until the reaction of ethyl quinoline-2-carboxylate is completely stopped; after the reaction solution is cooled to room temperature, it is concentrated under vacuum, and methanol and ethanol are added to the reaction system by alternating rotary evaporation until hydrazine hydrate is evaporated and removed, thereby obtaining quinoline-2-carboxylic acid hydrazide, wherein the molar ratio of ethyl quinoline-2-carboxylate to hydrazine hydrate is 1:15-30; (3) Dissolve quinoline-2-carboxylic acid hydrazide in anhydrous methanol and dissolve 2,4-dihydroxybenzaldehyde in anhydrous methanol. Slowly add the mixture dropwise to the methanol solution of quinoline-2-carboxylic acid hydrazide under stirring. Heat with stirring and condense the mixture under reflux for 9.5 h. After cooling the reaction solution to room temperature, filter and wash the precipitate. Dry under vacuum to obtain the reaction product, i.e., the quinoline hydrazide Schiff base fluorescent probe. The molar ratio of the feed materials of quinoline-2-carboxylic acid hydrazide and 2,4-dihydroxybenzaldehyde is 1:1.

[0008] The present invention further discloses the application of the quinoline hydrazide Schiff base fluorescent probe in the qualitative and quantitative detection of HSA, characterized in that the quinoline hydrazide Schiff base fluorescent probe forms a self-assembled nano fluorescent probe in a PBS buffer; the pH of the PBS buffer is 7-8. HSA is added to the probe solution, and under a 365 nm ultraviolet lamp, the solution changes from colorless to cyan, thereby realizing rapid detection of HSA. In addition, under 370-420 nm excitation light, there is almost no fluorescent signal itself. After reacting with HSA, a strong cyan fluorescent signal is generated at 495 nm, and the quantitative detection concentration range of HSA is 0-8 mM, and the detection limit is 8.49 nM. The experimental results show that the quinoline hydrazide Schiff base fluorescent probe has good detection ability for HSA, high sensitivity, good linear relationship between fluorescence intensity and concentration, and can accurately and quantitatively measure the concentration of HSA.

[0009] The present invention also discloses the application of quinoline hydrazide Schiff base fluorescent probe in detecting HSA in living cells and biological samples. The quinoline hydrazide Schiff base fluorescent probe and HSA form nanoparticles for detecting hemoglobin. Experimental results show that the nanoparticles formed by the fluorescent probe and HSA can quantitatively detect hemoglobin with low detection limit and good sensitivity.

[0010] The present invention also discloses the use of fluorescent tracer nanoparticles formed by quinoline hydrazide Schiff base fluorescent probe and HSA in the delivery of platinum drugs. The synthesis method thereof is prepared by the following reaction: 1 mM HSA aqueous solution was added to PBS buffer, and 1 mM probe molecule DMF solution was slowly added to HSA solution with a pipette gun under high-speed stirring. The above solution was placed in a dialysis bag that had been boiled and activated and filtered, and dialyzed with PBS buffer for 2 hours to remove single molecules and small particles from the system; then the sample was extracted from the dialysis bag with a pipette, and the bag was rinsed with a small amount of PBS buffer to ensure complete transfer of the sample, and the resulting solution was sealed in a refrigerator at 4°C. The experimental results show that the nanoparticles formed by the fluorescent probe and HSA have good fluorescence properties and can stably carry platinum drugs. The fluorescence disappears after the nanoparticles dissociate, which can realize the monitoring of drug carriers.

[0011] The present invention is more detailed as follows The application of the present invention as a fluorescent tracer carrier for HSA detection and platinum drugs is achieved through the following technical solutions: The nano fluorescent probe formed by self-assembly of quinoline hydrazide Schiff base for HSA detection has the following structural formula:

[0012] The self-assembled nano fluorescent probe formed by the quinoline hydrazide Schiff base is used for the qualitative and quantitative detection of HSA.

[0013] The synthesis of the self-assembled nano fluorescent probe formed by the quinoline hydrazide Schiff base is as follows: (1) Dissolve quinoline-2-carboxylic acid in anhydrous ethanol, then add an appropriate amount of concentrated sulfuric acid and heat under reflux for 5-7 h. After the reaction system is cooled, adjust the pH value to 6-9 with saturated NaHCO3 solution, extract with dichloromethane, and remove the solvent by rotary evaporation to obtain quinoline-2-carboxylic acid ethyl ester.

[0014] (2) Dissolve ethyl quinoline-2-carboxylate in methanol, then add hydrazine hydrate (N2H4·H2O) dropwise while stirring, heat and stir, and reflux for 1 h. Monitor the reaction by TLC until ethyl quinoline-2-carboxylate is completely reacted (petroleum ether: ethyl acetate = 5:1), then stop the reaction. After the reaction solution is cooled to room temperature, it is concentrated under reduced pressure in vacuo, and methanol and ethanol are added and alternately evaporated in a circular manner until hydrazine hydrate is evaporated away from the reaction system to obtain quinoline-2-carboxylic hydrazide.

[0015] The molar ratio of quinoline-2-carboxylic acid ethyl ester and hydrazine hydrate in the (2) is 1:(15-30).

[0016] (3) Dissolve quinoline-2-carboxylic acid hydrazide in anhydrous methanol and dissolve 2,4-dihydroxybenzaldehyde in methanol. Slowly add the two solutions to the methanol solution of quinoline-2-carboxylic acid hydrazide under stirring. Heat and stir, and condense and reflux for 9.5 h. After the reaction solution is cooled to room temperature, filter and wash the precipitate, and vacuum dry to obtain the reaction product, which is the quinoline hydrazide Schiff base fluorescent probe.

[0017] The molar ratio of quinoline-2-carboxylic acid hydrazide and 2,4-dihydroxybenzaldehyde in (3) is 1:1.

[0018] Described synthetic route is as follows:

[0019] The quinoline hydrazide Schiff base forms a self-assembled nano fluorescent probe in a PBS buffer.

[0020] The pH of the PBS buffer is 7-8.

[0021] The nano fluorescent probe formed by the self-assembly of the quinoline hydrazide Schiff base has almost no fluorescent signal when excited at an excitation wavelength range of 370-420 nm. After interacting with HSA, it generates a strong fluorescent signal at 495 nm. The quantitative detection concentration range of HSA is 0-8 mM, and the detection limit is 8.49 nM.

[0022] The nano fluorescent probe formed by self-assembly of the quinoline hydrazide Schiff base has an excitation wavelength range of 370-420 nm, an emission wavelength of about 495 nm, and a binding site with HSA at the FA1 site, which effectively overcomes the shortcomings of poor water solubility and interference from common drug molecules, has good selectivity for HSA, and can be used for the detection of HSA in biological systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is an electron microscope image of a nano fluorescent probe formed by self-assembly of the quinoline hydrazide Schiff base of the present invention; Figure 2The selective fluorescence spectra of the nano fluorescent probe formed by the self-assembly of the quinoline hydrazide Schiff base of the present invention to HSA; wherein (a) is the fluorescence response spectrum of the probe after mixing with various amino acids and proteins (10 μM, 1.0 equivalent); (b) is the fluorescence intensity at 495 nm after the probe is mixed with various amino acids and proteins; (c) is the fluorescence intensity at 495 nm after the probe is mixed with various metal cations; (d) is the fluorescence intensity at 495 nm after the probe is mixed with various anions; Figure 3 The fluorescence signal diagram of the nano fluorescent probe formed by the self-assembly of the quinoline hydrazide Schiff base of the present invention for recognizing HSA in the presence of other interferents; (a) is the fluorescence intensity of the probe-HSA at 495 nm in the presence of various amino acids and enzymes; (b) is the fluorescence intensity of the probe-HSA at 495 nm in the presence of various metal ions; (c) is the fluorescence intensity of the probe-HSA at 495 nm in the presence of various anions; Figure 4 The fluorescence intensity of the nano fluorescent probe formed by the self-assembly of the quinoline hydrazide Schiff base of the present invention and the fluorescence spectrum of the response to different concentrations of HSA (a) and the linear relationship diagram of the change value of the fluorescence intensity at 495 nm and the HSA concentration (b); Figure 5 The binding site displacement diagram of the probe-HSA of the present invention with warfarin, bilirubin, ibuprofen and hemin; Figure 6 Cell viability of H226 cells (a), HUVEC cells (b), and SW1990 cells (c) treated with different concentrations of probes for 6 h; Figure 7 The fluorescence imaging diagrams of the nano fluorescent probe of the present invention and HSA in H226 cells (a) and HUVEC cells (b); Figure 8 The fluorescence spectra of the probe-HSA conjugate as the hemin concentration increases (a) and the linear fitting diagram of the hemin titration probe-HSA conjugate in the concentration range of 0-4 μM (b); Fig. 9 Fluorescence images of probe-HSA detecting hemin in SW1990 cells; Row 1: SW1990 cells treated with probe molecule (10 μM) only; Row 2: SW1990 cells treated with probe molecule and HSA (10 μM) for 30 minutes; Row 3: SW1990 cells treated with probe molecule, HSA and hemin (10 μM) in sequence for 30 minutes; Fig.10Characterization of probe-HSA nanoparticles carrying cisplatin; (a) Effect of cisplatin on the fluorescence intensity of probe-HSA nanoparticles; (b) UV-visible spectrum of probe-HSA nanoparticles carrying cisplatin; (c) MALDI-TOF mass spectra of HSA, probe-HSA and cisplatin-probe-HSA conjugate. DETAILED DESCRIPTION

[0024] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are all commercially available. For example, quinoline-2-carboxylic acid, hydrazine hydrate, 2,4-dihydroxybenzaldehyde, anhydrous ethanol, concentrated sulfuric acid, dichloromethane, etc. are all commercially available. Example

[0025] Synthesis and characterization of fluorescent probes (1) Dissolve quinoline-2-carboxylic acid in anhydrous ethanol, then add an appropriate amount of concentrated sulfuric acid and heat under reflux for 5-7 h. After the reaction system is cooled, adjust the pH value to 6-9 with a saturated NaHCO3 solution, extract with dichloromethane, and remove the solvent by rotary evaporation from the dichloromethane layer to obtain quinoline-2-carboxylic acid ethyl ester; dissolve quinoline-2-carboxylic acid ethyl ester in methanol, then add hydrazine hydrate dropwise with stirring, heat and stir, and condense and reflux for 1 h. Monitor the reaction by TLC until the reaction of quinoline-2-carboxylic acid ethyl ester is complete (petroleum ether: ethyl acetate = 5:1), then stop the reaction. After the reaction solution is cooled to room temperature, vacuum concentrate, add methanol and ethanol, and cyclically rotate and evaporate until hydrazine hydrate is evaporated and removed from the reaction system to obtain quinoline-2-carboxylic acid hydrazide; wherein the molar ratio of quinoline-2-carboxylic acid ethyl ester to hydrazine hydrate is 1:(15-30) (2) Dissolve quinoline-2-carboxylic acid hydrazide in anhydrous methanol and dissolve 2,4-dihydroxybenzaldehyde in methanol. Slowly add the mixture dropwise to the methanol solution of quinoline-2-carboxylic acid hydrazide under stirring, heat and stir, and condense and reflux for 9.5 h. After the reaction solution is cooled to room temperature, it is filtered and the precipitate is washed and dried under vacuum to obtain the reaction product, which is the quinoline hydrazide Schiff base fluorescent probe; wherein the molar ratio of quinoline-2-carboxylic acid hydrazide to 2,4-dihydroxybenzaldehyde is 1:1. The yield is 79%. 1H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H), 11.57 (s, 1H), 10.02 (s, 1H), 8.82 (s, 1H), 8.63 (d, J = 8.5 Hz, 1H), 8.23 ​​(dd, J = 8.6, 2.3 Hz, 2H), 8.12 (d, J = 8.2 Hz, 1H), 7.93 (ddd, J = 8.4, 6.8, 1.5 Hz, 1H), 7.76 (t, J = 7.5 Hz, 1H), 7.32 (d,J = 8.4 Hz, 1H), 6.41 (dd, J = 8.4, 2.3 Hz, 1H), 6.37 (d, J = 2.3 Hz, 1H); 13 CNMR (101 MHz, DMSO-d6) δ 160.87, 160.22, 159.68, 150.91, 149.57, 146.02,137.99, 131.59, 130.64, 129.17, 128.94, 128.29, 128.17, 119.05, 110.57,107.77, 102.71; ESI-MS (m / z) calcd [GM-1+H] + = 308.1030, measured value 308.1033. Example

[0026] Study on the formation of self-assembled nanofluorescent probes Use a micropipette to add 20 μL of DMF solution (10 -3 M) was added to 2 mL of PBS buffer to prepare a solution of quinoline hydrazide Schiff base nanoparticles. Subsequently, the solution system was placed in an ultrasonic device and evenly mixed by ultrasound for 30 minutes to ensure that the quinoline hydrazide Schiff base nanoparticles were evenly distributed in the solution. The prepared quinoline hydrazide Schiff base self-assembled nanoparticle solution was placed in a cuvette, and the dispersion of the nanoparticle size was measured using DLS (dynamic light scattering) analysis at room temperature, and the average particle size was 339.4 nm. For morphological studies, the prepared quinoline hydrazide Schiff base self-assembled nanoparticle solution was slowly added to the surface of the copper mesh by using a micropipette to form droplets. After 10 minutes of precipitation, the excess solution was removed with a pipette and the copper mesh was allowed to dry naturally. Finally, the morphology was observed and the image was captured using a transmission electron microscope (TEM). The morphology of the formed nanoparticles appeared to be regular and well-dispersed spheres ( Figure 1 ). Example

[0027] Study on the Selectivity of Nano-fluorescent Probe to HSA Fluorescence detection of various common substances present in living systems and the environment, including metal ions (K + ,Ca 2+ , Na + , Mg 2+ , Al 3+ , Zn 2+ , Fe 3+ , Pb 2+ , Ag + , Ba 2+ , Mn 2+ , Cu 2+ , Co 2+ , Ni 2+ , Cr 3+ , Cd 2 + ), anions (F - , Cl - , Br - , HS - , NO3 - , CH3COO - , HPO4 2- , SO4 2- , HCO3 - ), amino acids and proteins (Ala, Lyr, Arg, Cys, His, Glu, Iso, Ser, Trp, Asp, Thr, Val, Lys, Met, Pro, bovine serum albumin, human serum albumin, pepsin, lipase, lysozyme, fibrinogen, hemoglobin) to demonstrate the specificity of the probe in HSA detection. In 2 mL of PBS buffer solution (pH 7.4), 20 μL of DMF solution of the probe molecule (10 -3 M), and then add equivalent amounts of various substances respectively, stir evenly at room temperature, and test their respective fluorescence emission spectra under excitation wavelength of 370 nm. Figure 2 As shown in the figure, the probe showed a significant fluorescence enhancement only after the addition of HSA. Under the same conditions, no obvious fluorescence response was observed for other substances. For bovine serum albumin BSA with a homologous structure, only a small fluorescence change was triggered when interacting with the probe, and the two similar proteins could be distinguished by fluorescence, confirming the uniqueness of the probe in HSA detection. Example

[0028] Study on the influence of different substances on the detection of HSA by nanofluorescent probe In 2 mL of PBS buffer solution (pH 7.4), add 10 μL of probe solution (10 -3 M) and other various metal ions, anions, amino acids and protein solutions (10 -3 M, and then 10 μL of HSA solution (10 -3 M), and then test the fluorescence signal changes after mixing evenly. Figure 3 As shown, the fluorescence intensity of the probe HSA conjugate changes little in the presence of various potential interfering substances, making it suitable for complex samples and biological environments. Example

[0029] Study on the response of nanofluorescent probe to different concentrations of HSA In 2 mL of PBS buffer solution (pH 7.4), add 20 μL of the probe DMF solution (10 -3 M), and then add different concentrations of HSA solution (0-10 μM) to test the changes in its fluorescence spectrum. Figure 4 As shown in the figure, in the absence of HSA, the probe NDQC has almost no fluorescence. As the HSA concentration increases, the fluorescence intensity at 495nm gradually increases. When the HSA concentration is in the range of 0 to 8μM, there is a linear relationship between the fluorescence intensity and the HSA concentration. This indicates that the nano fluorescent probe molecule can quantitatively detect HSA, and its detection limit is 8.49 nM (0.56 mg / L). Example

[0030] Study on the Binding Site of Fluorescent Probe to HSA A drug displacement experiment was conducted to determine the binding of specific drugs to HSA, where warfarin and bilirubin were selected as Sudlow site I specific binding drugs, ibuprofen was selected as Sudlow site II binding drug, and hemin was selected as heme site binding drug. Figure 5 As shown, when the concentrations of the four drugs in the probe-HSA system increased respectively, the effect of hemin on the fluorescence intensity was significantly greater than that of warfarin, bilirubin and ibuprofen, indicating that the probe prefers to bind to HSA through the heme site. Example

[0031] Cytotoxicity experiment of nanofluorescent probe H226 cells, HUVEC cells, and SW1990 cells were cultured in DMEM medium at 37°C, 95% air, and 5% CO2. The cells were then seeded into 96-well cell culture plates, treated with different concentrations of probe molecules (0–100 µM) at 37°C, and incubated for 6 hours. MTT solution (5 mg / mL, PBS, 10 µL / well) was added to the culture medium and incubated for 6 hours. Excess culture medium was removed, and 100 µL DMSO was added to each well. Finally, the absorbance of dissolved formazan was measured, and the cell survival rate was calculated. Figure 6 As shown, the three types of cells treated with different concentrations of the probe molecule all showed good survival rates (the survival rates were close to 100% from the low concentration to the highest detected concentration), and the probe molecule had basically no toxicity to various types of cells. Example

[0032] Study on imaging of HSA in living cells using nanofluorescent probes H226 cells and HUVEC cells were cultured in cell culture flasks containing DMEM and 10% FBS (fetal bovine serum) at 37°C and 5% CO2. The cells were then seeded in confocal culture dishes and allowed to attach overnight. HSA was added to one of the culture dishes and the cells were incubated for 30 min, and then the probe molecules were added to the culture dish and incubated for 30 min. Excess culture medium was removed, the surface was washed with PBS buffer, and the cells were observed under a laser confocal microscope. Figure 7 As shown, before the addition of HSA for culture, H226 and HUVEC cells cultured with only the probe molecule solution showed almost no fluorescence. However, after the probe molecule reacted with HSA, the cells showed bright blue fluorescence, mainly distributed in the cytoplasm. This indicates that NDQC has good cell membrane permeability and can interact with HSA in the cell, thus enabling the observation of the distribution of HSA at the cellular level by fluorescence imaging. Example

[0033] Study on the application of probe-HSA conjugate in the detection of hemin Take 2 mL of PBS buffer (pH = 7.4) in a 3.5 mL fluorescence cuvette and add 20 μL of the probe DMF solution (10 -3 M) and 20 μL HSA solution (10 -3 M), pipetting 30 times to complete the reaction, then adding hemin (0–6 μM) in a gradient manner, and measuring its fluorescence spectrum. Figure 8As shown, with the addition of hemin, the fluorescence intensity of the probe-HSA conjugate gradually decreased and reached the lowest value at 6 μM. In the concentration range of 0–4 μM, there is a linear relationship between the hemin concentration and the fluorescence intensity, so the probe-HSA conjugate can be used as a tool for heme content determination. We further evaluated the ability of the probe-HSA conjugate to detect exogenous hemin in SW1990 cells. When SW1990 cells were incubated with the probe molecule (5 μM) for 30 minutes, no obvious blue fluorescence was observed; in contrast, SW1990 cells stained with the probe molecule and HSA could emit bright blue fluorescence. After incubation with hemin (5 μM) for 30 minutes, the intracellular fluorescence was significantly reduced ( Fig. 9 ). The results showed that NDQC-HSA can be used to sensitively measure the level of free hemin in the cellular environment. Example

[0034] Preparation and characterization of cisplatin-loaded probe-HSA nanocarriers Add 50 μL of HSA aqueous solution (1 mM) to 10 mL of PBS buffer, and slowly add 50 μL of NDIC stock solution (1 mmol) to the above solution with a pipette under high-speed stirring. The above solution was loaded into a dialysis bag that had been boiled and activated for filtration, and dialyzed with PBS buffer for 2 hours to remove single molecules and small particles from the system. The sample was then extracted from the dialysis bag with a pipette, and the inside of the bag was rinsed with a small amount of ultrapure water to ensure complete transfer of the sample. The volume of the above solution was maintained at 5 mL, 50 μL of cisplatin DMF solution (1 mM) was added, and incubated for 2 hours. Then, the solution was added to the dialysis bag and dialyzed in ultrapure water for 24 hours (ultrapure water was replaced every 3 hours) to filter out the unreacted sample. The sample was extracted from the dialysis bag again using a pipette, and the inside of the dialysis bag was rinsed with a small amount of ultrapure water to obtain a sample volume of 8 mL. The resulting solution was sealed and kept in a refrigerator at 4°C. As Fig.10 As shown in a, the addition of cisplatin only resulted in a slight decrease in the fluorescence emission of the NDQC-HSA system, thus enabling fluorescent tracing of the nanocarrier. The UV-visible spectrum of the probe-HSA-cisplatin system in aqueous solution was measured and compared with that of HSA and the probe ( Fig.10 b). The UV absorption peak of HSA is around 280nm, and the absorption peak of NDQC is around 340nm. Correspondingly, for the probe-HSA cisplatin system, the absorption peak at 280nm remained basically unchanged, and the absorption peak at 340nm showed a slight red shift, which indicated that NDQC was labeled on HSA. We further verified the binding of the probe and cisplatin to HSA by MALDI-TOF mass spectrometry. Fig.10As shown in Figure c, when only HSA was present in the system, the maximum peak was observed at 66470 Da. After labeling with the probe, the maximum peak shifted to around 66919 Da, indicating an increase of 449 Da. After further addition of cisplatin, the maximum peak shifted to 67318 Da, resulting in an additional increase of 399 Da, indicating that NDQC-labeled HSA can carry cisplatin. ICP-MS detection confirmed that the platinum content in 1 ppb probe-HSA was 0.953 ppb. The results showed that in the co-incubation experiment, most of the added cisplatin molecules were retained in the probe-HSA system, indicating that cisplatin and probe-HSA have excellent binding effects and the NDQC-HSA system can successfully carry platinum drugs.

Claims

1. Quinoline hydrazide Schiff base fluorescent probe, characterized in that: It has the following structural formula: 。 2. The method for preparing the quinoline hydrazide Schiff base fluorescent probe as claimed in claim 1 is characterized in that Proceed as follows: (1) Dissolve quinoline-2-carboxylic acid in anhydrous ethanol, then add an appropriate amount of concentrated sulfuric acid and heat under reflux for 5-7 h. After the reaction system is cooled, adjust the pH value to 6-9 with saturated NaHCO3 solution, extract with dichloromethane, and remove the solvent by rotary evaporation of the dichloromethane layer to obtain solid quinoline-2-carboxylic acid ethyl ester; (2) dissolving ethyl quinoline-2-carboxylate in methanol, then adding hydrazine hydrate dropwise while stirring, heating and stirring, condensing and refluxing for 1 h, monitoring the reaction by TLC until the reaction of ethyl quinoline-2-carboxylate is complete, and then stopping the reaction; after the reaction solution is cooled to room temperature, concentrating under reduced pressure in vacuo, adding methanol and ethanol, and cyclically rotating and evaporating the solution alternately until hydrazine hydrate is evaporated and removed from the reaction system, thereby obtaining quinoline-2-carboxylic acid hydrazide, wherein the molar ratio of ethyl quinoline-2-carboxylate to hydrazine hydrate is 1:15-30; (3) Dissolve quinoline-2-carboxylic acid hydrazide in anhydrous methanol and dissolve 2,4-dihydroxybenzaldehyde in anhydrous methanol. Slowly add the mixture dropwise to the methanol solution of quinoline-2-carboxylic acid hydrazide under stirring. Heat with stirring and condense the mixture under reflux for 9.5 h. After cooling the reaction solution to room temperature, filter and wash the precipitate. Dry under vacuum to obtain the reaction product, i.e., the quinoline hydrazide Schiff base fluorescent probe. The molar ratio of the feed materials of quinoline-2-carboxylic acid hydrazide and 2,4-dihydroxybenzaldehyde is 1:

1.

3. Use of the quinoline hydrazide Schiff base fluorescent probe according to claim 1 in the qualitative and quantitative detection of HSA, characterized in that: The quinoline hydrazide Schiff base fluorescent probe forms a self-assembled nano fluorescent probe in PBS buffer; the pH of PBS buffer is 7-8.

4. The use according to claim 3, characterized in that: When HSA is added to the probe solution, the solution changes from colorless to cyan under a 365 nm ultraviolet lamp, thereby achieving rapid detection of HSA.

5. The use according to claim 3, characterized in that: Under 370-420 nm excitation light, it has almost no fluorescence signal. After interacting with HSA, it produces a strong cyan fluorescence signal at 495 nm. The quantitative detection concentration range of HSA is 0-8 mM, and the detection limit is 8.49 nM.

6. Use of the quinoline hydrazide Schiff base fluorescent probe according to claim 1 for detecting HSA in living cells and biological samples.

7. The quinoline hydrazide Schiff base fluorescent probe according to claim 1 forms nanoparticles with HSA for detecting hemoglobin.

8. Use of the fluorescent tracer nanoparticles formed by the quinoline hydrazide Schiff base fluorescent probe according to claim 1 and HSA in the delivery of platinum drugs.

9. The use as claimed in claim 8, wherein the synthesis method is prepared by the following reaction: 1 mM HSA aqueous solution was added into PBS buffer, 1 mM probe molecule DMF solution was slowly added into HSA solution with a pipette gun under high-speed stirring, the above solution was loaded into a dialysis bag that had been boiled and activated and filtered, and dialyzed with PBS buffer for 2 hours to remove single molecules and small particles from the system; then the sample was extracted from the dialysis bag with a pipette, and the bag was rinsed with a small amount of PBS buffer to ensure complete transfer of the sample, and the resulting solution was sealed and kept in a refrigerator at 4°C.

Images