Preparation and application of a fluorescent probe for specific detection of human serum albumin

Abstract

The application discloses a preparation and application of a fluorescent probe for specifically detecting human serum albumin, which can be used as a high-sensitivity and high-selectivity HSA fluorescent analysis method. The self-assembled nano fluorescent probe has the advantages of excellent selectivity, good anti-interference ability, rapid response, high sensitivity, good biocompatibility, strong stability, simple preparation, low synthesis cost and the like. The nanoparticles constructed by quinoline hydrazine Schiff base and HSA can be used as a visual carrier to carry platinum drugs. The nanoparticles have the advantages of emitting fluorescence under light excitation, thereby playing a tracing role on the nanoparticles. After the nanoparticles are dissociated, the fluorescence disappears, so that the release of the platinum drugs in a biological system can be monitored.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fluorescent probe detection and nano drug carrier application, more specifically, relates to the application of a quinoline hydrazine Schiff base self-assembled nano fluorescent probe in detecting HSA, and further to the application of the fluorescent labeling of HSA constituted nanoparticles to realize the tracing of drug molecule distribution and metabolism. BACKGROUND

[0002] Human serum albumin (HSA) is a heart-shaped single-chain protein with a molecular weight of 66.5 kDa, which is commonly used in clinical treatment of symptoms such as hemorrhage, shock, burns, polycythemia, and hypoalbuminemia. HSA can serve as a biomarker and play an important role in human health monitoring. Its normal concentration in blood is about 35-55 g / L, and the concentration in urine of healthy individuals should be less than 30 mg / L. Accurate tracking of the content and activity of disease-related proteins is crucial for clinical diagnosis, drug toxicity monitoring, and disease treatment. Microalbuminuria is an early indicator of diabetes, hypertension, cardiovascular disease, and kidney disease. Low HSA levels in plasma, i.e., hypoalbuminemia, can indicate 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 blood, accounting for 52%-60% of the total plasma protein content. It plays a crucial role in maintaining blood osmotic pressure, transporting various substances, and binding hormones, vitamins, fatty acids, calcium ions, and drug molecules. HSA as a drug carrier has excellent bioavailability. 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 therapeutic effect. HSA can bind to poorly water-soluble drugs, promote 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 platinum drug-loaded HSA nanoparticles in the biological body is of great significance for drug development, clinical application, and improving patient treatment outcomes. By combining fluorescent probe molecules with HSA, fluorescent tracing of HSA nano-carriers in biological systems can be achieved. Subsequently, as HSA decomposes, the fluorescent molecules will lose their fluorescent properties. Therefore, the purpose of detecting drug delivery and metabolism in the body can be achieved.

[0004] Traditional HSA detection and analysis methods include dye binding, fluorescence immunoassay, LC-MS, electrophoresis, etc. These methods usually require high material and instrument costs, involve complex sample preparation, poor specificity and anti-interference ability. Small molecule fluorescent probes have been proved to be a promising choice, which have the advantages of easy preparation, fast and convenient detection, non-invasive and non-destructive in biomolecule detection. So far, various HSA probes have been developed for selective and sensitive detection of HSA that may exist in biological samples, but there are still some limitations. For example, some HSA probes show poor water solubility, which hinders their application in biological environment. In addition, some probes are covalently bound to the amino or sulfhydryl group of the protein, causing HSA denaturation. Most HSA fluorescent probes bind to two classic HSA drug binding sites, site I and site II, so the commonly used drug molecules in vivo will cause serious interference to the fluorescence detection. It has great practical value and research significance to develop new small molecule probes for qualitative and quantitative detection of HSA. In recent years, molecular self-assembly HSA probes have become a powerful tool for constructing soft functional materials, and have wide applications in material science and biology. Small molecule organic dyes can be directly assembled into nanoparticles without nano-carriers, showing excellent light stability and biocompatibility, and providing wide flexibility in molecular design. Quinoline as a fluorophore has the advantages of high fluorescence quantum yield, large stokes shift, high light stability, good biocompatibility and adjustable fluorescence characteristics; the amide group can increase the binding affinity to HSA; the assembly design of the hydrazine Schiff base compound makes the different functional groups of the hydrazine 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

[0005] The present application aims to overcome the deficiencies of the prior art, and provides a new quinoline hydrazine Schiff base self-assembled nano fluorescent probe, which can be used as a high sensitivity and high selectivity HSA fluorescent analysis method. Compared with the probes (compounds) in the prior art, the self-assembled nano fluorescent probe of the present application has the advantages of excellent selectivity, good anti-interference ability, fast response, high sensitivity, good biocompatibility, strong stability, simple preparation, low synthesis cost, etc. The nanoparticles constructed by quinoline hydrazine 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 playing a tracking role for the nanoparticles. After the nanoparticles dissociate, the fluorescence disappears, which can monitor the release of platinum drugs in the biological system.

[0006] To achieve the above-mentioned purpose, the present application discloses the following technical content:

[0007] A quinoline hydrazine Schiff base fluorescent probe, characterized in that it has the following structural formula:

[0008] The application further discloses a preparation method of the quinoline hydrazide Schiff base fluorescent probe, and is characterized by the following steps:

[0009] (1) dissolving quinoline-2-carboxylic acid in anhydrous ethanol, then adding appropriate concentrated sulfuric acid and heating to reflux for 5-7 hours, adjusting the pH value to 6-9 with saturated NaHCO3 solution after the reaction system is cooled, extracting with dichloromethane, removing the solvent from the dichloromethane layer by rotary evaporation to obtain solid quinoline-2-carboxylic acid ethyl ester;

[0010] (2) dissolving quinoline-2-carboxylic acid ethyl ester in methanol, then adding hydrazine hydrate drop by drop under stirring, heating and stirring, condensing and refluxing for 1 hour, monitoring the reaction by TLC until the reaction of quinoline-2-carboxylic acid ethyl ester is completely stopped; after the reaction solution is cooled to room temperature, vacuum concentration is carried out, and methanol and ethanol are cycled and alternately rotary evaporated until hydrazine hydrate is evaporated and removed from the reaction system to obtain quinoline-2-formylhydrazine, wherein the molar ratio of quinoline-2-carboxylic acid ethyl ester to hydrazine hydrate is 1:15-30;

[0011] (3) dissolving quinoline-2-formylhydrazine in anhydrous methanol, dissolving 2,4-dihydroxybenzaldehyde in anhydrous methanol, slowly adding the 2,4-dihydroxybenzaldehyde methanol solution to the quinoline-2-formylhydrazine methanol solution under stirring, heating and stirring, condensing and refluxing for 9.5 hours, filtering and washing the precipitate after the reaction solution is cooled to room temperature, and vacuum drying to obtain the reaction product, i.e. the quinoline hydrazide Schiff base fluorescent probe; wherein the molar ratio of quinoline-2-formylhydrazine to 2,4-dihydroxybenzaldehyde is 1:1.

[0012] The application further discloses application of the quinoline hydrazide Schiff base fluorescent probe in qualitative and quantitative detection of HSA, and is characterized in that the quinoline hydrazide Schiff base fluorescent probe forms a self-assembled nanofluorescent probe in a PBS buffer solution; the pH of the PBS buffer solution is 7-8. HSA is added to the probe solution, under a 365 nm ultraviolet lamp, the solution changes from colorless to cyan, and rapid detection of HSA is realized. In addition, under 370-420 nm excitation light, the fluorescent signal of the probe itself is almost zero, after the probe reacts 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. Experimental results show that the quinoline hydrazide Schiff base fluorescent probe has good detection capacity for HSA, high sensitivity, good linear relationship between the fluorescent intensity and the concentration, and can accurately quantitatively measure the HSA concentration.

[0013] The application also discloses application of the quinoline acylhydrazone Schiff base fluorescent probe in detection of HSA in living cells and biological samples.

[0014] The application also discloses application of the quinoline acylhydrazone Schiff base fluorescent probe and HSA in forming fluorescent tracing nanoparticles in carrying platinum drugs.

[0015] 1 mM HSA aqueous solution is added into a PBS buffer solution, 1 mM probe molecule DMF solution is slowly added into the HSA solution under high-speed stirring by using a pipette gun, the solution is loaded into a dialysis bag which has been boiled and activated and filtered, and PBS buffer solution is used for dialysis for 2 hours to remove monomers and small particles from the system; then, a sample is extracted from the dialysis bag by using a pipette, and a small amount of PBS buffer solution is used for flushing the bag to ensure complete transfer of the sample, and the obtained solution is sealed in a refrigerator at 4 DEG C for preservation.

[0016] The application is more specifically as follows

[0017] The application of the quinoline acylhydrazone Schiff base fluorescent probe as HSA detection and platinum drug fluorescent tracing carrier is realized by the following technical scheme:

[0018] The self-assembled nanofluorescent probe formed by the quinoline acylhydrazone Schiff base for HSA detection has the following structural formula:

[0019]

[0020] The self-assembled nanofluorescent probe formed by the quinoline acylhydrazone Schiff base is used for qualitative and quantitative detection of HSA.

[0021] The synthesis of the self-assembled nanofluorescent probe formed by the quinoline acylhydrazone Schiff base is as follows:

[0022] (1) quinoline-2-carboxylic acid is dissolved in anhydrous ethanol, then an appropriate amount of concentrated sulfuric acid is added, and heating reflux is carried out for 5-7 hours; after the reaction system is cooled, the pH value is adjusted to 6-9 by using a saturated NaHCO3 solution, dichloromethane is used for extraction, the dichloromethane layer is rotary evaporated to remove the solvent, and quinoline-2-carboxylic acid ethyl ester is obtained.

[0023] (2) Dissolve ethyl quinoline-2-carboxylate in methanol, then add hydrazine hydrate (N2H4·H2O) drop by drop under stirring, heat and stir, and condense reflux for 1 h. Monitor the reaction by TLC until ethyl quinoline-2-carboxylate is completely reacted (petroleum ether: ethyl acetate = 5:1), and stop the reaction. After the reaction solution is cooled to room temperature, concentrate under vacuum and reduced pressure, and then add methanol and ethanol to be alternately circulated and rotary evaporated until hydrazine hydrate is evaporated and removed, to obtain quinoline-2-carboxylic acid hydrazide.

[0024] In the (2), the molar ratio of ethyl quinoline-2-carboxylate to hydrazine hydrate is 1: (15-30).

[0025] (3) Dissolve quinoline-2-carboxylic acid hydrazide in anhydrous methanol, and dissolve 2,4-dihydroxybenzaldehyde in methanol, then slowly add the solution of 2,4-dihydroxybenzaldehyde in methanol to the quinoline-2-carboxylic acid hydrazide solution under stirring, heat and stir, and condense reflux for 9.5 h. After the reaction solution is cooled to room temperature, filter and wash the precipitate, and then vacuum dry to obtain the reaction product, i.e. the quinoline hydrazide Schiff base fluorescent probe.

[0026] In the (3), the molar ratio of quinoline-2-carboxylic acid hydrazide to 2,4-dihydroxybenzaldehyde is 1:1.

[0027] The synthesis route is as follows:

[0028]

[0029] The quinoline hydrazide Schiff base forms a self-assembled nanofluorescent probe in a PBS buffer.

[0030] The pH of the PBS buffer is 7-8.

[0031] The nanofluorescent probe self-assembled from the quinoline hydrazide Schiff base has almost no fluorescence signal itself under excitation in the excitation wavelength range of 370-420 nm, but produces a very strong fluorescence signal at 495 nm after interacting with HSA, and the concentration range for the quantitative detection of HSA is 0-8 mM, and the detection limit is 8.49 nM.

[0032] The nanofluorescent probe self-assembled from the quinoline hydrazide Schiff base has an excitation wavelength range of 370-420 nm and an emission wavelength of about 495 nm, and the binding site with HSA is the FA1 site, effectively overcoming the shortcomings such as poor water solubility and interference of common drug molecules, and has good selectivity for HSA, and can be used for the detection of HSA in a biological system. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 It is an electron microscope image of the nanofluorescent probe self-assembled from the quinoline hydrazide Schiff base of the present application;

[0034] Figure 2 The selective fluorescence spectrum of the nano-fluorescent probe self-assembled from the quinoline hydrazine Schiff base of the application for 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 of the probe after mixing with various amino acids and proteins at 495 nm; (c) is the fluorescence intensity of the probe after mixing with various metal cations at 495 nm; (d) is the fluorescence intensity of the probe after mixing with various anions at 495 nm;

[0035] Figure 3 The fluorescence signal diagram of the nano-fluorescent probe self-assembled from the quinoline hydrazine Schiff base of the application 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 self-assembled from the quinoline hydrazine Schiff base of the application and the fluorescence spectrum diagram of the response of the fluorescence intensity to different concentrations of HSA (a) and the linear relationship diagram of the change value of the fluorescence intensity at 495 nm and the concentration of HSA (b);

[0036] Figure 5 The binding site displacement diagram of the probe-HSA of the application and warfarin, bilirubin, ibuprofen and chlorinated hematin;

[0037] Figure 6 The cell survival rate of H226 cells (a), HUVEC cells (b) and SW1990 cells (c) treated with different concentrations of the probe for 6 hours;

[0038] Figure 7 The fluorescence imaging diagram of the nano-fluorescent probe of the application and HSA in H226 cells (a) and HUVEC cells (b);

[0039] Figure 8 The fluorescence spectrum of the probe-HSA complex (a) and the linear fitting diagram of the probe-HSA complex titrated with chlorinated hematin in the concentration range of 0-4 μM (b) as the concentration of chlorinated hematin increases;

[0040] Figure 9Fluorescent images of probe-HSA detecting chlorin 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 min; Row 3: SW1990 cells treated with probe molecule, HSA and chlorin (10 μM) in turn for 30 min;

[0041] Figure 10 Characterization of probe-HSA nanoparticles carrying cisplatin; (a) Effect of cisplatin on fluorescence intensity of probe-HSA nanoparticles; (b) UV-Vis spectrum of probe-HSA nanoparticles carrying cisplatin; (c) MALDI-TOF mass spectrum of HSA, probe-HSA and cisplatin-probe-HSA conjugate. DETAILED DESCRIPTION

[0042] The present application is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present application are methods known to those skilled in the art. In addition, the embodiments are understood to be illustrative rather than limiting the scope of the present application, and the essence and scope of the present application are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and amounts in these embodiments without departing from the essence and scope of the present application also fall within the protection scope of the present application. The raw materials and reagents used in the present application are commercially available. For example, quinoline-2-carboxylic acid, hydrazine hydrate, 2,4-dihydroxybenzaldehyde, anhydrous ethanol, concentrated sulfuric acid, dichloromethane, etc. are commercially available. EXAMPLE

[0043] Synthesis and characterization of fluorescent probe

[0044] (1) Quinoline-2-carboxylic acid was dissolved in anhydrous ethanol, then an appropriate amount of concentrated sulfuric acid was added and heated to reflux for 5-7 h. After the reaction system was cooled, the pH value was adjusted to 6-9 with saturated NaHCO3 solution, extracted with dichloromethane, and the dichloromethane layer was rotary evaporated to remove the solvent to obtain quinoline-2-carboxylic acid ethyl ester; quinoline-2-carboxylic acid ethyl ester was dissolved in methanol, then hydrazine hydrate was added dropwise under stirring, heated and stirred, and condensed refluxed for 1 h. The reaction was monitored by TLC until quinoline-2-carboxylic acid ethyl ester was completely reacted (petroleum ether: ethyl acetate = 5:1), and the reaction was stopped. After the reaction liquid was cooled to room temperature, vacuum concentration was carried out, and methanol and ethanol were added and rotary evaporated alternately until hydrazine hydrate was evaporated and removed to obtain quinoline-2-carboxylic acid hydrazide; the molar ratio of quinoline-2-carboxylic acid ethyl ester and hydrazine hydrate was 1:(15-30)

[0045] (2) Dissolve quinoline-2-formylhydrazine in anhydrous methanol, and dissolve 2,4-dihydroxybenzaldehyde in methanol, slowly drop into the quinoline-2-formylhydrazine solution under stirring, heat and stir, and condense reflux for 9.5 h. After cooling the reaction solution to room temperature, filter and wash the precipitate, and dry under vacuum to obtain the reaction product, i.e. the quinoline hydrazide Schiff base fluorescent probe; wherein the molar ratio of quinoline-2-formylhydrazine and 2,4-dihydroxybenzaldehyde is 1:1. Yield 79%. 1 H 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 for [GM-1 + H] + = 308.1030, found 308.1033. Example

[0046] Study on the formation of self-assembled nanofluorescent probes

[0047] Using a micropipette, 20 μL of DMF solution (10 -3M) The quinoline hydrazine Schiff base nanoparticle solution was prepared by adding 2 mL of PBS buffer. Subsequently, the solution was systematically placed in an ultrasonic device for 30 minutes to ensure uniform distribution of the quinoline hydrazine Schiff base nanoparticles in the solution by ultrasonic mixing. The prepared quinoline hydrazine Schiff base self-assembled nanoparticle solution was placed in a cuvette and the dispersion of nanoparticle size was measured at room temperature using DLS (dynamic light scattering) analysis, which had an average particle size of 339.4 nm. For morphological studies, the prepared quinoline hydrazine Schiff base self-assembled nanoparticle solution was slowly added to the surface of the copper mesh 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 images were captured using a transmission electron microscope (TEM), which showed that the nanoparticles formed were regular and well-dispersed spheres Figure 1 ). Example

[0048] Selective study of the nanofluorescent probe for HSA

[0049] Various common substances present in life systems and the environment were subjected to fluorescence detection, 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 prove 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) was added, and then an equivalent amount of each substance was added, and after stirring uniformly at room temperature, the fluorescence emission spectrum of each was tested under excitation at an excitation wavelength of 370 nm. As shown in Figure 2 , only after the addition of HSA, the probe showed a significant fluorescence enhancement. Under the same conditions, no obvious fluorescence response was observed for other substances. For bovine serum albumin BSA with homologous structure, only a small fluorescence change was induced 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

[0050] Study of the effect of different substances on the detection of HSA by nano-fluorescent probe

[0051] In 2 mL of PBS buffer solution (pH 7.4), 10 μL of probe solution (10 -3 M) and other solutions of metal ions, anions, amino acids and proteins (10 -3 M) were added, and then 10 μL of HSA solution (10 -3 M) was added, and the fluorescence signal change was tested after mixing. As shown in Figure 3 , in the presence of various potential interfering substances, the fluorescence intensity of the probe HSA conjugate changed little, making it suitable for complex samples and biological environments. Example

[0052] Study of the response of nano-fluorescent probe to different concentrations of HSA

[0053] In 2 mL of PBS buffer solution (pH 7.4), 20 μL of DMF solution of the probe (10 -3 M) was added, and then different concentrations of HSA solution (0-10 μM) were added, and the fluorescence spectrum was tested. As shown in Figure 4As shown, the probe NDQC has almost no fluorescence in the absence of HSA. With the increase of HSA concentration, the fluorescence intensity at 495 nm gradually increases. When the HSA concentration is in the range of 0 to 8 μM, there is a linear relationship between the fluorescence intensity and the HSA concentration. It indicates that the nano-fluorescent probe molecule can quantitatively detect HSA, and the detection limit is 8.49 nM (0.56 mg / L) Example

[0054] Study on the binding site of the fluorescent probe and HSA

[0055] Drug displacement experiments were performed to study the binding of specific drugs to HSA, in which warfarin and bilirubin were selected as Sudlow site I specific binding drugs, ibuprofen was selected as Sudlow site II binding drug, and hematin was selected as hematin site binding drug. As shown in Figure 5 As shown, when the concentration of the four drugs in the probe-HSA system increases, the effect of hematin on fluorescence intensity is significantly greater than that of warfarin, bilirubin and ibuprofen, indicating that the probe is more inclined to bind to HSA through the hematin site. Example

[0056] Cell toxicity experiment of nano-fluorescent probe

[0057] H226 cells, HUVEC cells and SW1990 cells were respectively placed in DMEM medium at 37°C, 95% air and 5% CO2. Then the cells were inoculated 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 survival rate of cells was calculated. As shown in Figure 6 As shown, all three types of cells treated with different concentrations of probe molecules showed good survival rates (close to 100% from low concentration to the highest detection concentration), and the probe molecule had little toxicity to the cells. Example

[0058] Study on the imaging of nano-fluorescent probe for HSA in living cells

[0059] H226 cells and HUVEC cells were cultured in cell culture flasks with DMEM and 10% FBS (fetal bovine serum) at 37°C in a 5% CO2 environment. Then the cells were seeded in confocal dishes respectively and attached overnight. One of the dishes was added with HSA to incubate the cells for 30 min, and then the probe molecule was added to incubate for 30 min. After removing the extra medium, the surface was washed with PBS buffer, and the cells were observed under laser confocal microscope. As shown in Figure 7 , before adding HSA for incubation, H226 and HUVEC cells cultured with probe molecule solution only showed little fluorescence. However, after the reaction of probe molecule 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, so it can observe the distribution of HSA at the cell level by fluorescence imaging. Example

[0060] Application of probe-HSA conjugate in detection of chlorin

[0061] Take 2 mL PBS buffer (pH = 7.4) in 3.5 mL fluorescence cuvette, add 20 μL probe DMF solution (10 -3 M) and 20 μL HSA solution (10 -3 M), blow 30 times to make the reaction complete, and then add chlorin (0-6 μM) in gradient, measure the fluorescence spectrum. As shown in Figure 8 , with the addition of chlorin, the fluorescence intensity of probe-HSA conjugate gradually decreases, reaching the minimum value at 6 μM. In the concentration range of 0-4 μM, the concentration of chlorin is linearly related to the fluorescence intensity, so the probe-HSA conjugate can be used as a tool for determination of hematin content. We further evaluated the detection ability of probe-HSA conjugate for exogenous chlorin in SW1990 cells. When SW1990 cells were incubated with probe molecule (5 μM) for 30 minutes, no obvious blue fluorescence was observed; in contrast, SW1990 cells stained with probe molecule and HSA can emit bright blue fluorescence. After adding chlorin (5 μM) and incubating for 30 minutes, the intracellular fluorescence decreased significantly Figure 9 . The results show that NDQC-HSA can be used to sensitively determine the level of free chlorin in the cell environment. Example

[0062] Preparation and characterization of probe-HSA nanocarrier loaded with cisplatin

[0063] To 10 mL of PBS buffer, 50 μL of HSA aqueous solution (1 mM) was added, and 50 μL of NDIC stock solution (1 mmol) was slowly added into 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, and dialyzed for 2 hours with PBS buffer to remove monomers and small particles from the system. Then the sample was 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, and 50 μL of cisplatin DMF solution (1 mM) was added, and incubated for 2 hours. Then, the solution was added to a dialysis bag and dialyzed in ultrapure water for 24 hours (changed every 3 hours), to filter out unreacted samples. The sample was again extracted from the dialysis bag with 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 the refrigerator at 4 °C. As shown in (a) of FIG. 1, Figure 10 The addition of cisplatin only caused a slight decrease in the fluorescence emission of the NDQC-HSA system, so that fluorescence tracing of the nanocarrier could be achieved. The UV-Vis spectrum of the probe-HSA-cisplatin system in aqueous solution was measured and compared with that of HSA and the probe (b) of FIG. 1. Figure 10 The UV absorption peak of HSA is around 280 nm, and the absorption peak of NDQC is around 340 nm. Correspondingly, for the probe-HSA-cisplatin system, the absorption peak at 280 nm remains essentially unchanged, and the absorption peak at 340 nm shows a slight red shift, indicating that the NDQC label is on HSA. We further verified the binding of the probe and cisplatin to HSA by MALDI-TOF mass spectrometry. As shown in (c) of FIG. 1, Figure 10 When only HSA is present in the system, a maximum peak is observed at 66470 Da. After labeling with the probe, the maximum peak shifts to around 66919 Da, indicating an increase of 449 Da. After further addition of cisplatin, the maximum peak shifts to 67318 Da, resulting in an additional increase of 399 Da, which indicates that the NDQC-labeled HSA can carry cisplatin. Through ICP-MS detection, it was confirmed that the platinum content in 1 ppb of probe-HSA was 0.953 ppb. The results show that in the co-incubation experiment, most of the added cisplatin molecules remain in the probe-HSA system, indicating that cisplatin and probe-HSA have excellent binding effect, and the NDQC-HSA system can successfully carry platinum drugs.

Claims

1. Use of a quinoline hydrazide Schiff base fluorescent probe and HSA to form nanoparticles in the preparation of a reagent for detecting hemoglobin; the fluorescent probe has the following structure: .

2. Use of a quinoline hydrazide Schiff base fluorescent probe and HSA to form nanoparticles in the preparation of a drug carrier for cisplatin delivery. The structure of the fluorescent probe is as follows: .

Images