A fluorescent probe and a preparation method and application thereof

Abstract

The application provides a novel fluorescent probe Nap-NO2 for rapid and accurate detection of human serum albumin. A preparation method comprises the reaction of N-n-butyl-3-aldehyde-4-hydroxy-1,8-naphthalimide and pyridine salt. The fluorescent probe Nap-NO2 has the advantages of red light emission, rapid response, strong specific selectivity, low detection limit, good stability, simple detection process and the like, and has strong application value in the preparation of human serum albumin detection products and the screening or detection of human serum albumin IB domain related drugs.

Description

Technical Field

[0001] This invention relates to the field of fluorescence detection, specifically to a fluorescent probe for detecting human serum albumin, its preparation method, and its application. Background Technology

[0002] As is well known, human serum albumin (HSA) is the most abundant protein component in human plasma (40 kg / m³). 3 Human serum albumin (or 0.6 mM) possesses numerous physiological functions. It contains over 580 amino acids, including one tryptophan and 17 tyrosine residues. X-ray crystallography reveals that human serum albumin comprises three homologous domains (I-III), each containing two subdomains: A and B. The pharmacokinetic and pharmacodynamic characteristics of human serum albumin are altered due to the interactions of the A and B subdomains with metabolites and drugs in vivo, further influencing its distribution and activity at biological targets. Human serum albumin can bind to many endogenous and exogenous compounds, such as cholesterol, fatty acids, retinol, and retinoic acid, promoting their transport in the circulatory system. Urinary human serum albumin can also serve as an indicator for detecting chronic kidney disease. Furthermore, disturbances in the quantity and structure of human serum albumin are closely associated with life-threatening diseases such as cancer and liver failure.

[0003] To date, numerous methods have been developed for the detection of human serum albumin, including electrochemical and liquid chromatography-mass spectrometry (LC-MS). Meanwhile, detection methods using small-molecule fluorescent probes have attracted widespread attention due to their high sensitivity, good selectivity, rapid reaction speed, and ease of operation. Importantly, "on-hook" fluorescent probes play a crucial role in understanding the dynamics of biological processes. In recent years, many fluorescent probes have been reported for the detection of human serum albumin; however, these probes still have some inherent limitations, such as detection limits, short excitation and emission wavelengths, and difficulty in distinguishing between human serum albumin and bovine serum albumin.

[0004] Chinese patent document CN104341346A discloses a fluorescent probe, which is a bibenzoyl ester derivative with the C-4 hydroxyl group of the N-n-butyl-4-hydroxy-1,8-naphthalimide parent compound substituted, used for the quantitative determination of human serum albumin content. The excitation wavelength is 300-500 nm, and the emission wavelength is 410-600 nm. The detection principle of this fluorescent probe is based on the albumin pseudoesterase hydrolysis reaction. A pre-hydrolysis reaction is required before fluorescence detection, taking 5-120 minutes. The detection process requires simultaneous detection of the fluorescence of the probe substrate and the hydrolysis product, making the operation complex and time-consuming. Furthermore, there is an overlap between the excitation and emission wavelengths, affecting the accuracy of the results.

[0005] Therefore, developing fluorescent probes with long emission wavelengths, good selectivity, and high sensitivity for the detection of human serum albumin remains a challenge. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a novel fluorescent dye molecular probe for the rapid and accurate detection of human serum albumin (HSA). It can extend the emission wavelength, has good selectivity, high sensitivity, good stability, and can perform qualitative and quantitative detection. Moreover, the detection method is simple.

[0007] In the structural design of the fluorescent probe, this invention uses a carbon-carbon double bond as a link to combine the 4-hydroxy-1,8-naphthalimide fluorophore with a pyridine salt group that can improve water solubility, thus obtaining a novel fluorescent probe.

[0008] To achieve the objective of this invention, this invention provides a fluorescent probe having the structure shown in formula (Ⅰ).

[0009]

[0010] Wherein X is a halogen, selected from F, Cl, Br or I. Preferably, X is selected from Br or I.

[0011] The present invention also provides a method for preparing the fluorescent probe shown in formula (I), which is to obtain the fluorescent probe by reacting the compound shown in formula (II) with the compound shown in formula (III).

[0012]

[0013] Wherein X is a halogen, selected from F, Cl, Br or I. Preferably, X is selected from Br or I.

[0014] The fluorescent probe shown in formula (I) has the chemical name 4-[(N-n-butyl-4-hydroxy-1,8-naphthylimide)-3-vinyl]-1-(4-nitrobenzyl)pyridine-1-halonium halide, which is also named "Nap-NO2" in this invention. When X is Br, the fluorescent probe shown in formula (I) has the chemical name 4-[(N-n-butyl-4-hydroxy-1,8-naphthylimide)-3-vinyl]-1-(4-nitrobenzyl)pyridine-1-bromide, which is also named "Nap-NO2-a" in this invention; when X is I, the fluorescent probe shown in formula (I) has the chemical name 4-[(N-n-butyl-4-hydroxy-1,8-naphthylimide)-3-vinyl]-1-(4-nitrobenzyl)pyridine-1-iodide, which is also named "Nap-NO2-b" in this invention.

[0015] The chemical name of the compound shown in formula (II) is N-n-butyl-3-aldehyde-4-hydroxy-1,8-naphthalimide, which is also named "Nap-CHO" in this invention.

[0016] The chemical name of the compound shown in formula (III) is 4-methyl-1-(4-nitrobenzyl)pyridine-1-halogenium, which is also named "Py-NO2" in this invention. When X is Br, the chemical name of the compound shown in formula (III) is 4-methyl-1-(4-nitrobenzyl)pyridine-1-bromide, which is also named "Py-NO2-a" in this invention; when X is I, the chemical name of the compound shown in formula (III) is 4-methyl-1-(4-nitrobenzyl)pyridine-1-iodide, which is also named "Py-NO2-b" in this invention.

[0017] In the above method for preparing the fluorescent probe Nap-NO2:

[0018] Preferably, the molar ratio of the compound (Nap-CHO) shown in formula (II) to the compound (Py-NO2) shown in formula (III) is 1:1 to 1:2.

[0019] Preferably, the reaction is carried out under an inert gas atmosphere in the presence of pyridine.

[0020] Preferably, the reaction is carried out under reflux conditions.

[0021] In a further preferred embodiment, compound Nap-CHO can be prepared by reacting compound Nap-O with hexamethylenetetramine.

[0022]

[0023] In the preparation method of the above compound Nap-CHO:

[0024] Preferably, the molar ratio of compound Nap-O to hexamethylenetetramine is 1:1 to 1:3.

[0025] Preferably, the reaction is carried out in the presence of trifluoroacetic acid.

[0026] Preferably, the reaction temperature is 80–90°C and the reaction time is 5–8 hours.

[0027] In a further preferred embodiment, compound Nap-O can be prepared using 4-bromo-1,8-naphthalenedicarboxylic anhydride as a starting material, comprising the following steps: (a) reacting 4-bromo-1,8-naphthalenedicarboxylic anhydride with n-butylamine to obtain compound Nap-Br; (b) reacting compound Nap-Br with a methoxylating agent to obtain compound Nap-OCH3; (c) removing the methyl group from the methoxy group of compound Nap-OCH3 to obtain compound Nap-O.

[0028]

[0029] The chemical name of the compound Nap-Br is N-n-butyl-4-bromo-1,8-naphthalimide.

[0030] The chemical name of the compound Nap-OCH3 is N-n-butyl-4-methoxy-1,8-naphthalimide.

[0031] The chemical name of the compound Nap-O is N-n-butyl-4-hydroxy-1,8-naphthalimide.

[0032] In the preparation method of the above compound Nap-O:

[0033] Preferably, in step (a), the molar ratio of 4-bromo-1,8-naphthalenedicarboxylic anhydride to n-butylamine is 1:1 to 1:3, and the reaction is carried out under reflux.

[0034] Preferably, the methoxylating agent in step (b) includes, but is not limited to, methanol, sodium methoxide, and methyl sulfate, and the molar ratio of the methoxylating agent to the compound Nap-Br is at least 1:1.

[0035] Preferably, the reagents for demethylation in step (c) include, but are not limited to, hydroiodic acid and hydrobromic acid, and the molar ratio of the demethylation reagent to compound Nap-OCH3 is at least 1:1.

[0036] The fluorescent probe Nap-NO2 prepared in this invention was structurally confirmed by proton nuclear magnetic resonance (NMR) spectroscopy, carbon nuclear magnetic resonance (NMR) spectroscopy, and high-resolution mass spectrometry (HR-MS), proving that it has the structure of formula (I).

[0037] Studies have shown that under 500 nm excitation, Nap-NO2 solution alone exhibits only weak fluorescence emission. However, when human serum albumin is added to Nap-NO2 solution, the fluorescence emission intensity at 630 nm is significantly enhanced, with a fluorescence response time of only 5 seconds, maintaining high fluorescence intensity for up to 60 minutes after the response. Furthermore, when bovine serum albumin, adenosine triphosphate, heparin, protamine sulfate, concanavalin A, trypsin, various amino acids, and other substances are added to Nap-NO2 solution, the resulting solution shows no significant change in fluorescence emission intensity compared to Nap-NO2 solution alone. Therefore, Nap-NO2 can be used as a fluorescent probe for the detection of human serum albumin or for the preparation of human serum albumin detection products, offering advantages such as high fluorescence specificity and selectivity, rapid response, strong signal, low interference, accurate results, and good stability.

[0038] The present invention also provides a reagent, test strip or kit for detecting human serum albumin, wherein the reagent contains the fluorescent probe Nap-NO2.

[0039] The present invention also provides a visual fluorescence detection method, comprising the following steps: fully contacting the fluorescent probe Nap-NO2 with the sample to be tested, and if bright red fluorescence emission is observed under visible light irradiation, it indicates that the sample to be tested contains human serum albumin.

[0040] The present invention also provides a fluorescence detection method for human serum albumin, comprising the following steps: using 450-550 nm as the excitation wavelength, detecting the fluorescence emission intensity of a mixture of test samples containing the fluorescent probe Nap-NO2 at 620-640 nm.

[0041] Preferably, the working concentration of the fluorescent probe Nap-NO2 is 0.2 × 10⁻⁶. -6 mol / L~5×10 -6 mol / L.

[0042] Preferably, quantitative detection is performed by linear correlation between human serum albumin concentration and fluorescence emission intensity; more preferably, the quantitative detection uses 500 nm as the excitation wavelength to detect the fluorescence emission intensity at 630 nm.

[0043] Based on the linear correlation experiment of the fluorescent probe Nap-NO2 recognizing human serum albumin, under the excitation condition of 500 nm, the concentration of human serum albumin solution was linearly correlated with the fluorescence emission intensity at 630 nm, with a linear range of 0–20 μg / mL. The linear equation was y = 26595.15791 + 20537.43171x, and the fitting similarity was 0.99923. The calculated detection limit (3σ / k) was 0.264 μg / mL. This indicates that the fluorescent probe Nap-NO2 has the advantages of linear response, low detection limit, and high sensitivity, and has strong application value.

[0044] In the fluorescence detection method of the present invention, the sample to be tested is a water sample or a biological sample. The biological sample includes, but is not limited to, human serum samples, plasma samples, whole blood samples, urine samples, and tissue fluid samples.

[0045] In a drug fluorescence response experiment, the effect of drug addition on the fluorescent probe / human serum albumin complex solution allowed for the inference of the drug's binding site on human serum albumin. The fluorescent probe Nap-NO2 was confirmed to have entered the IB domain of human serum albumin, rather than its commonly used drug-targeting sites (IIA and IIIA domains). This allows the probe to avoid interference from commonly used drugs. This characteristic also suggests that the fluorescent probe Nap-NO2 can be used for screening or detecting drugs related to the IB domain of human serum albumin, demonstrating potential application value. These drugs related to the IB domain of human serum albumin include, but are not limited to, small molecule drugs, fusion protein drugs, antibody drugs, vaccines, and diagnostic reagents.

[0046] This invention synthesizes a novel fluorescence-enhanced probe, Nap-NO2, which exhibits excellent specificity and selectivity for human serum albumin. It shows no significant response to bovine serum albumin, adenosine triphosphate (ATP), trypsin, various amino acids, or other substances. It possesses advantages such as red light emission, rapid response (only 5 seconds), good stability (stable fluorescence intensity within 60 minutes), low detection limit (0.264 μg / mL), low interference, high sensitivity, and simple detection process. It has strong practical value in the preparation of detection products for human serum albumin. Further research suggests that Nap-NO2 enters the IB domain of human serum albumin, thus possessing potential application value in the screening or detection of related drugs. Attached Figure Description

[0047] Figure 1 This is a fluorescence selectivity diagram of the fluorescent probe Nap-NO2, with the horizontal axis representing wavelength (nm) and the vertical axis representing fluorescence intensity (au).

[0048] Figure 2 The graph shows the response time of the fluorescent probe Nap-NO2 to human serum albumin, with the horizontal axis representing time (s) and the vertical axis representing fluorescence intensity (au).

[0049] Figure 3 This is a graph showing the stability of the fluorescent probe Nap-NO2. The horizontal axis represents time (min), and the vertical axis represents fluorescence intensity (au).

[0050] Figure 4 The fluorescence titration diagram for the recognition of human serum albumin by the fluorescent probe Nap-NO2 is shown, with the horizontal axis representing wavelength (nm) and the vertical axis representing fluorescence intensity (au).

[0051] Figure 5 The graph shows the linear correlation between the fluorescent probe Nap-NO2 and human serum albumin (HSA). The x-axis represents the HSA solution concentration (μg / mL), and the y-axis represents the fluorescence intensity (au).

[0052] Figure 6 The graph shows the response of the fluorescent probe Nap-NO2 and the reference fluorescent probes Nap-C and Nap-OH to human serum albumin. The horizontal axis represents time (min) and the vertical axis represents fluorescence intensity (au).

[0053] Figure 7 A schematic diagram illustrating the mechanism by which the fluorescent probe Nap-NO2 recognizes human serum albumin.

[0054] Figure 8 The graph shows the fluorescence response of the fluorescent probe Nap-NO2 to the drug, with the horizontal axis representing the drug concentration (μM) and the vertical axis representing the normalized intensity.

[0055] Figure 9 The proton nuclear magnetic resonance spectrum of Nap-NO2 ( 1 H NMR).

[0056] Figure 10 The carbon NMR spectrum of Nap-NO2 ( 13 (C NMR).

[0057] Figure 11 High-resolution mass spectrometry (HR-MS) for Nap-NO2.

[0058] Figure 12 The proton nuclear magnetic resonance spectrum of Nap-OH ( 1 H NMR).

[0059] Figure 13 The carbon NMR spectrum of Nap-OH ( 13 (C NMR).

[0060] Figure 14 High-resolution mass spectrometry (HR-MS) for Nap-OH.

[0061] Figure 15 The proton nuclear magnetic resonance spectrum of Nap-C ( 1 H NMR).

[0062] Figure 16 The carbon NMR spectrum of Nap-C ( 13 (C NMR).

[0063] Figure 17 High-resolution mass spectrometry (HR-MS) for Nap-C. Detailed Implementation

[0064] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the claims of this application.

[0065] The models of the detection equipment are: fluorescence spectrometer (Edinburgh FS-5, UK), ultraviolet-visible spectrometer (Varian Cary-300, USA), nuclear magnetic resonance spectrometer (Bruker advanced II (400M Hz), Germany), and high-resolution mass spectrometer (Shimadzu LCMS-IT-TOF, Japan).

[0066] Example 1: Preparation and structural confirmation of compounds

[0067] The synthesis route is as follows:

[0068]

[0069] I. Preparation of Pyridine Salts

[0070] Take 0.50 mL of 4-methylpyridine and iodoethanol (5.05 mmol), 4-methylpyridine and iodoethane (5.05 mmol), and 4-methylpyridine and p-nitrobenzyl bromide (5.05 mmol) and add them to 50 mL round-bottom flasks respectively. Use acetonitrile as solvent and heat under nitrogen protection at 70 °C for reflux for 5 hours. After the reaction is completed, distill the reaction solution under reduced pressure to obtain the corresponding pyridine salts, named Py-OH, Py-C and Py-NO2-a.

[0071] Py-NO2-b was prepared using the method described above.

[0072] II. Preparation of the fluorescent probe Nap-NO2

[0073] (1) 2.77 g (10 mmol) of 4-bromo-1,8-naphthalenedicarboxylic anhydride was added to a 50 mL round-bottom flask. 20 mL of anhydrous ethanol was used as the solvent, and 2 mL (20 mmol) of n-butylamine was added. The mixture was heated under nitrogen protection in an oil bath at 80 °C for 5 hours. After the reaction was completed, the mixture was filtered under reduced pressure and dried to obtain N-n-butyl-4-bromo-1,8-naphthalimide (named Nap-Br).

[0074] (2) Take methanol (30 mL), N-n-butyl-4-bromo-1,8-naphthalimide (1.60 g, 4.802 mmol) and anhydrous potassium carbonate (2.00 g, 14.5 mmol) and add them to a 50 mL round-bottom flask. Heat under nitrogen protection at 80 °C for reflux for 8 hours. After the reaction is complete, filter under reduced pressure to obtain N-n-butyl-4-methoxy-1,8-naphthalimide (named Nap-OCH3).

[0075] (3) Add 666 mg (2.35 mmol) of N-n-butyl-4-methoxy-1,8-naphthalimide and 8 mL of 55% HI solution to a 50 mL round-bottom flask and heat overnight at 140 °C in the dark under nitrogen protection. After the reaction is complete, slowly pour the solution into a beaker containing 400 mL of ice water and stir continuously until a precipitate is observed. Filter under reduced pressure and purify by silica gel column chromatography to obtain N-n-butyl-4-hydroxy-1,8-naphthalimide (named Nap-O).

[0076] (4) Take N-n-butyl-4-hydroxy-1,8-naphthalimide (1.29 g, 4.785 mmol), hexamethylenetetramine (1.43 g, 9.57 mmol) and trifluoroacetic acid (10 mL) and add them to a 100 mL round bottom flask. Heat at 85 °C for 6 hours under nitrogen protection. After the reaction is complete, pour it into 500 mL of ice water, filter under reduced pressure, and purify by silica gel column chromatography to obtain N-n-butyl-3-aldehyde-4-hydroxy-1,8-naphthalimide (named Nap-CHO).

[0077] (6) Dissolve N-n-butyl-3-aldehyde-4-hydroxy-1,8-naphthimide (100 mg, 0.34 mmol) and pyridinium salt Py-NO2-a (0.34 mmol) in ethanol (10 mL), add piperidine (0.1 mL), and heat under nitrogen protection in an oil bath at 80 °C for 8 hours under reflux. After the reaction is complete, filter under reduced pressure to obtain crude product, recrystallize three times with anhydrous ethanol to obtain the fluorescent probe Nap-NO2-a.

[0078] (7) Referring to the method in (6), the fluorescent probe Nap-NO2-b was prepared using pyridine salt Py-NO2-b as the raw material.

[0079] III. Preparation of Reference Fluorescent Probes Nap-OH and Nap-C

[0080] N-n-butyl-3-aldehyde-4-hydroxy-1,8-naphthimide (100 mg, 0.34 mmol) and pyridinium salt Py-OH (0.34 mmol) were dissolved in ethanol (10 mL), and piperidine (0.1 mL) was added. The mixture was heated under nitrogen protection in an oil bath at 80 °C for 8 hours under reflux. After the reaction was complete, the crude product was obtained by vacuum filtration and recrystallized three times with anhydrous ethanol to obtain the reference fluorescent probe Nap-OH.

[0081] By replacing Py-OH with Py-C and keeping other conditions the same, the reference fluorescent probe Nap-C was obtained.

[0082] IV. Structural Confirmation of Compounds

[0083] By nuclear magnetic resonance hydrogen spectrum ( 1 H NMR, carbon nuclear magnetic resonance (NMR) 13 The structures of Nap-NO2, Nap-OH, and Nap-C were confirmed by C10 NMR and high-resolution mass spectrometry (HR-MS). Figure 9-17 ).

[0084] Nap-NO2, m / z: 508.1859 (calculated: 508.1867); Nap-OH, m / z: 417.1804 (calculated: 417.1809); Nap-C, m / z: 401.1854 (calculated: 401.1860).

[0085] Example 2: Selectivity of the fluorescent probe Nap-NO2

[0086] Prepare 1×10 using DMSO -3 mol / L Nap-NO2 solution.

[0087] Prepare a 2 mg / mL human serum albumin (HSA) solution using distilled water.

[0088] Prepare 2 mg / mL solutions of other substances using distilled water. The other substances are selected from bovine serum albumin (BSA), adenosine triphosphate (ATP), heparin (Hep), protamine sulfate (PRTM), concanavalin A (Con A), trypsin, histidine (His), aspartic acid (Asp), isoleucine (Ile), phenylalanine (Phe), methionine (Met), valine (Val), serine (Ser), cysteine ​​(Cys), alanine (Ala), acetylcysteine ​​(NAC), leucine (Leu), threonine (Thr), proline (Pro), tyrosine (Tyr), glutamine (Gln), and tryptophan (Trp).

[0089] 2 mL of water and 2 μL of the above Nap-NO2 solution were added to each cuvette, followed by 20 μL each of the above human serum albumin solution and other substance solutions. The selectivity of the probe Nap-NO2 for human serum albumin and other substances was investigated using fluorescence spectroscopy. The fluorescence emission intensity at 630 nm was detected under 500 nm excitation conditions. The results are as follows: Figure 1 As shown, the Nap-NO2 solution (Blank) alone has a weak fluorescence emission intensity at 630 nm. When human serum albumin is added, the fluorescence emission intensity at 630 nm is significantly enhanced. However, when other substances are added, the fluorescence emission intensity of the resulting solution system does not change significantly compared with the fluorescence emission intensity of the probe solution alone.

[0090] The above experimental results show that the fluorescent probe Nap-NO2 has good fluorescence specificity and selectivity for human serum albumin, but no obvious response to bovine serum albumin, adenosine triphosphate, heparin, protamine sulfate, concanavalin A, trypsin, various amino acids and other substances.

[0091] Example 3: Response time of fluorescent probe Nap-NO2 to human serum albumin

[0092] Prepare 1×10 using DMSO -3 mol / L Nap-NO2 solution.

[0093] Prepare a 2 mg / mL human serum albumin solution using distilled water.

[0094] Add 2 mL of water, 2 μL of the above Nap-NO2 solution, and 18 μL of the above human serum albumin solution to a cuvette. Use a fluorescence spectrometer to investigate the response time of the fluorescent probe Nap-NO2 to human serum albumin. The excitation wavelength is 500 nm, and the fluorescence emission intensity at 630 nm is detected.

[0095] Figure 2 The results showed that Nap-NO2 responded to human serum albumin within 5 seconds. This demonstrates that the fluorescent probe Nap-NO2 can respond rapidly to human serum albumin, enabling rapid detection. Therefore, the fluorescent probe Nap-NO2 is suitable for preparing detection products for human serum albumin, such as reagents, test strips, or kits, offering rapid, accurate, and strong signal response.

[0096] Example 4: Stability of the fluorescent probe Nap-NO2

[0097] Prepare 1×10 using DMSO -3 mol / L Nap-NO2 solution.

[0098] Prepare a 2 mg / mL human serum albumin solution using distilled water.

[0099] Add 2 mL of water and 2 μL of the above Nap-NO2 solution to a cuvette and incubate for 60 min. Detect the fluorescence emission intensity at 630 nm using a fluorescence spectrometer with an excitation wavelength of 500 nm and a detection interval of 5 min.

[0100] Add 2 mL of water, 2 μL of the above Nap-NO2 solution, and 18 μL of the above human serum albumin solution to a cuvette and incubate for 60 min. Detect the fluorescence response of Nap-NO2 to human serum albumin using a fluorescence spectrometer. The excitation wavelength is 500 nm, and the fluorescence emission intensity at 630 nm is measured at 5 min intervals.

[0101] Test results as follows Figure 3 As shown, the Nap-NO2 probe emitted weak fluorescence over 60 minutes. However, the fluorescence intensity emitted by the Nap-NO2 probe in recognizing human serum albumin remained at a high level with almost no change over the same period. These results indicate that the Nap-NO2 probe exhibits good stability.

[0102] Example 5: Fluorescence titration and linear correlation plot of the fluorescent probe Nap-NO2 recognizing human serum albumin.

[0103] Prepare a 1×10⁻³ mol / L Nap-NO₂ solution using DMSO.

[0104] Prepare a 2 mg / mL human serum albumin solution using distilled water.

[0105] 2 mL of water and 2 μL of the above Nap-NO2 solution were added to 11 clean fluorescence cuvettes, respectively. Volumes of the above human serum albumin solution were gradually added to each cuvette at levels of 0 μL, 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, 12 μL, 14 μL, 16 μL, 18 μL, and 20 μL. The fluorescence emission intensity of each sample was measured using a fluorescence spectrometer with an excitation wavelength of 500 nm.

[0106] A fluorescence titration diagram of the fluorescent probe Nap-NO2 recognizing human serum albumin was obtained by plotting the fluorescence emission wavelength on the x-axis and the fluorescence emission intensity on the y-axis. Figure 4 ). Figure 4 The results showed that as the concentration of human serum albumin solution in the cuvette gradually increased to 20 μg / mL, the fluorescence emission intensity of the probe Nap-NO2 increased accordingly; and, at different concentrations of human serum albumin solution, the main peak of the fluorescence emission curve was basically located at 630 nm.

[0107] A linear correlation graph was obtained by plotting the HSA solution concentration on the x-axis and the fluorescence emission intensity at 630 nm on the y-axis to obtain the recognition of human serum albumin by the fluorescent probe Nap-NO2. Figure 5 ). Figure 5 The results showed that the HAS solution concentration was linearly correlated with the fluorescence emission intensity, with a linear range of 0–20 μg / mL. The linear equation was y = 26595.15791 + 20537.43171x, and the fitting similarity was 0.99923. Calculation (3σ / k) showed that the detection limit of the fluorescent probe Nap-NO2 for human serum albumin was 0.264 μg / mL. This indicates that the probe has the advantages of linear response, low detection limit, and high sensitivity, and has strong application value.

[0108] Example 6: Response of Nap-NO2, Nap-OH, and Nap-C to Human Serum Albumin

[0109] Prepare solutions of Nap-NO2, Nap-OH, and Nap-C at a concentration of 1×10-3 mol / L using DMSO.

[0110] Prepare a 2 mg / mL human serum albumin solution using distilled water.

[0111] Take 2 μL each of the above Nap-NO2, Nap-OH, and Nap-C solutions and add them to a clean fluorescent cuvette. Then add 2 mL of water and 20 μL of the above human serum albumin solution, respectively. Measure the fluorescence emission intensity on a fluorescence spectrometer using excitation wavelengths of 478 nm, 472 nm, and 500 nm to obtain the response graphs of Nap-NO2, Nap-OH, and Nap-C to human serum albumin. Figure 6 ).

[0112] Figure 6 The results showed that Nap-NO2, Nap-OH, and Nap-C all responded to human serum albumin in the red region, but compared with Nap-OH and Nap-C, Nap-NO2 itself had the weakest fluorescence emission intensity, thus causing less interference with HSA detection and resulting in more accurate detection results.

[0113] Example 7: Mechanism Discussion

[0114] The detection mechanism of the fluorescent probe Nap-NO2 of this invention was explored using reference fluorescent probes Nap-OH and Nap-C.

[0115] Nap-OH and Nap-C serve as controls, possessing the same donor-π-acceptor structure as Nap-NO2. In the synthesis and structure-activity relationship studies of dye molecular probes, different electron-pulling and electron-pulling groups can alter the probe structure to adjust the properties of fluorescent probes. Compared to Nap-OH with the electron-pulling group -OH and Nap-C without the electron-pulling and electron-pulling group -C, Nap-NO2 with the electron-pulling group -NO2 has a different electronic conjugated structure than Nap-OH and Nap-C, enabling a torsional intramolecular charge transfer (TICT) mechanism.

[0116] Figure 7 This diagram illustrates the mechanism by which the fluorescent probe Nap-NO2 recognizes human serum albumin. The proposed mechanism suggests that the Nap-NO2 probe is in a free-rotating state in solution. Upon the addition of human serum albumin, the probe's rotation is inhibited, causing it to enter a distorted intramolecular charge-transfer state. This state is excited and releases strong fluorescence, resulting in a significantly enhanced fluorescence signal in the solution system, thus enabling the detection of human serum albumin.

[0117] Example 8: Fluorescence response of the fluorescent probe Nap-NO2 to the drug

[0118] Prepare 1×10 using DMSO -3 mol / L Nap-NO2 solution.

[0119] Prepare a 2 mg / mL human serum albumin (HSA) aqueous solution using distilled water.

[0120] Prepare 1×10 using distilled water. -4 A mol / L solution of heme (the binding site of HSA is located in the IB domain).

[0121] Prepare 1×10 using distilled water. -4 A mol / L solution of warfarin (the binding site of HSA is located in the IIA domain).

[0122] Prepare 1×10 using distilled water. -4 A mol / L solution of ibuprofen (the binding site of HSA is located in the IIIA domain).

[0123] Add 2 mL of water and 2 μL of the above Nap-NO2 solution to each clean fluorescent cuvette, and then add 20 μL of the above human serum albumin solution to form a Nap-NO2 / HSA complex solution. Add 0 μL, 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, and 15 μL of the above heme solution, warfarin solution, and ibuprofen solution to the Nap-NO2 / HSA complex solution, respectively. Under an excitation wavelength of 500 nm, the fluorescence emission intensity at 630 nm is measured using a fluorescence spectrometer. Plot the drug solution concentration on the x-axis and the normalized intensity on the y-axis to obtain the fluorescence response diagram of the fluorescent probe Nap-NO2 to the drug. Figure 8 ).

[0124] Depend on Figure 8 Only heme was observed to cause a significant change in the fluorescence of the Nap-NO2 / HSA complex solution, while the addition of warfarin and ibuprofen did not result in significant fluorescence changes. This confirms that Nap-NO2 enters the IB domain of human serum albumin, rather than the commonly used drug action sites (IIA and IIIA domains). This characteristic indicates that the fluorescent probe Nap-NO2 can avoid interference from commonly used drugs and also suggests that the fluorescent probe Nap-NO2 has potential value in the screening and detection of drugs related to the IB domain of human serum albumin.

[0125] Examples 2-8 above use Nap-NO2-a as an example to perform relevant tests on the fluorescent probe Nap-NO2. When Nap-NO2 with other coordinated anions of the present invention is used to perform the tests as in Examples 2-8, the test results are consistent with the test results of Nap-NO2-a.

Claims

1. A fluorescent probe, characterized by, It has the structure shown in equation (Ⅰ), Where X is a halogen, selected from F, Cl, Br or I.

2. The method of claim 1, wherein the fluorescent probe is prepared by the steps of: The fluorescent probe is obtained by reacting the compound shown in formula (II) with the compound shown in formula (III). Where X is a halogen, selected from F, Cl, Br or I.

3. The production method according to claim 2, characterized by, The molar ratio of the compound shown in formula (II) to the compound shown in formula (III) is 1:1 to 1:2, and the reaction is carried out under inert gas protection in the presence of pyridine.

4. The use of the fluorescent probe of claim 1 in the detection of human serum albumin for non-diagnostic purposes or in the preparation of non-diagnostic detection products of human serum albumin.

5. A reagent, test strip or kit for detecting human serum albumin, characterized in that, It contains the fluorescent probe as described in claim 1.

6. A fluorescent method for the visualization of human serum albumin for non-diagnostic purposes, comprising the steps of: If the fluorescent probe described in claim 1 is brought into full contact with the sample to be tested and obvious red fluorescence emission is observed under visible light irradiation, it indicates that the sample to be tested contains human serum albumin.

7. A method for fluorescent detection of human serum albumin for non-diagnostic purposes, characterized by, Includes the following steps: The fluorescence emission intensity of the sample mixture containing the fluorescent probe of claim 1 at 620-640 nm was detected using an excitation wavelength of 450-550 nm.

8. The method of fluorescence detection of human serum albumin for non-diagnostic purposes according to claim 7, characterized in that, Quantitative detection was performed using the linear correlation between human serum albumin concentration and fluorescence emission intensity.

9. The fluorescence detection method according to any one of claims 6 to 8, characterized in that, The sample to be tested is a water sample or a biological sample.

10. The use of the fluorescent probe of claim 1 in the preparation of reagents for in vitro screening or in vitro detection of drugs related to the IB domain of human serum albumin.

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