Application of camphor-based fluorescent probe in albumin detection

Abstract

The application discloses a fluorescent probe based on camphor derivative structure, takes camphor derivative as a fluorescent group, takes phenolic hydroxyl as a recognition group, and a structural formula is shown as (I). The probe molecule has no fluorescence in a solution, the solution fluorescence is enhanced after response to human albumin, and red fluorescence is emitted. The fluorescent probe can be used for detection of human albumin in water samples and biological samples, and can also be used for detection of human albumin in real urine and blood samples. The fluorescent probe has high sensitivity, good selectivity and anti-interference performance, can rapidly detect human serum albumin, shortens response time (less than 1 min), realizes real-time and effective detection of human albumin, and can realize human albumin imaging in cells and real samples, and is expected to play a role in biological medicine production and clinical medical detection, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to the application of a fluorescent probe using camphor derivatives as fluorescent groups and phenolic hydroxyl groups as recognition groups in albumin detection. Background Technology

[0002] Human serum albumin (HSA) is the most abundant protein produced by the liver. It participates in many important physiological processes, including plasma osmotic pressure balance, immune regulation, drug transport, and enzyme catalysis, playing a crucial role in maintaining normal human physiological functions. Furthermore, HSA is composed of a large number of water-soluble amino acids, exhibiting excellent water solubility and stability, making it a primary carrier for many endogenous biomolecules (such as fatty acids, thyroxine, iron sulfate, and bilirubin). In addition, many therapeutic drugs (such as paclitaxel and all-trans retinoic acid) or probes can be loaded into the hydrophobic cavity of HSA through non-covalent adsorption and hydrophobic interactions, significantly improving drug uptake efficiency.

[0003] In healthy adults, the normal level of human serum albumin in plasma is between 35-55 g / L. Changes in plasma albumin levels are closely related to various diseases, and abnormal concentrations are often considered physiological indicators of early signs in many diseases. Lower than normal plasma albumin levels are called hypoalbuminemia, often associated with liver damage, cirrhosis, liver failure, chronic hepatitis, liver cancer, and other liver diseases. Furthermore, compared to plasma concentrations, normal urinary serum albumin levels are below 30 mg / L. When the glomerular filtration barrier is damaged or renal tubular reabsorption function is abnormal, albumin leaks into the urine, a condition known as albuminuria. Based on severity, it is classified as microalbuminuria (30-300 mg / day, commonly seen in early diabetic nephropathy or hypertensive nephropathy) and overt albuminuria (>300 mg / day, indicating significant renal impairment, requiring further investigation of the cause). Furthermore, some chronic wasting diseases (such as hyperthyroidism, severe tuberculosis, and malignant tumors), malnutrition, and protein-losing enteropathies (such as Crohn's disease and ulcerative colitis) are all accompanied by decreased serum albumin levels. Therefore, accurate detection of changes in human serum albumin levels can reflect the body's health status, assess an individual's health condition, and screen for diseases affecting liver metabolic function, which is of great significance for clinical diagnosis and biological research.

[0004] To date, numerous methods exist for detecting human serum albumin, including capillary electrophoresis, LC-MS / MS proteomics, immunoturbidimetry, radioimmunoassay, and enzyme-linked immunosorbent assay (ELISA). While these methods meet the needs of human serum albumin detection, several drawbacks remain, such as high cost, long detection time, low sensitivity, complex sample processing, expensive equipment, and the need for specialized analysts. Currently, the most commonly used clinical method for determining human serum albumin concentration is the bromocresol green colorimetric method. However, the complex formed by human serum albumin and bromocresol green has poor stability, and the colorimetric method has low sensitivity, making it suitable only for detecting biological samples with high levels of human serum albumin, and not for detecting trace amounts. In recent years, fluorescence detection technology based on small molecule fluorescent probes has been widely recognized as one of the most attractive molecular detection techniques, offering advantages such as simplicity, convenience, and high sensitivity, and is widely used for the visual detection of target analytes in chemistry, biology, and the environment. Fluorescent probe molecules, through changes in light color and fluorescence intensity, can be effectively used for the analysis and real-time imaging monitoring of biomarkers in complex biological microenvironments, providing possibilities for in-depth exploration of the intermolecular interaction mechanisms in the microscopic world.

[0005] Meanwhile, many fluorescent probes used for human serum albumin detection employ fluorescent groups that can be structurally classified into rhodamine, naphthimide, BODIPY, hemicyanine, benzopyranone, dicyanoisophorone, tricyanofuran, and coumarin groups, among others. However, reports on constructing fluorescent groups through structural modification of renewable natural camphor are still very few. Camphor, as an important source of natural terpenoids, is widely distributed in Lauraceae plants and can serve as an ideal starting material for the preparation of various compounds, such as antitumor drugs, anesthetics, antibacterial agents, and catalysts. Furthermore, camphor derivatives exhibit good biocompatibility and low cytotoxicity.

[0006] Chinese patent document CN117263958A discloses a fluorescent probe based on a camphor derivative structure. Using camphor derivatives as the fluorescent group and 2,4-dinitrophenol as the recognition group, the fluorescent probe can respond to thiophenol compounds with high selectivity and high sensitivity. In the presence of thiophenols, the fluorescence intensity decreases significantly. It can be used for the qualitative and quantitative detection of thiophenol compounds without interference from aliphatic thiols such as glutathione (GSH), cysteine ​​(Cys), and homocysteine ​​(Hcy).

[0007] Chinese patent document CN119591619A discloses a fluorescent probe based on an acryloyl camphor derivative structure. The fluorescent probe uses an acryloyl group as a recognition group, which can specifically react with three biothiols: cysteine, homocysteine, and glutathione, and distinguish between the three biothiols, without being interfered with by other amino acids, ions, or other substances in the sample.

[0008] In the aforementioned patent documents, It participates as an intermediate in the preparation of the aforementioned fluorescent probes. Summary of the Invention

[0009] Based on previous research, this invention provides a novel fluorescent probe based on camphor derivatives and its applications.

[0010] Unlike existing fluorescent probes based on camphor derivative structures that use 2,4-dinitrophenol and acryloyl groups as recognition groups for the detection of thiophenol, the probe of this invention uses a phenolic hydroxyl group as a recognition group, which can specifically detect human albumin. It has the characteristics of high selectivity and high sensitivity, and can respond to human albumin very quickly (< 1 min).

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] A fluorescent probe based on camphor derivatives, using phenolic hydroxyl groups as recognition groups.

[0013] A specific example of the present invention is that the fluorescent probe has the structural formula shown in formula (Ⅰ):

[0014] .

[0015] The present invention also discloses the application of the above-mentioned fluorescent probe in the preparation of albumin detection reagents.

[0016] Test samples include liquid and solid samples. Liquid samples are selected from blood, urine, cerebrospinal fluid, ascites or other body cavity fluid, saliva, amniotic fluid, and synovial fluid. Solid samples are cells, tissues, organs, and living organisms. Test samples can come from humans, cattle, horses, sheep, pigs, monkeys, dogs, cats, rabbits, or mice.

[0017] Preferably, the test sample is serum or urine. The albumin is serum albumin or urine albumin. More preferably, the serum albumin is human serum albumin or / or urine albumin.

[0018] The fluorescent probe described in this invention has a maximum absorption wavelength of 360 nm in the ultraviolet spectrum of aqueous solution, and the fluorescence of the probe itself is very weak. When human serum albumin is present in the sample, the ultraviolet spectrum of the solution shows a slight red shift. After the fluorescent probe binds to human serum albumin, the fluorescence intensity of the solution at 582 nm increases by 125 times. Under a 365 nm ultraviolet lamp, the color of the solution changes from colorless to red.

[0019] The fluorescent probe described in this invention can be used for the qualitative or quantitative detection of albumin.

[0020] The albumin content in the sample can be detected or the sample can be fluorescently imaged at an excitation wavelength of 360 nm and a detection wavelength of 582 nm.

[0021] The fluorescent probe described in this invention is used as a diagnostic reagent in the diagnosis of diseases related to abnormal human serum albumin levels. These diseases are selected from liver damage, cirrhosis, liver failure, chronic hepatitis, liver cancer, nephrotic syndrome, glomerulonephritis, coronary heart disease, Crohn's disease, ulcerative colitis, hyperthyroidism, malignant tumors, and malnutrition.

[0022] The fluorescent probe described in this invention is also used as a detection reagent to further study the kinetic mechanism of human serum albumin transport and accumulation in organisms.

[0023] A specific example of the application of the fluorescent probe (Ⅰ) described in this invention includes the following steps:

[0024] (1) Preparation of fluorescent probe system;

[0025] (2) Prepare a solution of human serum albumin;

[0026] (3) Add the solution of human serum albumin to the fluorescent probe system to prepare a mixed solution with different concentrations of human serum albumin;

[0027] (4) Test the fluorescence intensity of the mixed solution at 582 nm, and plot a standard curve based on the relationship between the fluorescence intensity and the concentration of human serum albumin;

[0028] (5) Quantitatively detect the content of human albumin in the sample solution to be tested according to the standard curve.

[0029] The steps for preparing the fluorescent probe system described above include:

[0030] (1) Dissolve the fluorescent probe (Ⅰ) in an organic solvent to obtain a mother liquor, and make the concentration of the probe in the mother liquor 5 × 10⁻⁶. -7 ~5×10 -3 mol / L, the organic solvent is one or more of methanol, ethanol, acetonitrile, tetrahydrofuran, acetone, acetic acid, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide.

[0031] (2) Dilute the concentration of the mother solution with a buffer solution to obtain the fluorescent probe system. The buffer solution for diluting the mother solution is one or more of PBS buffer, HEPES buffer, and Tris buffer.

[0032] Another object of the present invention is to provide a method for preparing the fluorescent probe (Ⅰ) described in the present invention, comprising the following steps:

[0033] S1: Compound (II) was prepared by reacting camphorquinone with 4,7-dibromobenzo[c][1,2,5]thiadiazole-5,6-diamine in an organic solvent.

[0034] ;

[0035] The organic solvents mentioned above are selected from one or more of formic acid, acetic acid, propionic acid, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, toluene, xylene, 1,4-dioxane, and 2-methylpyrrolidone.

[0036] The preferred reaction temperature is 0 ℃ to 200 ℃.

[0037] S2: Compound (II) and pinacol ester of 4-hydroxyphenylboronic acid are dissolved in an organic solvent and reacted with a catalyst and base under nitrogen protection to obtain fluorescent probe (I):

[0038] .

[0039] The organic solvents mentioned above are selected from one or more of acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, toluene, xylene, 1,4-dioxane, 2-methylpyrrolidone, ethanol, methanol, ethylene glycol dimethyl ether, and water.

[0040] The selected catalyst is one or more of Pd(PPh3)4, Pd(PPh3)2Cl2, Pd(dppf)Cl2, Pd(dba)2-PCy3, PdCl2, Pd(OAc)2, Pd / C, and NiCl2(dppf).

[0041] The selected base is selected from one or more of K2CO3, K3PO4, Na2CO3, CsF, Cs2CO3, t-BuONa, t-BuOK, Ba(OH)2, NaOH, NaHCO3, triethylamine, N,N-diisopropylethylamine, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0042] The preferred reaction temperature is 0 ℃ to 200 ℃.

[0043] A specific preparation method of the present invention is as follows:

[0044] S1: Camphorquinone and 4,7-dibromobenzo[c][1,2,5]thiadiazole-5,6-diamine were dissolved in a reaction solvent and heated with stirring. After the reaction was complete, the mixture was cooled to room temperature and filtered to obtain the crude product. Product (II) was obtained by column chromatography.

[0045] S2: Compound (II), pinacol 4-hydroxyphenylboronic acid, base, and catalyst were dissolved in the reaction solvent and heated and stirred under a nitrogen atmosphere. After the reaction was complete, the reaction mixture was cooled to room temperature, most of the solvent was removed by rotary evaporation, and then extracted with ethyl acetate. The combined organic layers were washed with saturated brine and dried over anhydrous Na2SO4. The fluorescent probe (I) was obtained by column chromatography.

[0046] For specific reaction conditions described above, please refer to the examples.

[0047] The advantages of this invention are:

[0048] (1) The fluorescent probe provided by the present invention introduces natural camphor molecules into the dye as a fluorescent parent and uses the simplest phenolic hydroxyl group as a recognition group. It can respond to human albumin with high selectivity and high sensitivity and will not be interfered with by other amino acids, ions, DNA, RNA, proteins and other substances in the sample.

[0049] (2) The fluorescent probe provided by the present invention has low toxicity to cells and good biocompatibility. It can detect human albumin in solutions (including water samples, urine samples and serum samples), as well as endogenous and exogenous human albumin in cells. It can be used to study the dynamic mechanism of human albumin transport and accumulation in organisms, and also helps to elucidate the biological role and mechanism of abnormal human albumin content in the pathogenesis process. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0051] Figure 1 The UV spectrum (a), fluorescence spectrum (b), and color change of the solution (inset) are shown after adding fluorescent probe (Ⅰ) (10 μM) to human serum albumin (1.25 mg / mL) for 0.5 h. The detection system was PBS (10 mM, pH=7.4), and the detection temperature was 37 ℃. The excitation wavelength of the fluorescence spectrum was 360 nm, and the slit width was 5 / 5 nm.

[0052] Figure 2This study compares the changes in fluorescence spectra of control probes 1 and 2 (10 μM) after 0.5 h of addition of human serum albumin (1.25 mg / mL). The detection system was PBS (10 mM, pH=7.4), and the detection temperature was 37 ℃. The excitation wavelength of control probe 1 was 450 nm, with a slit width of 2.5 / 5 nm, while the excitation wavelength of control probe 2 was 440 nm, with a slit width of 2.5 / 5 nm.

[0053] Figure 3 The fluorescence spectra (a) and fluorescence intensity (b) at 582 nm of the fluorescent probe (I) (10 μM) after adding different concentrations of human serum albumin (0–1.6 mg / mL) are shown as changes in human serum albumin concentration. Inset: Linear fitting graph of fluorescence intensity versus concentration in the range of 0–0.4 mg / mL. The detection system was PBS (10 mM, pH=7.4), the detection temperature was 37 ℃, incubation time was 0.5 h, excitation wavelength was 360 nm, and slit width was 5 / 5 nm.

[0054] Figure 4 (a) Schematic diagram of human serum albumin detection using fluorescent probe (I) (10 μM) combined with a universal colorimeter and color picker; the fluorescence color of fluorescent probe (I) changes with human serum albumin concentration under 365 nm UV light irradiation. (b) Linear graph of RGB values ​​(R / B) triggered by fluorescence color change versus human serum albumin concentration in the range of 0 to 0.2 mg / mL. The detection system was PBS (10 mM, pH=7.4).

[0055] Figure 5 The fluorescence intensity at 582 nm changes over time after adding fluorescent probe (Ⅰ) (10 μM) to human serum albumin (0.1, 0.5, 1.0 mg / mL). The detection system was PBS (10 mM, pH=7.4), the detection temperature was 37 ℃, the excitation wavelength was 360 nm, and the slit width was 5 / 5 nm.

[0056] Figure 6The fluorescence spectrum (a) and the change in fluorescence intensity at 582 nm (b) are shown after adding 1 mg / mL human serum albumin or 1 mg / mL of various analytes (Glucose oxidase, proteinase K, lysozyme, RNA, DNA, pepsin, uric acid, trypsin, hemoglobin, collagens, creatinine, chymotrypsin) to fluorescent probe (Ⅰ) (10 μM). The detection system was PBS (10 mM, pH=7.4), the detection temperature was 37 ℃, the excitation wavelength was 360 nm, and the slit width was 5 / 5 nm.

[0057] Figure 7 The fluorescence spectrum (a) and the change in fluorescence intensity at 582 nm (b) are shown after adding 1 mg / mL human serum albumin or 1 mg / mL of various amino acids and ions to fluorescent probe (Ⅰ) (10 μM). The detection system was PBS (10 mM, pH=7.4), the detection temperature was 37 ℃, the excitation wavelength was 360 nm, and the slit width was 5 / 5 nm. The following are the fluorescent probes 1-25 in order: Probe, HSA, Ala, Arg, Asp, Cys, Hcy, GSH, Gln, His, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, NaSH, NaNO2, MgSO4, Na2S2O3, KI, Na2SO4.

[0058] Figure 8 This is the color of the solution after adding 1 mg / mL human serum albumin or 1 mg / mL of various analytes to the fluorescent probe (Ⅰ) (10 μM) under 365 nm ultraviolet light. From left to right, they are: HSA, glucose oxidase, lysozyme, RNA, DNA, pepsin, uric acid, trypsin, collagen, creatinine, and chymotrypsin. The detection system is PBS (10 mM, pH=7.4).

[0059] Figure 9 The color of the solution after adding 1 mg / mL human serum albumin or 1 mg / mL of various amino acids and ions to the fluorescent probe (Ⅰ) (10 μM) under 365 nm ultraviolet light is measured. The detection system is PBS (10 mM, pH=7.4).

[0060] Figure 10This describes the change in fluorescence intensity at 582 nm after adding 1 mg / mL of HSA and 1 mg / mL of various other analytes (glucose oxidase, lysozyme, RNA, DNA, pepsin, uric acid, trypsin, collagen, creatinine, and chymotrypsin) to fluorescent probe (Ⅰ) (10 μM). The detection system was PBS (10 mM, pH=7.4). The excitation wavelength was 360 nm, and the slit width was 5 / 5 nm.

[0061] Figure 11 This describes the change in fluorescence intensity at 582 nm after adding 1 mg / mL of HSA and 1 mg / mL of various other amino acids and ions to fluorescent probe (Ⅰ) (10 μM). The detection system was PBS (10 mM, pH=7.4). The values ​​1-25 were: Probe, HSA, HSA + Ala, HSA + Arg, HSA + Asp, HSA + Cys, HSA + Hcy, HSA + GSH, HSA + Gln, HSA + His, HSA + Leu, HSA + Met, HSA + Phe, HSA + Pro, HSA + Ser, HSA + Thr, HSA + Trp, HSA + Tyr, HSA + Val, HSA + NaSH, HSA + NaNO2, HSA + MgSO4, HSA + Na2S2O3, HSA + KI, HSA + Na2SO4. The excitation wavelength was 360 nm, and the slit width was 5 / 5 nm.

[0062] Figure 12 The color of the fluorescent probe (I) (10 μM) solution under 365 nm UV light after the addition of human serum albumin (1 mg / mL) and various analytes (1 mg / mL) is shown. From left to right, the colors are: HSA, HSA + glucose oxidase, HSA + lysozyme, HSA + RNA, HSA + DNA, HSA + pepsin, HSA + uric acid, HSA + trypsin, HSA + collagen, HSA + creatinine, and HSA + chymotrypsin. The detection system is PBS (10 mM, pH=7.4).

[0063] Figure 13 The color of the fluorescent probe (I) (10 μM) solution under 365 nm ultraviolet light is measured after the addition of human serum albumin (1 mg / mL) and various amino acids and ions (1 mg / mL). The detection system is PBS (10 mM, pH=7.4).

[0064] Figure 14 This is the Job's plot image of fluorescent probe (I). The fluorescence intensity at 582 nm was plotted against [Probe] / ([Probe + HSA]) in an experiment with continuous equimolar variations of fluorescent probe (I) and human serum albumin, where [Probe + HSA] = 10 μM. The detection system was PBS (10 mM, pH = 7.4). The excitation wavelength was 360 nm, and the slit width was 5 / 5 nm.

[0065] Figure 15 This is a laser confocal microscope image of the fluorescent probe (Ⅰ) (5 μM) used to detect exogenous human serum albumin in HepG2 cells.

[0066] Figure 16 This is a laser confocal microscope image of a fluorescent probe (Ⅰ) (5 μM) used to detect endogenous human serum albumin in HepG2 cells. Detailed Implementation

[0067] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention and should not be used to limit the scope of protection of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0068] Example 1: Preparation of fluorescent probes

[0069]

[0070] Preparation of S1: 4,11-dibromo-6,12,12-trimethyl-6,7,8,9-tetrahydro-6,9-methyl[1,2,5]thiadiazole[3,4-b]phenazine (II)

[0071] Camphorquinone (1.66 g, 10 mmol) and 4,7-dibromobenzo[c][1,2,5]thiadiazole-5,6-diamine (3.24 g, 10 mmol) were dissolved in a reaction solvent (120 mL) and stirred at 120 °C for 12 h. After the reaction was complete (monitored by TLC), the mixture was cooled to room temperature, the solvent was removed by evaporation under reduced pressure, the residue was dissolved in dichloromethane (300 mL), washed three times with saturated NaHCO3 solution (100 mL), the organic layer was washed with saturated brine, and dried over anhydrous Na2SO4. The product was separated by column chromatography (eluting ethyl acetate: petroleum ether = 1:100 to elute the target compound). 2.41 g of yellow solid (II) was given, yield 53%.

[0072] Structural determination:

[0073] 1 H NMR (600 MHz, CDCl3) δ 3.26 (d, J = 4.6 Hz, 1H), 2.44 – 2.33 (m,1H), 2.15 (dd, J = 16.8, 5.4 Hz, 1H), 1.59 (t, J = 8.5 Hz, 2H), 1.50 (s, 3H),1.20 (s, 3H), 0.73 (s, 3H); 13 C NMR (150 MHz, CDCl3) δ 169.4, 167.7, 151.6,151.5, 139.7, 139.6, 114.0, 113.4, 54.4, 53.2, 52.6, 31.8, 24.7, 20.9, 18.2,9.9; HRMS (ESI + calcd for C 16 H 14 Br2N4S [M+H] + 452.9379, found 452.9378.

[0074] S2: Preparation of fluorescent probe (Ⅰ)

[0075] 4,11-Dibromo-6,12,12-trimethyl-6,7,8,9-tetrahydro-6,9-methyl[1,2,5]thiadiazole[3,4-b]phenazine (II, 454 mg, 1.0 mmol), pinacol 4-hydroxyphenylboronic acid (880 mg, 4.0 mmol), and Na₂CO₃ (420 mg, 4.0 mmol) were dissolved in a mixed solvent of toluene / ethanol / H₂O (10 / 6 / 5 mL). PdCl₂(dppf) (74 mg, 0.1 mmol) was added to the reaction system, and the system was heated to 70 °C and stirred for 6 h under a nitrogen atmosphere. After the reaction was complete (monitored by TLC), the reaction mixture was cooled to room temperature, the solvent was removed by rotary evaporation, and then extracted with dichloromethane (3 × 15 mL). The combined organic layers were washed with saturated brine and dried over anhydrous Na₂SO₄. The product was separated by column chromatography (eluting with ethyl acetate:petroleum ether = 1:5 to elute the target compound). 306 mg of red solid (V) was obtained, with a yield of 63.6%.

[0076] Structural determination:

[0077] 1 H NMR (600 MHz, DMSO) δ 9.66 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 7.9Hz, 2H), 7.58 (d, J = 7.8 Hz, 2H), 6.94 (d, J = 7.5 Hz, 4H), 2.95 (s, 1H), 2.27 (s, 1H), 2.04 (t, J = 10.1 Hz, 1H), 1.41 (t, J = 10.5 Hz, 2H), 1.23 (s, 3H), 1.08 (s, 3H), 0.63 (s, 3H); 13 C NMR (150 MHz, DMSO) δ 166.3, 164.8,157.7, 152.7, 152.4, 137.6, 137.2, 134.5, 134.1, 129.2, 129.0, 126.3, 126.0,114.8, 114.6, 53.8, 52.8, 52.5, 31.9, 31.4, 24.8, 22.5, 20.7, 18.3, 14.4,10.1; HRMS (ESI + calcd for C 28 H 24 N4O2S [M+H] + 481.1693, found 481.1697.

[0078] Fluorescent probes were prepared according to Chinese patent documents CN117263958A and CN119591619A, respectively. and As a comparison probe, probes 1 and 2 are used.

[0079] Example 2: Basic experimental conditions for detecting human serum albumin using fluorescent probe (Ⅰ).

[0080] Fluorescent probe (I) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM probe stock solution, which was stored in a cool pharmaceutical cabinet at 4 ℃. During testing, the probe stock solution was diluted with DMSO to a concentration of 1 mM to be used as the stock solution for spectral analysis to investigate the changes in the UV and fluorescence spectral properties of the probe in response to human serum albumin.

[0081] Human serum albumin was dissolved in PBS (10 mM, pH 7.4) buffer to a concentration of 10 mg / mL. The human serum albumin stock solution was prepared fresh for use immediately.

[0082] Various proteins, enzymes, amino acids, and other molecules were dissolved in PBS (10 mM, pH 7.4) buffer to a concentration of 10 mg / mL. These molecules included glucose oxidase, lysozyme, RNA, DNA, pepsin, uric acid, trypsin, collagen, creatine, and chymotrypsin.

[0083] To simulate physiological experiments, all experiments were conducted in PBS buffer (10 mM, pH=7.4) at 37 ℃. The excitation wavelength for fluorescence spectra was 360 nm, and the slit width was 5 nm / 5 nm.

[0084] During probe performance testing, 3 μL of the fluorescent probe (I) stock solution was accurately pipetted into 2700 μL of PBS buffer (10 mM, pH 7.4), followed by 300 μL of the analyte stock solution as required by the experiment. After thorough mixing, the mixture was incubated in a 37°C water bath for a period of time, and then UV and fluorescence spectroscopy were performed. At this point, the concentration of the fluorescent probe (I) in the system was 10 μM.

[0085] Example 3: Spectral response of fluorescent probe (I) to human serum albumin

[0086] 3 μL of probe (I) stock solution and 300 μL of human serum albumin stock solution were accurately measured using a pipette. 2700 μL of PBS (10 mM, pH 7.4) buffer was added sequentially to make the concentration of probe (I) 10 μM and the concentration of human serum albumin 1 mg / mL. After the solution was shaken well, it was incubated in a 37 ℃ water bath for 30 min. Then the ultraviolet and fluorescence spectra were measured.

[0087] The changes in the UV absorption spectrum of probe (Ⅰ) before and after the addition of human serum albumin are as follows: Figure 1 As shown in a. From Figure 1 It can be seen that after the addition of human serum albumin, the maximum absorption wavelength of the solution near 475 nm red-shifts to 500 nm, while the maximum absorption wavelength near 360 nm remains basically unchanged. Figure 1 a).

[0088] The fluorescence spectrum changes of probe (Ⅰ) before and after the addition of human serum albumin are as follows: Figure 1 As shown in b. From Figure 1 As shown in b, before the addition of human serum albumin, the fluorescence of the detection solution was very weak; after the addition of human serum albumin, the fluorescence intensity of the solution at 582 nm increased by 125 times. Under a 365 nm ultraviolet lamp, the solution color changed significantly, from the initial colorless state to a bright red. Figure 1 b. Illustration).

[0089] The spectral responses of probes 1 and 2 to human serum albumin were measured using the method described above. The changes in fluorescence spectra before and after the addition of human serum albumin are shown below. Figure 2 As shown. From Figure 2 As can be seen, before and after the addition of human serum albumin, there was almost no change in the fluorescence intensity and emission wavelength of probes 1 and 2.

[0090] The above research results demonstrate that, through changes in ultraviolet and fluorescence spectra and visual observation, the fluorescent probe (I) of this invention exhibits a good response to human serum albumin. Comparative probes 1 and 2, which have similar structures to the fluorescent probe (I) of this invention, do not respond to human serum albumin.

[0091] Example 4: Detection limit of fluorescent probe (I) for human serum albumin

[0092] 3 μL of 10 mM probe stock solution was precisely pipetted into PBS (10 mM, pH 7.4) buffer to maintain the probe (I) concentration at 10 μM. A series of human serum albumin solutions of different concentrations were prepared by adding different volumes of human serum albumin stock solution. After thorough mixing, the solutions were incubated in a 37 ℃ water bath for 30 minutes, followed by fluorescence spectroscopy. The excitation wavelength for the fluorescence spectrum was 360 nm, and the slit width was 5 nm / 5 nm. The detection limit of probe (I) for human serum albumin was calculated using the 3σ / k method, where σ represents the standard deviation of the emission intensity values ​​of ten blank samples, and k represents the slope of the fitted line between fluorescence intensity and human serum albumin concentration.

[0093] When different concentrations (0–1.6 mg / mL) of human serum albumin were added to the system, the fluorescence spectrum of probe (Ⅰ) and the change in fluorescence intensity at 582 nm were as follows: Figure 3 As shown: with the increase of human serum albumin concentration, the fluorescence intensity of the solution shows a sharp increasing trend ( Figure 3 ).like Figure 3 As shown in the illustration, when the concentration of human serum albumin is in the range of 0-0.4 mg / mL, the fluorescence intensity of the solution shows a linear correlation with the concentration. The linear regression equation is y=3600.2x+19.213, and the correlation coefficient R is 1 / 2. 2 = 0.9946. According to the formula 3σ / k, the detection limit of probe (Ⅰ) for human serum albumin is as low as 0.9580 μg / mL, which is far lower than the concentration of human serum albumin of 30 μg / mL in healthy urine, proving that this fluorescent probe can be used to quantitatively analyze human serum albumin.

[0094] Example 5: Fluorescent probe (I) combined with universal color recognition and color picker to analyze fluorescence signals

[0095] 3 μL of 10 mM probe stock solution was precisely pipetted into PBS (10 mM, pH 7.4) buffer to maintain the probe (I) concentration at 10 μM. A series of human serum albumin solutions of different concentrations were prepared by adding different volumes of human serum albumin stock solution. After thorough mixing, the solutions were incubated in a 37 ℃ water bath for 30 minutes. The solutions were then photographed under 365 nm UV light, and the fluorescence signal was analyzed using a universal colorimeter and color picker (ColorDesk (Android / iOS), a smartphone color recognition application).

[0096] The fluorescence color of the solution changed from purple to red as the concentration of human serum albumin increased (0 to 0.2 mg / mL). Figure 4 Meanwhile, the red / blue ratio (R / B) of the test strips was positively correlated with the concentration of human serum albumin. Figure 4 b). Therefore, the fluorescent probe (I) combined with a smartphone color recognition application can overcome the limitations of professional fluorescence / colorimetric analysis software in large laboratory instruments and has the potential to become an effective tool for on-site detection of human serum albumin.

[0097] Example 6: Response time of fluorescent probe (I) to human serum albumin

[0098] Using a pipette, 3 μL of fluorescent probe (I) stock solution and a certain amount of human serum albumin stock solution were precisely transferred, and PBS (10 mM, pH 7.4) buffer was added sequentially to maintain the concentration of probe (I) at 10 μM. The concentrations of human serum albumin were 0.1, 0.5, and 1.0 mg / mL, respectively. The change in fluorescence intensity of the solution over time after the addition of human serum albumin was observed. The excitation wavelength of the fluorescence spectrum was 360 nm, and the slit width was 5 nm / 5 nm.

[0099] The fluorescence intensity change at 582 nm of probe (Ⅰ) solution after the addition of human serum albumin is as follows: Figure 5 As shown, the fluorescence intensity of the solution increased sharply after the addition of human serum albumin, and quickly reached a stable plateau in just 30 seconds. This rapid response characteristic indicates that the fluorescent probe (I) has the ability to rapidly detect human serum albumin.

[0100] Example 7: Selectivity of fluorescent probe (I) for human serum albumin recognition

[0101] Generally, high selectivity for the target analyte is a core requirement for all detection methods. According to Example 2, solutions of various proteins, enzymes, amino acids, and ions were prepared using PBS (10 mM, pH=7.4) as the solvent, with a concentration of 10 mg / mL. Specific proteins included glucose oxidase, lysozyme, RNA, DNA, pepsin, uric acid, trypsin, collagen, creatine, and chymotrypsin; amino acids included Ala, Arg, Asp, Cys, Hcy, GSH, Gln, His, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val; and ions included NaSH, NaNO2, MgSO4, Na2S2O3, KI, and Na2SO4.

[0102] To investigate the selectivity of probe (I) for human serum albumin, a reaction mixture of 10 μM probe (I) and 1 mg / mL human serum albumin or 1 mg / mL of other analytes was prepared using PBS buffer (10 mM, pH=7.4). The prepared reaction mixture was incubated at 37 ℃ for 0.5 h, and the fluorescence spectrum was measured. The excitation wavelength of the fluorescence spectrum was 360 nm, and the slit width was 5 nm / 5 nm.

[0103] The fluorescence spectrum and fluorescence intensity changes of probe (I) after adding 1 mg / mL human serum albumin or 1 mg / mL of other analytes to the reaction mixture are as follows: Figure 6 , 7As shown, only human serum albumin caused a significant change in the fluorescence spectrum and fluorescence intensity at 582 nm of the probe, while other analytes did not, indicating that probe (I) has high selectivity for human serum albumin.

[0104] The selectivity of probe (I) can also be demonstrated by observing changes in the color of the solution with the naked eye. For example... Figure 8 , 9 As shown, under a 365nm UV lamp, the color of the probe solution remained purple when other analytes were added (the color of the RNA solution was bright white); after the addition of human serum albumin, the color of the solution rapidly changed to red and the fluorescence was enhanced.

[0105] The above results indicate that the probe has high selectivity for human serum albumin, and can effectively distinguish human serum albumin from other analytes when applied, thus having great application value.

[0106] Example 8: Competitive Experiment of Fluorescent Probe (I)

[0107] To demonstrate the promising application potential of the fluorescent probe, its anti-interference performance needs to be tested. Analytes that may interfere with the detection of the fluorescent probe (I) are added to the detection system to examine whether the detection capability of the fluorescent probe (I) is affected in the presence of these analytes.

[0108] A reaction mixture containing 10 μM probe (I), 1 mg / mL human serum albumin, and 1 mg / mL of other analytes was prepared using PBS buffer (10 mM, pH 7.4). The prepared reaction mixture was incubated at 37 °C for 0.5 h, and the fluorescence spectrum was measured. The excitation wavelength for the fluorescence spectrum was 360 nm, and the slit width was 5 nm / 5 nm. Figure 10 , 11 As shown, in the presence of the other analytes, the fluorescence of the solution was enhanced after the probe (Ⅰ) detected human serum albumin, proving that the presence of the other analytes does not interfere with the detection of human serum albumin by the probe.

[0109] The ability of probe (Ⅰ) to resist interference can also be demonstrated by observing changes in the color of the solution with the naked eye. For example, Figure 12 , 13 As shown, under a 365 nm UV lamp, even in the presence of other analytes, the solution color still turns red after the addition of human serum albumin.

[0110] The above results indicate that the probe has excellent anti-interference performance and has the potential to be applied to detection in complex environments.

[0111] Example 9: Continuous variation experiment of isoprotons of fluorescent probe (I) (Job's Plot)

[0112] The rapid and highly selective detection of human serum albumin by fluorescent probe (I) is based on the twisted intramolecular charge transfer (TICT) mechanism. When the phenolic hydroxyl group of the fluorescent probe enters the hydrophobic cavity of human serum albumin, the microenvironment surrounding the probe molecule changes. The steric hindrance and low polarity of the microenvironment inhibit the free rotation of the probe molecule, thereby increasing the fluorescence intensity of the solution. Therefore, it is necessary to use an experiment with continuous changes in the amount of substance to determine the optimal binding ratio of fluorescent probe (I) to human serum albumin.

[0113] In a PBS buffer (pH=7.4) system, the total molar concentration of probe (I) and HSA was fixed at 10 μM. The ratio of probe (I) to human serum albumin was changed to 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0. The fluorescence spectrum of the solution was measured and Job's plot was generated.

[0114] like Figure 14 As shown, the fluorescence intensity is the highest when the mole fraction of probe (Ⅰ) in the solution is close to 0.5, therefore the stoichiometric ratio of the binding of probe (Ⅰ) to human serum albumin is 1:1.

[0115] Example 10: Fluorescent probe (I) for quantitative detection of albumin in human urine samples

[0116] Urine samples were collected from healthy adults without any pretreatment. Urine was diluted 10-fold with PBS buffer (10 mM, pH 7.4) (urine:PBS = 1:9, v / v), and then different concentrations of HSA (0–100 μg / mL) and a 10 μM probe (I) were added. The prepared mixtures were incubated at 37 °C for 0.5 h, and then fluorescence spectra were measured. The excitation wavelength for the fluorescence spectra was 360 nm, and the slit width was 5 nm / 5 nm. Simultaneously, the HSA content in the samples was detected using an albumin assay kit (bromocresol green colorimetric method, BCG) purchased from Adamas Life, and the results were compared. Methods reference: Analytica Chimica Acta, 2021, 1188: 339201.

[0117] As shown in Table 1, fluorescent probe (I) exhibited high sensitivity and accuracy in detecting human albumin in urine samples, with a good recovery rate (99.17%-102.78%). Furthermore, the ratio of fluorescent probe (I) to BCG data was close to 1, further confirming the effectiveness of fluorescent probe (I) in detecting human albumin. These results collectively demonstrate the application potential of fluorescent probe (I) in urine diagnosis and other biomedical assays using urine as a sample matrix.

[0118] Table 1. Determination of albumin content in human urine samples

[0119]

[0120] Example 11: Fluorescent probe (I) for quantitative detection of serum albumin in human serum samples

[0121] Four human serum samples were purchased from Sigma-Aldrich, Solarbio, and two other companies. They were stored at -20 °C without any additional treatment for further analysis. For analysis, the serum samples were diluted 100-fold with PBS buffer (10 mM, pH 7.4) (serum:PBS = 1:99, v / v). The prepared solutions were incubated at 37 °C for 0.5 h before fluorescence spectra were measured. The excitation wavelength for the fluorescence spectra was 360 nm, and the slit width was 5 nm / 5 nm. Simultaneously, the HSA content in the samples was detected using an albumin assay kit (bromocresol green colorimetric method, BCG) purchased from Adamas Life, and the results were compared. Methods were referenced in Sensors and Actuators: B. Chemical, 2024, 414: 135950 and Dyesand Pigments, 2024, 231: 112416.

[0122] As shown in Table 2, fluorescent probe (I) exhibits high sensitivity and accuracy in detecting albumin in human serum samples, requiring only a small amount of serum for assay. Furthermore, the ratio of fluorescent probe (I) to BCG data is close to 1, further confirming the effectiveness of fluorescent probe (I) in detecting human serum albumin. These results collectively demonstrate the application potential of fluorescent probe (I) in human serum diagnostics and other biomedical assays using human serum as a sample matrix.

[0123] Table 2. Determination of albumin content in human serum samples

[0124]

[0125] Example 12: Fluorescent probe (I) for the detection of exogenous human serum albumin in cells

[0126] HepG2 cells were resuscitated and passaged using serum-containing medium, then plated into confocal dishes and incubated with 1.0 mL of serum-containing medium until the cells reached 70-80% growth. Subsequently, the cells were starved with 1.0 mL of serum-free medium for 24 h, and the cell growth status was observed to eliminate the influence of residual serum albumin in the serum-containing medium.

[0127] The treated HepG2 cells were imaged using a Nikon ECLIPSE Ti2-U confocal laser scanning microscope. DAPI dye was used for nuclear labeling, with an excitation wavelength of 405 nm, and fluorescence was collected from 420 to 470 nm. Fluorescent probe (I) was used, with an excitation wavelength of 488 nm, and fluorescence was collected from 570 to 616 nm.

[0128] The first group of HepG2 cells (control group) was incubated in 1.0 mL of serum-free medium for 120 min, washed three times with PBS, fixed with 1 mL of 4% paraformaldehyde for 15 min, washed three times with PBS, and stained with DAPI for 5 min. The second group of HepG2 cells was incubated in 1.0 mL of serum-free medium (containing 5 μM fluorescent probe (I)) for 120 min, washed three times with PBS, fixed, and stained with DAPI for 5 min. The third group of HepG2 cells was incubated in 1.0 mL of serum-free medium (containing 5 μM fluorescent probe (I)) for 60 min, washed three times with PBS, then incubated in 1.0 mL of serum-free medium containing 0.5 mg / mL HSA for 60 min, fixed, and stained with DAPI for 5 min. The fourth group of HepG2 cells was incubated in 1.0 mL of serum-free medium (containing 5 μM fluorescent probe (I)) for 60 min, washed three times with PBS, then incubated in 1.0 mL of serum-free medium containing 1.0 mg / mL HSA for 60 min. Cells were incubated in serum-free medium containing mg / mL HSA for 60 min to fix them, and nuclei were stained with DAPI for 5 min. The treated cells were then imaged under a laser confocal microscope.

[0129] Figure 15 This is the imaging result of using fluorescent probe (I) in HepG2 cells for exogenous human serum albumin. No fluorescent probe (I) was added to the control group, therefore no yellow fluorescence was observed, and no fluorescence interference was seen. Because the expression level of HSA in HepG2 cells was excessive, the yellow fluorescence signal was enhanced after co-incubating the second group of cells with fluorescent probe (I). When fluorescent probe (I) was pre-incubated with cells, and then different doses of exogenous HSA were added for co-incubation, a positive correlation between yellow fluorescence and HSA concentration was observed (third and fourth groups of cells). Statistical analysis confirmed that fluorescent probe (I) can be used for imaging exogenous HSA in cells.

[0130] Example 13: Fluorescent probe (I) for the detection of endogenous human serum albumin in cells

[0131] The first group of HepG2 cells was incubated in 1.0 mL of serum-free medium (containing 5 μM fluorescent probe (I)) for 120 min, washed three times with PBS, fixed with 1 mL of 4% paraformaldehyde for 15 min, washed three times with PBS, and stained with DAPI for 5 min. The second group of HepG2 cells was incubated in 1.0 mL of serum-free medium (containing 10 μM simvastatin) for 24 h, washed three times with PBS, then incubated in 1.0 mL of serum-free medium containing 5 μM fluorescent probe (I) for 60 min, washed three times with PBS, fixed, and stained with DAPI for 5 min. The third group of HepG2 cells was incubated in 1.0 mL of serum-free medium (containing 100 μM chloramphenicol) for 60 min, washed three times with PBS, then incubated in 1.0 mL of serum-free medium containing 5 μM fluorescent probe (I) for 60 min, washed three times with PBS, fixed, and stained with DAPI for 5 min. The cells treated in the above ways were imaged under a laser confocal microscope.

[0132] Figure 16 This is the imaging result of using fluorescent probe (I) to detect endogenous human serum albumin in HepG2 cells. Because HSA expression was excessive in HepG2 cells, the control group cells showed a strong yellow fluorescence signal after co-incubation with fluorescent probe (I). Simvastatin can induce HSA overexpression in HepG2 cells. After pre-incubating the second group of HepG2 cells with simvastatin for 24 hours, followed by co-incubation with fluorescent probe (I), the yellow fluorescence signal was significantly enhanced. Chloramphenicol is an antibiotic that inhibits HSA synthesis and has hepatotoxicity. When the third group of cells was pre-incubated with chloramphenicol for 60 minutes before co-incubation with fluorescent probe (I), the fluorescence intensity decreased significantly. Statistical analysis confirmed that fluorescent probe (I) can be used for imaging endogenous HSA in cells.

[0133] It should be noted that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. The application of a camphor derivative-based fluorescent probe in the preparation of albumin detection reagents, characterized in that... The structural formula of the fluorescent probe is shown in formula (Ⅰ): 。 2. The application according to claim 1, characterized in that... The test samples include liquid samples and solid samples. Liquid samples are selected from blood, urine, cerebrospinal fluid, ascites or other body cavity fluid, saliva, amniotic fluid, and synovial fluid. Solid samples are cells, tissues, organs, and living organisms.

3. The application according to claim 2, characterized in that... The test sample is serum or urine.

4. The application according to claim 2, characterized in that... The test samples came from humans, cattle, horses, sheep, pigs, monkeys, dogs, cats, rabbits, or mice.

5. The application according to claim 1, characterized in that... The detection is either qualitative or quantitative.

6. The application according to claim 5, characterized in that... The excitation wavelength was 360 nm, and the detection wavelength was 582 nm.

7. The application according to claim 6, characterized in that... The application is to detect the albumin content in a sample or to perform fluorescence imaging on the sample.

8. The application according to claim 1, characterized in that... The fluorescent probe is a diagnostic reagent for diseases caused by abnormal albumin levels.

9. The application according to claim 8, characterized in that... The diseases caused by abnormal albumin levels are selected from liver damage, cirrhosis, liver failure, chronic hepatitis, liver cancer, nephrotic syndrome, glomerulonephritis, coronary heart disease, Crohn's disease, ulcerative colitis, hyperthyroidism, malignant tumors, and malnutrition.

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