An enzymatic serum albumin fluorescent probe and its synthesis and application

Abstract

The present invention provides an enzymatic serum albumin fluorescent probe, a synthesis method, and an application thereof. The fluorescent probe has a structure represented by formula (I), and its preparation process is as follows: (1) 3-hydroxy-1,8-naphthylic anhydride and (S)-1-(4-pyridyl)ethylamine are added to an organic solvent, heated to reflux, and after the reaction is complete, an intermediate product represented by formula (II) is isolated; (2) the intermediate product represented by formula (II) and a base are added to an organic solvent, stirred in an ice-water bath until uniformly mixed, cyclopropylcarbonyl chloride is added dropwise, stirring is continued until the reaction terminates, and the enzymatic serum albumin fluorescent probe of the present invention is isolated. The fluorescent probe of the present invention has simple synthesis steps, high sensitivity, and good selectivity, and can achieve accurate quantification of HSA in serum and urine, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to an enzymatic serum albumin fluorescent probe and its synthesis and application. Background Art

[0002] Human serum albumin (HSA) is the most abundant protein in plasma, accounting for over 50% of total plasma protein. It performs numerous physiological functions, including transporting endogenous and exogenous substances, maintaining plasma colloid osmotic pressure, scavenging free radicals, and participating in immune regulation. The HSA concentration in healthy adults is maintained between 35 and 50 g / L. Decreased HSA concentrations reflect conditions such as nutritional deficiencies or malabsorption, chronic hepatitis, liver damage, cirrhosis, diabetes, chronic glomerulonephritis, and systemic lupus erythematosus. Under normal metabolic conditions, urinary HSA concentrations are below 30 mg / L due to glomerular filtration and tubular reabsorption. However, when renal vascular disease develops, increased glomerular permeability leads to leakage of HSA from the blood into the urine, significantly increasing urinary HSA concentrations and causing proteinuria. Low concentrations of albumin in the urine (30–300 mg / L) are known as microalbuminuria and are a sign of early kidney damage. They are the most sensitive and reliable diagnostic marker for early detection of kidney disease. Therefore, accurate detection of HSA content is of key significance in medical diagnosis, clinical research, and biological sciences.

[0003] The main methods for detecting HSA include colorimetry, immunochemistry, size exclusion chromatography, proteomics, Raman scattering spectroscopy, electrochemistry, and fluorescence analysis. The first two methods are the most widely used in biochemical detection. Colorimetry typically uses bromocresol green (BCG) and bromocresol purple (BCP) as colorants. BCG binds simultaneously to HSA and globulins, which can easily lead to overestimation of measurement values, while BCP is subject to interference from endogenous ligands, which can easily lead to underestimation of measurement values. Both methods are also susceptible to interference from heparin. Furthermore, BCG and BCP methods have low sensitivity and are not suitable for the determination of urinary microalbumin. Furthermore, the stability of the BCG / BCP-HSA conjugate is poor, resulting in a strong time-dependent measurement value and low test accuracy. Immunochemistry has good specificity, but low sensitivity, cumbersome procedures, long time consumption, and high cost. Size exclusion chromatography, proteomics, and Raman scattering spectroscopy rely on instrument platforms, require high operator expertise, are costly, and time-consuming, making them unsuitable for high-throughput and rapid detection of HSA. The electrochemical method involves the preparation of special electrodes, has poor stability and selectivity, and is difficult to apply to high-throughput detection of HSA.

[0004] In recent years, fluorescence analysis based on molecular probes has shown great potential for high-throughput rapid detection of HSA due to its combined advantages of strong selectivity, high sensitivity, rapid response, simple operation, low cost, and ease of design. Reported HSA fluorescent probes can be divided into two categories based on their detection principles: binding and enzymatic. Binding HSA fluorescent probes are designed based on the change in fluorescence signal following non-covalent binding of the probe molecule to HSA. However, these probes are prone to nonspecific binding with other biomacromolecules, resulting in poor selectivity and susceptible to interference from coexisting substances, making accurate quantification of HSA difficult in complex matrices such as serum and urine.

[0005] Enzymatic HSA probes are typically designed based on the pseudoesterase activity of HSA. Upon encountering HSA, these probe molecules undergo hydrolysis of their ester bonds under the catalysis of the HSA pseudoesterase activity, generating highly fluorescent enzymatic hydrolysis products. Due to the specificity of the enzymatic reaction, these probes exhibit high selectivity for HSA. However, existing reported enzymatic HSA fluorescent probes still have several drawbacks, including insufficient sensitivity, making it difficult to accurately measure HSA in samples with low HSA content, such as urine; poor interference resistance, making it difficult to accurately quantify HSA in complex matrices such as serum; and complex synthesis steps, hindering commercialization. Summary of the Invention

[0006] In response to the shortcomings of the prior art, the present invention aims to provide an enzymatic serum albumin fluorescent probe, its synthesis, and application. The enzymatic serum albumin fluorescent probe has simple synthesis steps, high sensitivity, and high selectivity, and can accurately quantify HAS in serum and urine.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] An enzymatic serum albumin fluorescent probe, the structural formula of the fluorescent probe is shown in formula (I):

[0009]

[0010] The preparation method of the enzymatic serum albumin fluorescent probe comprises the following steps:

[0011] Step 1: Add 3-hydroxy-1,8-naphthalene anhydride and (S)-1-(4-pyridyl)ethylamine to an organic solvent, heat under reflux, and after the reaction is completed, separate and obtain an intermediate product with a structure shown in formula (II):

[0012]

[0013] Step 2: Add the intermediate product represented by formula (II) and a base to an organic solvent, stir in an ice-water bath until uniformly mixed, add cyclopropylcarbonyl chloride dropwise, continue stirring until the reaction terminates, and isolate the enzymatic serum albumin fluorescent probe represented by formula (I):

[0014]

[0015] Furthermore, in step 1, the molar ratio of 3-hydroxy-1,8-naphthalene anhydride to (S)-1-(4-pyridyl)ethylamine is 1:(1-2).

[0016] Furthermore, in step 1, the organic solvent is at least one of ethanol, tetrahydrofuran, acetonitrile, acetone, and N,N-dimethylformamide, and the amount thereof is 20-60 ml.

[0017] Furthermore, in step 1, the heating reflux reaction temperature is 60° C.-100° C.; and the reaction time is 2-8 hours.

[0018] Furthermore, in step 2, the molar ratio of the intermediate product, cyclopropylcarbonyl chloride and the base is 1:(1-6):(1-12).

[0019] Furthermore, in step 2, the base is at least one of cesium carbonate, potassium carbonate, sodium carbonate, sodium bicarbonate, pyridine, piperazine, triethylamine, and N,N-dimethylaminopyridine.

[0020] Furthermore, in step 2, the organic solvent is at least one of dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran, acetonitrile, acetone, N,N-dimethylformamide or 1,2-dichloroethane, and the amount thereof is 20-60 ml.

[0021] Furthermore, in step 2, the mixture is stirred in an ice-water bath for 30 to 120 minutes, cyclopropylcarbonyl chloride is added dropwise, and stirring is continued for 1 to 8 hours to terminate the reaction.

[0022] The present invention also provides the use of the enzymatic serum albumin fluorescent probe in the quantitative detection of HSA.

[0023] The principle by which the fluorescent probe of the present invention can achieve quantitative detection of HSA is that during the incubation process, the fluorescent probe reacts with HSA in the sample to be tested. Under the action of the pseudoesterase activity of HSA, the cyclopropanecarboxylate bond in the fluorescent probe molecule is hydrolyzed and broken to remove the cyclopropanecarboxylate group, resulting in a significant enhancement of fluorescence.

[0024] The present invention provides a novel enzymatic HSA fluorescent probe designed based on the pseudoesterase activity of HSA. Compared with existing HSA fluorescent probes, it has the following advantages:

[0025] (1) High sensitivity, suitable for accurate determination of samples with low HSA content, such as urine; the fluorescent probe of the present invention has an excitation wavelength of 300-550 nm and an emission wavelength of 400-800 nm. Its fluorescence intensity is linearly related to HSA concentration in the range of 0.32-500 μg / mL, with a detection limit of 0.095 μg / mL;

[0026] (2) It has good selectivity and strong resistance to interference from coexisting substances such as ions, amino acids, carbohydrates, and proteins, making it suitable for the precise quantification of HSA in complex matrices such as serum;

[0027] (3) The synthesis steps are simple, easy to separate and purify, and have good stability, making them suitable for mass production and conducive to commercial promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 For the intermediate product (Formula (II)) 1 H NMR spectrum.

[0029] Figure 2 For the intermediate product (Formula (II)) 13 C NMR spectrum.

[0030] Figure 3 The enzymatic serum albumin fluorescent probe (Formula (I)) of the present invention is 1 H NMR spectrum.

[0031] Figure 4 The enzymatic serum albumin fluorescent probe (Formula (I)) of the present invention is 13 C NMR spectrum.

[0032] Figure 5 The absorption spectrum (Figure a) and fluorescence spectrum (Figure b) of the enzymatic serum albumin fluorescent probe (Formula (I)) of the present invention after reacting with HSA.

[0033] Figure 6 The fluorescence spectrum of the enzymatic serum albumin fluorescent probe (Formula (I)) of the present invention changes with HSA concentration (Figure a) and the linear relationship between the fluorescence intensity at 585 nm and HSA concentration (Figure b).

[0034] Figure 7 It is the fluorescence intensity of the enzymatic serum albumin fluorescent probe (Formula (I)) of the present invention at 585 nm after adding different analytes. DETAILED DESCRIPTION

[0035] The present invention will be further described below with reference to the following examples. The following examples are only for illustration and are not intended to limit the scope of protection of the present invention in any way.

[0036] Example 1 Synthesis of Enzymatic Serum Albumin Fluorescent Probe

[0037] Step 1: Add 3-hydroxy-1,8-naphthalene anhydride (0.43 g, 2.00 mmol) and (S)-1-(4-pyridyl)ethanamine (288 μL, 2.40 mmol) to anhydrous ethanol (20 mL). Heat to 80°C, reflux for 4 hours, and cool naturally to room temperature. The reaction solution is concentrated under reduced pressure to remove the solvent, and the residue is separated by column chromatography (silica gel, 200-300 mesh, eluent: a 40:1 volume ratio of dichloromethane / methanol mixture) to obtain the intermediate product, whose structure is shown in Formula (II):

[0038]

[0039] Step 2: The intermediate product obtained in Step 1 (0.32 g, 1.00 mmol) and cesium carbonate (1.95 g, 6.00 mmol) were added to anhydrous dichloromethane (20 mL). After stirring in an ice-water bath for 30 minutes, cyclopropylcarbonyl chloride (272 μL, 3.0 mmol) was added dropwise. After stirring for 3 hours, the reaction was terminated. The reaction solution was concentrated under reduced pressure to remove the solvent, and the residue was separated by column chromatography (silica gel, 200-300 mesh, eluent: a dichloromethane / methanol mixture with a volume ratio of 40:1) to obtain the enzymatic serum albumin fluorescent probe of the present invention, whose structure is shown in Formula (I):

[0040]

[0041] Intermediate product (Formula (II)) 1 H NMR spectrum Figure 1 As shown, 13 C NMR spectrum Figure 2 As shown, 1 H NMR spectra and 13 The C NMR spectrum corresponded to the chemical structure of the intermediate product, indicating its successful synthesis.

[0042] 1 H NMR (500 MHz, DMSO-d 6 )δ10.55(s,1H),8.49–8.44(m,2H),8.25–8.19(m,2H),7.99(d,J=2.4Hz,1H),7.72(t,J=7.8H z,1H),7.65(d,J=2.4Hz,1H),7.36–7.31(m,2H),6.29(q,J=6.9Hz,1H),1.85(d,J=7.0Hz,3H).

[0043] 13 C NMR (126 MHz, DMSO-d6 )δ163.99,163.64,156.64,150.69,149.86,133.75,133.11,128.22,127.83,124.02,122.82,122.51,121.68,116.32,55.32,48.79,16.11.

[0044] The enzymatic serum albumin fluorescent probe (Formula (I)) of the present invention 1 H NMR spectrum Figure 3 As shown, 13 C NMR spectrum Figure 4 As shown, 1 H NMR spectra and 13 The C NMR spectrum corresponded to the chemical structure of the fluorescent probe, indicating its successful synthesis.

[0045] 1 H NMR (500MHz, DMSO-d6) δ8.58–8.53(m,3H),8.32–8.25(m,2H),8.18(d,J=2.3Hz,1H),7.85(t,J=7.8Hz,1H),7. 62–7.57(m,2H),5.36(q,J=6.6Hz,1H),2.00(tt,J=7.1,5.4Hz,1H),1.51(d,J=6.7Hz,3H),1.18–1.10(m,4H).

[0046] 13 C NMR (126 MHz, DMSO-d 6 )δ173.42,159.44,154.31,150.13,148.84,147.40,132.92,131.67,128.68,127 .72,127.31,126.31,126.24,122.40,122.25,121.16,53.87,24.20,13.14,9.84.

[0047] Example 2 Fluorescence responsiveness of the enzymatic serum albumin fluorescent probe of the present invention to HSA

[0048] The enzymatic serum albumin fluorescent probe (Formula (I)) prepared in Example 1 was prepared into a DMSO stock solution (0.2 mM) and added to an EP tube. Deionized water, PBS buffer (200 mM, pH 7), and HSA stock solution (1 mg / mL) were then added to the tube in sequence. The total volume of the solution was 1 mL, with a final concentration of 10 μM for the probe, 20 mM for PBS, and 200 μg / mL for HSA. The above solution was placed in a 37°C incubator for 1 hour, and its absorption and fluorescence spectra were then measured.

[0049] like Figure 5 As shown in Figure a, the fluorescent probe (Formula (I)) has an absorption peak at 340 nm. After incubation with HSA at 37°C for 1 hour, the absorption of the probe at 340 nm weakened, but the absorption at 380 nm and 450 nm increased.

[0050] like Figure 5 As shown in Figure b, under excitation at 370 nm, the probe exhibits weak fluorescence at wavelengths ≥500 nm. After incubation with HSA, a strong fluorescence signal is generated at 585 nm. Under the test conditions, HSA exhibits no absorption or fluorescence signal at wavelengths ≥500 nm.

[0051] The above results show that the fluorescent probe of the present invention has a significant fluorescence response to HSA.

[0052] Example 3 Sensitivity of fluorescent probe to HSA

[0053] To a series of EP tubes, add a DMSO stock solution (0.2 mM) of the enzymatic serum albumin fluorescent probe (Formula (I)) prepared in Example 1, deionized water, PBS buffer (200 mM, pH 7), and varying volumes of HSA stock solution (1 mg / mL). The total volume of the solution was 1 mL, with a final concentration of 10 μM probe, 20 mM PBS, and 0-800 μg / mL HSA. The solutions were incubated at 37°C for 1 hour, and their fluorescence spectra were then measured.

[0054] like Figure 6 As shown, the fluorescence of the fluorescent probe (Formula (I)) is weak, and as the concentration of HSA increases, the fluorescence gradually increases ( Figure 6 a). When 800 μg / mL HSA was added, the fluorescence intensity of the probe at 585 nm increased by approximately 160-fold, indicating its high sensitivity to HSA.

[0055] like Figure 6As shown in Figure b, there is a linear relationship between the fluorescence intensity of the probe at 585 nm and the HSA concentration, with a linear range of 0.32-500 μg / mL and a detection limit of 0.095 μg / mL (the detection limit is calculated as 3σ / k, where σ is the standard deviation of 11 parallel measurements of the fluorescence intensity of the probe at 585 nm, and k is the slope of the linear equation).

[0056] The above results indicate that the fluorescent probe of the present invention can be used for highly sensitive quantitative detection of HSA.

[0057] Example 4 Selectivity and Anti-interference of the Enzymatic Serum Albumin Fluorescent Probe of the Present Invention for HSA

[0058] The DMSO stock solution (concentration of 0.2 mM) of the enzymatic serum albumin fluorescent probe (Formula (I)) prepared in Example 1, deionized water, PBS buffer (200 mM, pH 7), and various analytes were added to an EP tube, mixed thoroughly, and incubated at 37°C for 1 hour before measuring the fluorescence spectrum. The total volume of the solution was 1 mL, the final concentration of the probe was 10 μM, the final concentration of PBS was 20 mM, and the various analytes and their final concentrations were: 1 HSA (100 μg / mL), 2 Na + (1mM), 3K + (1mM), 4Ba 2+ (1mM), 5Fe 2+ (1mM), 6NH4 + (1mM), 7Ca 2+ (1mM), 8Mg 2+ (1mM), 9Zn 2+ (1mM), 10Mn 2+ (1mM), 11Cu 2+ (1mM), 12PO4 3- (1mM), 13S2O3 2- (1mM), 14Br - (1mM), 15SO4 2- (1mM), 16Cl - (1mM), 17I - (1mM), 18NO3 - (1mM), 19CO3 2-(1mM), 20 glutamic acid (Glu, 1mM), 21 serine (Ser, 1mM), 22 isoleucine (Ile, 1mM), 23 threonine (Thr, 1mM), 24 methionine (Met, 1mM), 25 phenylalanine (Phe, 1mM), 26 histidine (His, 1mM), 27 asparagine (Asn, 1mM), 28 tryptophan (Trp, 1mM), 29 tyrosine (Tyr, 1mM), 30 lysine (Lys, 1mM), 31 alanine (Ala, 1mM), 32 valine (Val, 1mM), 33 leucine ( 1mM), 34 arginine (Arg, 1mM), 35 proline (Pro, 1mM), 36 aspartic acid (Asp, 1mM), 37 urea (Urea, 1mM), 38 uric acid (UA, 1mM), 39 lactic acid (LA, 1mM), 40 creatinine (Cr, 1mM), 41 creatine (Crea, 1mM), 42 hyaluronic acid (HA, 100μg / mL), 43 chondroitin sulfate (Chs, 100μg / mL), 44 heparin (Hep, 100μg / mL), 45 dextran (Dex, 100μg / mL), 46 glucose ( Glu, 100μg / mL), 47 sucrose (Suc, 100μg / mL), 48 D-fructose (Fruc, 100μg / mL), 49 vitamin C (Vc, 100μg / mL), 50 D-biotin (Biotin, 100μg / mL), 51 pepsin (Pep, 100μg / mL), 52 acetylcholinesterase (AChE, 100μg / mL), 53 chymotrypsin (CT, 100μg / mL), 54 β-galactosidase (β-Gal, 100μg / mL), 55 lipase (LPS, 100μg / mL), 56 Trypsin (TPS, 100 μg / mL), 57 Carboxylesterase (CE, 100 μg / mL), 58 Proteinase K (PK, 100 μg / mL), 59 Transferrin (Tf, 100 μg / mL), 60 Myoglobin (Mb, 100 μg / mL), 61 Bovine serum albumin (BSA, 100 μg / mL), 62 Immunoglobulin G (IgG, 100 μg / mL), 63 γ-globulin (GG, 100 μg / mL), 64 Insulin (Ins, 100 μg / mL), 65 Bovine fibrinogen (Fbg, 100 μg / mL).

[0059] like Figure 7 As shown, the fluorescence of the fluorescent probe (Formula (I)) is significantly enhanced after the addition of HSA, but the change is relatively small after the addition of cations, anions, amino acids, carbohydrates, proteins and other substances, indicating that the fluorescent probe has good selectivity for HSA.

[0060] Example 5 Quantitative analysis of HSA in serum samples

[0061] To an EP tube, add a DMSO stock solution (0.2 mM) of the fluorescent probe (Formula (I)), deionized water, PBS buffer (200 mM, pH 7), and serum samples pre-spiked with varying concentrations of HSA. Mix thoroughly, incubate at 37°C for 1 hour, and then measure the fluorescence spectrum. The total volume of the solution is 1 mL, the final concentration of the probe is 10 μM, the final concentration of PBS is 20 mM, and the final concentrations of HSA are 0 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL. The final volume fraction of serum is 1%.

[0062] As shown in Table 1, using the fluorescence analysis method based on fluorescent probe (I), the HSA concentration in the serum sample was determined to be 16.59 g / L, and the spike recovery rate was 106.9-109.9%, indicating that the fluorescent probe of the present invention can be used for the quantitative analysis of HSA in serum.

[0063] Table 1 Quantitative analysis of HSA in serum samples using fluorescent probe (Formula (I))

[0064]

[0065] Example 6 Quantitative analysis of HSA in urine samples

[0066] To an EP tube, a DMSO stock solution (0.2 mM) of the fluorescent probe (Formula (I)), deionized water, PBS buffer (200 mM, pH 7), and urine samples pre-spiked with varying concentrations of HSA were added. The mixture was thoroughly mixed and incubated at 37°C for 1 hour before fluorescence spectra were measured. The total volume of the solution was 1 mL, the final concentration of the probe was 10 μM, the final concentration of PBS was 20 mM, and the final concentrations of HSA were 0 μg / mL, 1.75 μg / mL, 3 μg / mL, and 5 μg / mL. The final volume fraction of urine was 5%.

[0067] As shown in Table 2, using the fluorescence analysis method based on fluorescent probe (I), the measured value of HSA concentration in urine samples was 30.78 mg / L, and the spiked recovery rate was 91.9-100.7%, indicating that the fluorescent probe of the present invention can be used for the quantitative analysis of HSA in urine.

[0068] Table 2 Quantitative analysis of HSA in urine samples using fluorescent probe (Formula (I))

[0069]

Claims

1. An enzymatic serum albumin fluorescent probe, characterized in that: The structural formula of the fluorescent probe is shown in formula (I):

2. The method for preparing the enzymatic serum albumin fluorescent probe according to claim 1, characterized in that: The following steps are involved: Step 1: Add 3-hydroxy-1,8-naphthalene anhydride and (S)-1-(4-pyridyl)ethylamine to an organic solvent, heat under reflux, and after the reaction is completed, separate and obtain an intermediate product with a structure shown in formula (II): Step 2: Add the intermediate product represented by formula (II) and a base to an organic solvent, stir in an ice-water bath until uniformly mixed, add cyclopropylcarbonyl chloride dropwise, continue stirring until the reaction terminates, and isolate the enzymatic serum albumin fluorescent probe represented by formula (I):

3. The preparation method according to claim 2, characterized in that In step 1, the molar ratio of 3-hydroxy-1,8-naphthalene anhydride to (S)-1-(4-pyridyl)ethylamine is 1:(1-2).

4. The preparation method according to claim 2, characterized in that In step 1, the organic solvent is at least one of ethanol, tetrahydrofuran, acetonitrile, acetone, and N,N-dimethylformamide, and the amount thereof is 20-60 ml.

5. The preparation method according to claim 2, characterized in that In step 1, the heating reflux reaction temperature is 60° C.-100° C.; and the reaction time is 2-8 hours.

6. The preparation method according to claim 2, characterized in that In step 2, the molar ratio of the intermediate product, cyclopropylcarbonyl chloride and the base is 1:(1-6):(1-12).

7. The preparation method according to claim 2, characterized in that In step 2, the base is at least one of cesium carbonate, potassium carbonate, sodium carbonate, sodium bicarbonate, pyridine, piperazine, triethylamine, and N,N-dimethylaminopyridine.

8. The preparation method according to claim 2, characterized in that In step 2, the organic solvent is at least one of dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran, acetonitrile, acetone, N,N-dimethylformamide and 1,2-dichloroethane, and the amount thereof is 20-60 ml.

9. The preparation method according to claim 2, characterized in that In step 2, the mixture is stirred in an ice-water bath for 30 to 120 minutes, cyclopropylcarbonyl chloride is added dropwise, and stirring is continued for 1 to 8 hours to terminate the reaction.

10. Use of the enzymatic serum albumin fluorescent probe according to claim 1 in the quantitative detection of HSA, wherein the purpose of the use is not the diagnosis and treatment of diseases.

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