Enzymatic serum albumin fluorescent probe as well as synthesis and application thereof

Abstract

The invention provides an enzymatic serum albumin fluorescent probe as well as a synthesis method and application thereof. The fluorescent probe has a structure as shown in a formula (I), and the preparation process of the fluorescent probe comprises the following steps: (1) adding 3-hydroxy-1, 8-anhydride naphthalene and (S)-1-(4-pyridyl) ethylamine into an organic solvent, heating and refluxing, and after the reaction is completed, separating to obtain an intermediate product with a structure as shown in a formula (II); and (2) adding the intermediate product as shown in the formula (II) and alkali into an organic solvent, stirring in an ice-water bath until the mixture is uniformly mixed, dropwise adding cyclopropylformyl chloride, continuously stirring until the reaction is ended, and separating to obtain the enzymatic serum albumin fluorescent probe. The fluorescent probe disclosed by the invention is simple in synthesis step, high in sensitivity and good in selectivity, can realize accurate quantification of HSA in serum and urine, and has a very good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, 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 more than 50% of the total plasma protein. It plays many physiological functions, such as transporting endogenous and exogenous substances, maintaining plasma colloidal osmotic pressure, scavenging free radicals, and participating in immune regulation. The HSA content in the blood of healthy adults is maintained at 35-50g / L. Its reduced concentration reflects symptoms such as nutrition or malabsorption, chronic hepatitis, liver damage, cirrhosis, diabetes, chronic glomerulonephritis, and systemic lupus erythematosus. Under normal metabolic conditions, due to glomerular filtration and tubular reabsorption, the concentration of HSA in urine is less than 30mg / L. However, after renal vascular lesions occur, glomerular permeability increases, causing HSA in the blood to leak into the urine, significantly increasing the concentration of HSA in the urine and causing proteinuria. Low concentrations of albumin in urine (30–300mg / L) are called microalbuminuria, which is a manifestation of early kidney damage and the most sensitive and reliable diagnostic indicator for early detection of kidney disease. Therefore, accurate detection of HSA content is of key significance in fields such as 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. The colorimetric method usually uses bromocresol green (BCG) and bromocresol purple (BCP) as colorimetric agents. BCG binds to HSA and globulin at the same time, which easily leads to high measured values, while BCP is interfered by endogenous ligands, which easily leads to low measured values, and both methods are also susceptible to interference from heparin. In addition, the BCG method and the BCP method have low sensitivity and are not suitable for the determination of urinary microalbumin, and the stability of the BCG / BCP and HSA conjugates is poor, the measured values ​​have a strong time correlation, and the accuracy of the test results is low. The immunochemical method has good specificity, but low sensitivity, cumbersome operation, long time consumption and high cost. Size exclusion chromatography, proteomics and Raman scattering spectroscopy rely on instrument platforms, require high professional skills of operators, are costly and time-consuming, and are not suitable for high-throughput rapid detection of HSA. The electrochemical method involves the preparation of special electrodes, which have poor stability and selectivity and are difficult to apply to high-throughput detection of HSA.

[0004] In recent years, the fluorescence analysis method based on molecular probes has shown great potential in the high-throughput rapid detection of HSA due to its comprehensive advantages of strong selectivity, high sensitivity, fast response, simple operation, low cost and easy design. The reported HSA fluorescent probes can be divided into two categories: binding type and enzymatic type according to the detection principle. Among them, the design of the binding type HSA fluorescent probe is based on the change of the fluorescence signal after the probe molecule non-covalently binds to HSA. However, this type of probe is prone to non-specific binding with other biomacromolecules, resulting in poor selectivity, and the detection results are easily interfered by coexisting substances, making it difficult to achieve accurate quantification of HSA in complex matrices such as serum and urine.

[0005] Enzymatic HSA probes are usually designed based on the pseudoesterase activity of HSA. When such probe molecules encounter HSA, the ester bonds in their molecules can be hydrolyzed under the catalysis of HSA pseudoesterase activity to generate highly fluorescent enzymatic hydrolysis products. Due to the specificity of the enzymatic reaction, such probes have high selectivity for HSA. However, the existing reported enzymatic HSA fluorescent probes still have some defects, such as insufficient sensitivity, making it difficult to accurately measure samples with low HSA content such as urine; poor anti-interference ability, making it difficult to accurately quantify HSA in complex matrices such as serum; and complex synthesis steps, which are not conducive to commercial promotion. Summary of the invention

[0006] In view of the defects of the prior art, the present invention aims to provide an enzymatic serum albumin fluorescent probe and its synthesis and application. The enzymatic serum albumin fluorescent probe has simple synthesis steps, high sensitivity and high selectivity, and can achieve accurate quantification of 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 to 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 the mixture is uniformly mixed, add cyclopropylcarbonyl chloride dropwise, continue stirring until the reaction stops, and separate 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 application of the enzymatic serum albumin fluorescent probe in 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, and 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, and the fluorescence is significantly enhanced.

[0024] The present invention provides a novel enzymatic HSA fluorescent probe, which is designed based on the pseudoesterase activity of HSA and has the following beneficial effects compared with the existing HSA fluorescent probe:

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

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

[0027] (3) The synthesis steps are simple, easy to separate and purify, have good stability, are suitable for mass production, and are 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 the HSA concentration (Figure a) and the linear relationship between the fluorescence intensity at 585 nm and the 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 is further described below in conjunction with 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)ethylamine (288 μL, 2.40 mmol) to anhydrous ethanol (20 mL), heat to 80°C, heat under 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 (silica gel, 200-300 mesh, eluent is a dichloromethane / methanol mixture with a volume ratio of 40:1) to obtain an intermediate product, whose structure is shown in formula (II):

[0038]

[0039] Step 2: The intermediate product (0.32 g, 1.00 mmol) obtained in step 1 and cesium carbonate (1.95 g, 6.00 mmol) were added to anhydrous dichloromethane (20 mL), stirred in an ice-water bath for 30 minutes, and then 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 (silica gel, 200-300 mesh, eluent was 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] The 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 corresponds 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), added to an EP tube, and then deionized water, PBS buffer (200 mM, pH 7) and HSA stock solution (1 mg / mL) were added to the tube in sequence. 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 concentration of HSA was 200 μg / mL. The above solution was placed in a constant temperature incubator at 37° C. for 1 hour, and then its absorption spectrum and fluorescence spectrum were measured.

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

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

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

[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 about 160 times, indicating that it has a high sensitivity to HSA.

[0055] like Figure 6As shown in 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 the EP tube, mixed thoroughly, incubated at 37°C for 1 hour, and then the fluorescence spectrum was 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 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 ( 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 adding HSA, but the change range is relatively small after adding 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] Add DMSO stock solution (concentration of 0.2 mM) of the fluorescent probe (Formula (I)), deionized water, PBS buffer (200 mM, pH 7) and serum samples pre-added with different concentrations of HSA to the EP tube, mix thoroughly, incubate at 37°C for 1 hour, and 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, the final concentration of HSA is 0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and the final volume fraction of serum is 1%.

[0062] As shown in Table 1, using the fluorescence analysis method based on the fluorescent probe (I), the measured value of HSA concentration in the serum sample was 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 by fluorescent probe (Formula (I))

[0064]

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

[0066] Add DMSO stock solution (concentration of 0.2 mM) of the fluorescent probe (Formula (I)), deionized water, PBS buffer (200 mM, pH 7), and urine samples pre-added with different concentrations of HSA to the EP tube, 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, the final concentration of HSA is 0 μg / mL, 1.75 μg / mL, 3 μg / mL, 5 μg / mL, and the final volume fraction of urine is 5%.

[0067] As shown in Table 2, using the fluorescence analysis method based on the fluorescent probe (I), the measured value of HSA concentration in the urine sample was 30.78 mg / L, and the spike 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 to 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 the mixture is uniformly mixed, add cyclopropylcarbonyl chloride dropwise, continue stirring until the reaction stops, and separate 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.

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