A class of HSA fluorescent probes and their synthesis and applications

Abstract

The present invention provides a class of HSA fluorescent probes and their synthesis and applications. The fluorescent probe is an enzymatic HSA fluorescent probe having a structure represented by formula (I). The fluorescent probe of the present invention has high selectivity and high sensitivity, capable of distinguishing HSA from other enzymes (such as chymotrypsin, lipase, protease, β-galactosidase, pepsin, acetylcholinesterase, butyrylcholinesterase, carboxylesterase, trypsin, DNA enzyme, alkaline phosphatase, lysozyme, etc.), with a detection limit as low as 0.27 μg / mL. Therefore, the fluorescent probe of the present invention can achieve qualitative and quantitative detection of HSA in serum, urine, or cells.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to a type of HSA fluorescent probe and its synthesis and application. Background Art

[0002] Human serum albumin (HSA), composed of 585 amino acid residues, is the most abundant biomacromolecule in plasma. It plays a key role in maintaining colloid osmotic pressure, drug transport, and immune regulation. Under normal physiological conditions, HSA concentrations in urine are typically below 30 mg / L, while those in blood range from 35 to 50 g / L. Abnormal changes in HSA concentration are a sign of physiological imbalance in the body and an early warning sign of conditions such as vascular disease, liver disease, diabetes, and kidney disease. A drop in plasma HSA concentrations below 35 g / L may indicate pathological conditions such as chronic dehydration, increased adipose tissue, or kidney damage. In diabetic patients, renal vascular damage increases glomerular permeability, leading to leakage of HSA from plasma into urine, resulting in elevated urinary HSA concentrations. This phenomenon, known as microalbuminuria, is a key diagnostic indicator for chronic kidney disease. Therefore, the development of HSA detection technology has important application value in the diagnosis of liver and kidney diseases.

[0003] In recent years, HSA detection methods have primarily included dye-binding, immunochemical, chromatographic, nanoparticle, electrophoretic, and fluorescent probe methods. Dye-binding methods, such as bromocresol green (BCG) and bromocresol purple (BCP), can detect low concentrations of HSA, but are susceptible to interference from globulins and heparin, resulting in poor selectivity and stability. While immunochemical methods offer good selectivity, they are also costly, time-consuming, and have high background values. Chromatographic methods offer high precision, but are complex, time-consuming, and costly. Nanoparticle detection methods offer excellent specificity and sensitivity, but require high sample preparation, requiring precise control of sample handling steps and conditions, and are susceptible to interference from various factors, such as sample impurities and environmental noise. Electrophoretic methods can distinguish HSA from other plasma proteins, but are complex, potentially inactivating proteins, and require specialized operator skills. Compared to these methods, fluorescent probe methods offer advantages such as high sensitivity, selectivity, rapid response, ease of use, and low cost, showing broad application prospects for HSA detection in biological matrices such as serum and urine.

[0004] Existing HSA fluorescent probes primarily perform qualitative and quantitative analysis by measuring changes in fluorescence signals (e.g., intensity, wavelength, lifetime, polarity, etc.) after binding to HSA. However, since HSA may nonspecifically bind to other biomolecules (e.g., globulins, heparin, etc.), affecting the fluorescence signal, this method has difficulty achieving accurate quantification of HSA in complex biological matrices.

[0005] To improve selectivity, researchers have developed new enzymatic fluorescent probes based on HSA pseudoesterase activity. These probes, under the action of HSA, hydrolyze ester bonds to produce highly fluorescent products, thereby enabling the detection of HSA. However, despite the substrate specificity of enzymatic reactions, existing HSA enzymatic probes lack suitable ester recognition groups and are easily hydrolyzed by other enzymes such as acetylcholinesterase, butyrylcholinesterase, carboxylesterase, and lipase. This results in low selectivity for HSA and is susceptible to interference from other enzymes. Furthermore, existing HSA enzymatic probes have low sensitivity, which limits their application in the precise qualitative and quantitative detection of HSA. For example, Jin's group developed an enzymatic fluorescent probe DDAP based on the pseudoesterase activity and substrate selection of HSA. DDAP has high detection limit (6.51 μg / mL) for lipase, proteinase K, β-galactosidase, DNAse, alkaline phosphatase, lysozyme, transferrin, myoglobin, bovine serum albumin, immunoglobulin G, γ-globulin, insulin, bovine fibrinogenase, and protein (Jin Q, Feng L, Zhang SJ, et al., 2017). Summary of the Invention

[0006] In view of the low selectivity and sensitivity of HSA fluorescent probes in the prior art, the present invention aims to provide a class of HSA fluorescent probes and their synthesis and application. The fluorescent probes have high selectivity and high sensitivity and can realize the qualitative and quantitative detection of HSA in serum, urine and cells.

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

[0008] A type of HSA fluorescent probe, the HSA fluorescent probe comprises a total of 8 molecules, abbreviated as DDHB, DDHC, DDHD, DDHE, DDHF, DDHG, DDHH, and DDHI, and its structure is shown in formula (I):

[0009]

[0010] The preparation method of the above-mentioned HSA fluorescent probe comprises the following steps:

[0011] Step 1: Add 3-hydroxy-1,8-naphthalene anhydride and 4-(2-aminoethyl)morpholine to an organic solvent, heat under reflux, and after the reaction is complete, separate and obtain the intermediate product DDHE-OH, the structural formula of which is shown below:

[0012]

[0013] Step 2: Dissolve the intermediate product DDHE-OH obtained in step 1 and the base in an organic solvent, stir in an ice-water bath until the mixture is uniform, and then add Continue stirring until the reaction is terminated, and separate the HSA fluorescent probe represented by formula (I):

[0014]

[0015] Furthermore, in step 1, the molar ratio of 3-hydroxy-1,8-naphthalene anhydride to 4-(2-aminoethyl)morpholine 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] Further, in step 2, the intermediate product DDHE-OH, The molar ratio of the base used 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] Further, in step 2, stir in an ice water bath for 30 to 120 minutes, add Continue stirring for 1-8 hours to terminate the reaction.

[0022] The present invention also provides application of the HSA fluorescent probe in HSA detection.

[0023] The present invention provides a class of enzymatic HSA fluorescent probes by regulating the ester recognition group of the HSA fluorescent probe. The HSA fluorescent probe has high selectivity and can distinguish HSA from other enzymes, such as chymotrypsin, lipase, protease, β-galactosidase, pepsin, acetylcholinesterase, butyrylcholinesterase, carboxylesterase, trypsin, DNA enzyme, alkaline phosphatase, lysozyme, etc., and has high sensitivity, with a detection limit as low as 0.27 μg / mL. Therefore, the HSA fluorescent probe of the present invention can be used for the qualitative and quantitative detection of HSA in serum, urine and cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1The intermediate product DDHE-OH 1 H NMR spectrum (Figure a) and 13 C NMR spectrum (Figure b).

[0025] Figure 2 For the fluorescent probe DDHB 1 H NMR spectrum (Figure a) and 13 C NMR spectrum (Figure b).

[0026] Figure 3 For the fluorescent probe DDHC 1 H NMR spectrum (Figure a) and 13 C NMR spectrum (Figure b).

[0027] Figure 4 For the fluorescent probe DDHD 1 H NMR spectrum (Figure a) and 13 C NMR spectrum (Figure b).

[0028] Figure 5 For the fluorescent probe DDHE 1 H NMR spectrum (Figure a) and 13 C NMR spectrum (Figure b).

[0029] Figure 6 For the fluorescent probe DDHF 1 H NMR spectrum (Figure a) and 13 C NMR spectrum (Figure b).

[0030] Figure 7 DDHG is a fluorescent probe 1 H NMR spectrum (Figure a) and 13 C NMR spectrum (Figure b).

[0031] Figure 8 DDHH is a fluorescent probe 1 H NMR spectrum (Figure a) and 13 C NMR spectrum (Figure b).

[0032] Figure 9 For the fluorescent probe DDHI 1 H NMR spectrum (Figure a) and 13 C NMR spectrum (Figure b).

[0033] Figure 10 Figure 2 shows the absorption spectrum (Figure a) and fluorescence spectrum (Figure b) of the fluorescent probe DDHE after reacting with HSA.

[0034] Figure 11The fluorescence spectrum of the fluorescent probe DDHE changes with the HSA concentration (Figure a) and the linear relationship between the fluorescence intensity at 585 nm and the HSA concentration (Figure b).

[0035] Figure 12 is the fluorescence intensity of the fluorescent probe DDHE after adding different analytes.

[0036] Figure 13 The fluorescence intensities of the fluorescent probes DDHB (Figure a), DDHC (Figure b), DDHD (Figure c), DDHF (Figure d), DDHG (Figure e), DDHH (Figure f), and DDHI (Figure g) after adding different analytes.

[0037] Figure 14 Fluorescence images of A549 cells after pretreatment with acetylsalicylic acid (0, 0.5, 1, 2 mM) for 2 h and subsequent incubation with DDHE (10 μM) for 20 min (Figure a) and the relationship between the mean fluorescence intensity in A549 cells and the acetylsalicylic acid concentration (Figure b) (FL field: λex = 488 nm, λem = 520-650 nm; scale bar: 20 μm). DETAILED DESCRIPTION

[0038] 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.

[0039] Example 1 Synthesis of HSA fluorescent probe of the present invention

[0040] The synthetic route of the fluorescent probe of the present invention is as follows:

[0041]

[0042] Taking DDHE as an example, the specific steps of synthesizing the HSA fluorescent probe of the present invention are described in detail.

[0043] Step 1: Dissolve 3-hydroxy-1,8-naphthalene anhydride (0.43 g, 2.00 mmol) and 4-(2-aminoethyl)morpholine (262 μL, 2.00 mmol) in 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 DDHE-OH, whose structural formula is shown below:

[0044]

[0045] Step 2: The intermediate product DDHE-OH (0.326 g, 1.0 mM) obtained in Step 1 and Cs2CO3 (1.30 g, 4 mM) were dissolved in anhydrous dichloromethane (15 mL) and stirred in an ice bath for 30 min. Benzothiophene-2-carbonyl chloride (1.04 g, 4.0 mM) was then added and stirred for 8 hours. 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 fluorescent probe DDHE for detecting HSA. Its structural formula is shown below:

[0046]

[0047] Compounds DDHB, DDHC, DDHD, DDHF, DDHG, DDHH and DDHI were prepared respectively according to the above method.

[0048] Intermediate DDHE-OH 1 H NMR spectra and 13 C NMR spectra are shown in Figure 1 a and Figure 1 As shown in b, 1 HNMR spectra and 13 The C NMR spectrum corresponded to the chemical structure of DDHE-OH, indicating its successful synthesis.

[0049] 1 H NMR (500 MHz, Methanol-d 4 )δ8.34(d,J=7.2Hz,1H),8.16–8.09(m,2H),7.71(t,J=7.8Hz,1H),7.59(d,J=2.5Hz,1H), 4.34(t,J=6.8Hz,2H), 3.68(t,J=4.7Hz,4H), 2.73(t,J=6.8Hz,2H), 2.63(t,J=4.7Hz,4H).

[0050] 13C NMR (126MHz, DMSO_D6) δ163.99,163.65,156.63,133.82,133.08,128.00,127.84,123.85,122.62,122.33,116.24,66.69,56.03,53.88,37.27.

[0051] Fluorescent probe DDHB 1 H NMR spectra and 13 C NMR spectra are shown in Figure 2 a and Figure 2 As shown in b, 1H NMR spectra and 13 The C NMR spectrum corresponded to the chemical structure of the fluorescent probe DDHB, indicating its successful synthesis.

[0052] 1 H NMR (500 MHz, DMSO-d 6 )δ8.53–8.41(m,4H),8.32–8.26(m,2H),7.98–7.94(m,2H),7.92(dd,J=8.2,7.3Hz,1H),7.83–7.78(m,2H),7.54(dd,J =8.2,6.8Hz,2H),7.51–7.44(m,1H),4.20(t,J=6.9Hz,2H),3.54(t,J=4.7Hz,4H),2.59(t,J=7.0Hz,2H),2.47(s,4H). 13 C NMR (126 MHz, DMSO-d 6 )δ164.97,163.71,163.24,149.42,146.12,139.12,134.43,132.70,131.19,131.02,129.66,129.15,128 .58,127.78,127.65,127.57,126.25,125.96,124.20,122.67,66.67,56.50,55.98,53.87,37.40,19.01.

[0053] Fluorescent probe DDHC 1 H NMR spectra and 13 C NMR spectra are shown in Figure 3 a and Figure 3 As shown in b, 1 H NMR spectra and 13 The C NMR spectrum corresponded to the chemical structure of the fluorescent probe DDHC, indicating its successful synthesis.

[0054] 1 H NMR (500 MHz, DMSO-d 6)δ8.50(dd,J=7.2,1.1Hz,1H),8.46(dd,J=8.4,1.2Hz,1H),8.41(d,J=2.4Hz,1H),8.37(d,J=2.3Hz,1H),8.00(d,J=5.0Hz,1H), 7.92(dd,J=8.2,7.3Hz,1H),7.22(d,J=5.0Hz,1H),4.20(t,J=6.9Hz,2H),3.53(t,J=4.7Hz,4H),2.60(s,6H),2.48–2.44(m,3H).

[0055] 13 C NMR (126 MHz, DMSO-d 6 )δ163.69,163.21,160.88,148.88,134.43,133.97,132.93,132.71,131.03,128.58,126.20,126.00,124.70,12 4.17,122.67,66.68,55.98,53.87,40.48,40.40,40.31,40.23,40.14,39.97,39.81,39.64,39.48,37.40,16.37.

[0056] Fluorescent probe DDHD 1 H NMR spectra and 13 C NMR spectra are shown in Figure 4 a and Figure 4 As shown in b, 1 H NMR spectra and 13 The C NMR spectrum corresponded to the chemical structure of the fluorescent probe DDHD, indicating its successful synthesis.

[0057] 1 H NMR (500MHz, CDCl3) δ8.59(d,J=7.2Hz,1H),8.42(d,J=2.3Hz,1H),8.20(d,J=8.2Hz,1H),8.09(d,J=2.4Hz,1H),7.86(d,J=4.0Hz, 1H), 7.80 (t, J = 7.8Hz, 1H), 7.07 (d, J = 4.1Hz, 1H), 4.36 (t, J = 6.8Hz, 2H), 3.70 (t, J = 4.6Hz, 4H), 2.73 (t, J = 6.8Hz, 2H), 2.62 (s, 4H).

[0058] 13C NMR (126MHz, CDCl3) δ163.95,163.35,159.31,148.83,139.61,135.08,133.67,132.48,131.10,129.9 8,127.90,126.35,125.91,124.96,124.47,122.77,77.29,77.04,76.78,66.91,56.11,53.77,37.25.

[0059] Fluorescent probe DDHE 1 H NMR spectra and 13 C NMR spectra are shown in Figure 5 a and Figure 5 As shown in b, 1 H NMR spectra and 13 The C NMR spectrum corresponds to the chemical structure of the fluorescent probe DDHE, indicating its successful synthesis.

[0060] 1 H NMR (500MHz, CDCl3) δ8.60(dd,J=7.3,1.0Hz,1H),8.49(d,J=2.3Hz,1H),8.34(s,1H),8.22(d,J=8.2Hz,1H),8.16(d,J=2.3Hz,1H),7.96(dd,J= 14.3,8.1Hz,2H),7.81(t,J=7.8Hz,1H),7.58–7.52(m,1H),7.52–7.45( m,1H),4.37(t,J=6.8Hz,2H),3.79–3.63(m,4H),2.68(d,J=58.8Hz,6H).

[0061] 13 C NMR (126MHz, CDCl3) δ163.98,163.38,161.03,149.06,142.92,138.62,133.70,132.74,132.53,131.63,131 .08,127.89,127.76,126.38,126.01,125.35,125.02,124.49,122.94,122.80,66.95,56.13,53.79,37.27.

[0062] Fluorescent probe DDHF 1 H NMR spectra and 13 C NMR spectra are shown in Figure 6 a and Figure 6 As shown in b, 1 H NMR spectra and 13The C NMR spectrum corresponds to the chemical structure of the fluorescent probe DDHF, indicating its successful synthesis.

[0063] 1 H NMR (500 MHz, DMSO-d 6 )δ8.50(d,J=7.2Hz,1H),8.46(d,J=7.0Hz,3H),8.13(s,1H),7.95–7.87(m,2H),7.81(d,J=8.4Hz,1H),7.64–7.57(m, 1H), 7.44 (t, J = 7.6Hz, 1H), 4.19 (t, J = 7.0Hz, 2H), 3.54 (t, J = 4.5Hz, 4H), 2.59 (t, J = 6.9Hz, 2H), 2.48 (d, J = 4.5Hz, 4H). 13 C NMR (126 MHz, DMSO-d 6 )δ163.64,163.15,157.68,155.97,148.68,144.36,134.46,132.65,131.13,129.13,128.63,127.10, 126.06,125.98,125.85,124.81,124.31,124.06,122.66,116.89,112.78,66.69,55.98,53.88,37.43.

[0064] Fluorescent probe DDHG 1 H NMR spectra and 13 C NMR spectra are shown in Figure 7 a and Figure 7 As shown in b, 1 H NMR spectra and 13 The C NMR spectrum corresponds to the chemical structure of the fluorescent probe DDHG, indicating its successful synthesis.

[0065] 1 H NMR(500MHz, CDCl3)δ8.58(dt,J=7.3,1.3Hz,1H),8.43(d,J=2.3Hz,1H),8.23–8.16(m,3H),8.08(d,J=2.2Hz,1H),7.82–7.75(m ,1H),7.61–7.55(m,2H),4.35(t,J=6.8Hz,2H),3.68(t,J=4.6Hz,4H),2.71(t,J=6.8Hz,2H),2.60(t,J=4.5Hz,4H),1.39(s,9H). 13C NMR (126MHz, CDCl3) δ164.99,164.00,163.43,158.11,149.55,133.61,132.54,130.86,130.28,127.7 3,126.35,126.22,125.93,125.81,125.11,124.34,122.75,67.04,56.15,53.83,37.34,35.31,31.11.

[0066] Fluorescent probe DDHH 1 H NMR spectra and 13 C NMR spectra are shown in Figure 8 a and Figure 8 As shown in b, 1 H NMR spectra and 13 The C NMR spectrum corresponded to the chemical structure of the fluorescent probe DDHH, indicating its successful synthesis.

[0067] 1 H NMR (500MHz, CDCl3) δ8.61(dd,J=7.3,1.1Hz,1H),8.46(d,J=2.4Hz,1H),8.39(d,J=8.1Hz,2H),8.22(d,J=8.2Hz,1H),8.12(d,J=2. 3Hz, 1H), 7.87–7.82 (m, 2H), 7.81 (d, J = 7.8Hz, 1H), 4.36 (t, J = 6.8Hz, 2H), 3.68 (t, J = 4.7Hz, 4H), 2.72 (t, J = 6.8Hz, 2H), 2.60 (s, 4H).

[0068] 13C NMR (126MHz, CDCl3) δ163.93,163.86,163.35,149.12,133.64,132.54,132.05,131.16 ,130.77,127.97,126.42,125.89,124.95,124.61,122.85,67.04,56.14,53.83,37.38.

[0069] Fluorescent probe DDHI 1 H NMR spectra and 13 C NMR spectra are shown in Figure 9 a and Figure 9 As shown in b, 1 H NMR spectra and 13 The C NMR spectrum corresponds to the chemical structure of the fluorescent probe DDHI, indicating its successful synthesis.

[0070] 1 H NMR (500MHz, CDCl3) δ8.56 (dd, J=7.3, 1.1Hz, 1H), 8.29 (d, J=2.3Hz, 1H), 8.16 (dd, J=8.4, 1.1Hz,1H),7.93(d,J=2.3Hz,1H),7.77(dd,J=8.3,7.3Hz,1H),4.34(t,J=6.9Hz,2H),3.6 8(t,J=4.7Hz,4H),2.73–2.63(m,3H),2.59(s,3H),2.13(dt,J=12.6,3.6Hz,2H),1.87(dt ,J=12.9,3.7Hz,2H),1.73(dt,J=12.4,3.8Hz,1H),1.70–1.60(m,3H),1.47–1.27(m,3H).

[0071] 13 C NMR (126MHz, CDCl3) δ174.38,164.01,163.48,149.42,133.54,132.52,130.82,127.70,126. 27,126.17,124.93,124.27,122.74,67.06,56.15,53.84,43.19,37.33,28.95,25.67,25.33.

[0072] Examples 2-3 take DDHE as an example to illustrate in detail the fluorescence responsiveness and sensitivity of the fluorescent probe of the present invention to HSA.

[0073] Example 2 Fluorescence responsiveness of DDHE to HSA

[0074] The fluorescent probe DDHE prepared in Example 1 was prepared into a DMSO stock solution (1 mM) and added to an EP tube. Then, primary water, PBS buffer (200 mM, pH 7.4), and HSA stock solution (1 mg / mL) were added to the tube in sequence. After thorough mixing, the tube was placed in a 37°C constant temperature incubator for 70 minutes before absorption and fluorescence spectra were measured. The final concentration of PBS was 10 mM, the final concentration of DDHE was 10 μM, and the final concentration of HSA was 200 μg / mL. The excitation wavelength λex = 460 nm.

[0075] like Figure 10 As shown in a, the fluorescent probe DDHE has two absorption peaks at 305nm and 345nm. After incubation with HSA at 37℃ for 1h, the absorption of the probe at 345nm is enhanced, and two new absorption peaks appear at 380nm and 450nm.

[0076] like Figure 10 As shown in Figure b, under excitation at 460 nm, the probe exhibited no fluorescence signal in the wavelength range ≥500 nm. After incubation with HSA, the probe's fluorescence was significantly enhanced, with a peak emission at 585 nm. Under the test conditions, HSA exhibited no absorption or fluorescence signal in the wavelength range ≥500 nm.

[0077] The above results show that the fluorescent probe of the present invention has a significant fluorescence response to HSA.

[0078] Example 3 Sensitivity of fluorescent probe to HSA

[0079] Prepare a series of EP tubes and add a 1 mM DMSO stock solution of the fluorescent probe DDHE, grade 1 water, PBS buffer (200 mM, pH 7.4), and varying volumes of HSA stock solution (1 mg / mL). Mix thoroughly and place in a 37°C incubator for 70 minutes before measuring fluorescence spectra. The final concentrations of PBS, DDHE, and HSA were 10 mM, 10 μM, and 0-900 μg / mL, respectively, at λex = 460 nm.

[0080] like Figure 11 As shown in Figure 2, DDHE's own fluorescence is weak, and as the concentration of HSA increases, its fluorescence gradually increases ( Figure 11 a). When 900 μg / mL HSA was added, the fluorescence intensity of the probe at 585 nm increased by approximately 340-fold, indicating its high sensitivity to HSA.

[0081] like Figure 11 As shown in b, there is a linear relationship between the fluorescence intensity of the probe at 585 nm and the HSA concentration.

[0082] (F 585 =0.0972×[HSA]+1.6133, linear range is 0-40μg / mL; F 585 =0.6799×[HSA]-45.548, linear range: 40-900 μg / mL), and detection limit: 0.27 μg / mL.

[0083] The above results indicate that the probe of the present invention can be used for the quantitative analysis of HSA.

[0084] Example 4 Selectivity of the fluorescent probe of the present invention for HSA

[0085] The DMSO stock solution (1 mM) of DDHE prepared in Example 1, first-grade water, PBS buffer (200 mM, pH 7.4), and various test substances were added to the EP tube in sequence, mixed thoroughly, incubated at 37°C for 70 min, and then the fluorescence spectrum was measured.The total volume of the solution was 1 mL, the final concentration of DDHE was 10 μM, the final concentration of PBS was 10 mM, and the final concentrations of the various analytes were: 1 HSA (200 μg / mL), 2 chymotrypsin (CT, 200 μg / mL), 3 lipase (LPS, 200 μg / mL), 4 proteinase K (PK, 200 μg / mL), 5 β-galactosidase (β-Gal, 200 μg / mL), 6 pepsin (Pep, 200 μg / mL), 7 acetylcholinesterase (AChE, 200 μg / mL), 8 butyrylcholinesterase (BChE, 200 μg / mL), 9 carboxylesterase (CE, 200 μg / mL), 10 trypsin (TPS, 200 μg / mL). μg / mL), 11 DNAse (DNase, 200μg / mL), 12 alkaline phosphatase (ALP, 200μg / mL), 13 lysozyme (LYZ, 200μg / mL), 14 transferrin (Tf, 200μg / mL), 15 myoglobin (Mb, 200μg / mL), 16 bovine serum albumin (BSA, 200μg / mL), 17 immunoglobulin G (IgG, 200μg / mL), 18 gamma-globulin (GG, 200μg / mL), 19 insulin (Ins, 200μg / mL), 20 bovine fibrinogen (Fbg, 200μg / mL), 21 glutamic acid (Glu, 2mM), 22 serine (Ser, 2 mM)、23 Isoleucine (Ile, 2mM)、24 Threonine (Thr, 2mM)、25 Methionine (Met, 2mM)、26 Phenylalanine (Phe, 2mM)、27 Histidine (His, 2mM)、28 Asparagine (Asn, 2mM)、29 Tryptophan (Trp, 2mM)、30 Tyrosine (Tyr, 2mM)、31 Lysine (Lys, 2mM)、32 Alanine (Ala, 2mM)、33 Valine (Val, 2mM)、34 Leucine (Leu, 2mM)、35 Arginine (Arg, 2mM)、36 Proline (Pro, 2mM)、37 Aspartic acid (Asp, 2mM)、38 Lactic acid (LA, 2mM)、 39 Urea (Urea, 2mM), 40 Uric acid (UA, 2mM), 41 Creatinine (Cr, 2mM), 42 Creatine (Crea, 2mM), 43 Hyaluronic acid (HA, 200μg / mL), 44 Chondroitin sulfate (Chs, 200μg / mL), 45 Heparin (Hep, 200μg / mL), 46 Dextran (Dex, 200μg / mL), 47 Glucose (Glu, 200μg / mL), 48 Sucrose (Suc, 200μg / mL), 49 D-fructose (Fruc, 200μg / mL), 50 Vitamin C (Vc, 200μg / mL), 51 D-biotin (Biotin, 200μg / mL), 52Na. +(2mM), 53K + (2mM), 54Ba 2+ (2mM), 55Fe 2+ (2mM), 56NH4 + (2mM), 57Ca 2+ (2mM), 58Mg 2+ (2mM), 59Zn 2+ (2mM), 60Mn 2+ (2mM), 61Cu 2+ (2mM), 62PO4 3- (2mM), 63S2O3 2- (2mM), 64Br - (2mM), 65SO4 2- (2mM), 66Cl - (2mM), 67I - (2mM), 68NO3 - (2mM), 69CO3 2- (2mM).

[0086] like Figure 12 As shown in the figure, the fluorescence of the fluorescent probe DDHE is significantly enhanced after the addition of HSA, but the change is relatively small after the addition of proteins, amino acids, carbohydrates, cations, anions and other substances, indicating that the fluorescent probe has good selectivity for HSA.

[0087] The selectivity of other fluorescent probes (DDHB, DDHC, DDHD, DDHF, DDHG, DDHH and DDHI) of the present invention to HSA was tested according to the above method. The test results are as follows: Figure 13 shown.

[0088] Depend on Figure 13 a As can be seen from Figure 5, the fluorescence of the fluorescent probe DDHB is significantly enhanced after the addition of HSA, but the change is relatively small after the addition of proteins, amino acids, carbohydrates, cations, anions and other substances, indicating that the fluorescent probe has good selectivity for HSA.

[0089] Depend on Figure 13 b As can be seen, the fluorescence of the fluorescent probe DDHC is significantly enhanced after the addition of HSA, but the change is relatively small after the addition of proteins, amino acids, carbohydrates, cations, anions and other substances, indicating that the fluorescent probe has good selectivity for HSA.

[0090] Depend on Figure 13c It can be seen that the fluorescence of the fluorescent probe DDHD is significantly enhanced after the addition of HSA, but the change range is relatively small after the addition of proteins, amino acids, carbohydrates, cations, anions and other substances, indicating that the fluorescent probe has good selectivity for HSA.

[0091] Depend on Figure 13 d As can be seen, the fluorescence of the fluorescent probe DDHF is significantly enhanced after the addition of HSA, but the change is relatively small after the addition of proteins, amino acids, carbohydrates, cations, anions and other substances, indicating that the fluorescent probe has good selectivity for HSA.

[0092] Depend on Figure 13 e It can be seen that the fluorescence of the fluorescent probe DDHG is significantly enhanced after the addition of HSA, but the change range is relatively small after the addition of proteins, amino acids, carbohydrates, cations, anions and other substances, indicating that the fluorescent probe has good selectivity for HSA.

[0093] Depend on Figure 13 f As can be seen, the fluorescence of the fluorescent probe DDHH is significantly enhanced after the addition of HSA, but the change is relatively small after the addition of proteins, amino acids, carbohydrates, cations, anions and other substances, indicating that the fluorescent probe has good selectivity for HSA.

[0094] Depend on Figure 13 g It can be seen that the fluorescence of the fluorescent probe DDHI is significantly enhanced after the addition of HSA, but the change range is relatively small after the addition of proteins, amino acids, carbohydrates, cations, anions and other substances, indicating that the fluorescent probe has good selectivity for HSA.

[0095] The above results indicate that the eight fluorescent probes described in the present invention all have good selectivity for HSA and can be used for the specific detection of HSA.

[0096] Example 5 Quantitative analysis of HSA in serum samples

[0097] The DMSO stock solution (1 mM) of DDHE prepared in Example 1, grade 1 water, PBS buffer (200 mM, pH 7.4), and serum samples pre-spiked with varying concentrations of HSA were added to an EP tube and thoroughly mixed. After incubation at 37°C for 70 minutes, fluorescence spectra were measured. The final concentration of DDHE was 10 μM, the final concentration of PBS was 1 mM, the final volume fraction of serum was 2%, and the final HSA concentrations were 0, 10, 20, and 40 μg / mL, respectively. λex = 460 nm.

[0098] As shown in Table 1, using the fluorescence analysis method based on the fluorescent probe DDHE, the HSA concentration in the serum sample was determined to be 20.4 g / L, and the spike recovery rate was 96.5-112.1%, indicating that the fluorescent probe can be used for the quantitative analysis of HSA in serum.

[0099] Table 1 Quantitative analysis of HSA in serum samples using fluorescent probe DDHE

[0100]

[0101]

[0102] Example 6 Quantitative analysis of HSA in urine samples

[0103] To an EP tube, a DMSO stock solution (1 mM) of the DDHE prepared in Example 1, grade 1 water, PBS buffer (200 mM, pH 7.4), and urine samples pre-spiked with varying concentrations of HSA were added and thoroughly mixed. After incubation at 37°C for 70 minutes, the fluorescence spectrum was measured. The total volume of the solution was 1 mL, the final concentration of the probe DDHE was 10 μM, the final concentration of PBS was 10 mM, the concentrations of HSA were 0 μg / mL, 35 μg / mL, 70 μg / mL, and 100 μg / mL, the final volume fraction of urine was 10%, and λex = 460 nm.

[0104] As shown in Table 2, using the fluorescence analysis method based on the fluorescent probe DDHE, the HSA concentration in the urine sample was determined to be 34.6 mg / L, and the spike recovery rate was 103.4-114.9%, indicating that the fluorescent probe of the present invention can be used for the quantitative analysis of HSA in urine.

[0105] Table 2 Quantitative analysis of HSA in urine samples using fluorescent probe DDHE

[0106]

[0107] Example 7

[0108] A549 cells were cultured at 3 × 10 5 Cells were seeded at a density of 100 cells / dish into confocal microplates and cultured for 24 hours to allow them to adhere to the wall. Subsequently, 1 mL of cell culture medium was aspirated and 1 mL of cell culture medium containing acetylsalicylic acid was added. The cells were then cultured at 37°C for 2 hours. Next, 1 mL of cell culture medium was aspirated and 1 mL of cell culture medium containing DDHE was added. After incubation for 20 minutes, the cells were washed twice with PBS and then observed using a confocal fluorescence microscope for fluorescence imaging. The final concentrations of acetylsalicylic acid were 0 mM, 0.5 mM, 1 mM, and 2 mM, and the final concentration of DDHE was 10 μM.

[0109] like Figure 14 As shown, when A549 cells were co-incubated with acetylsalicylic acid, the fluorescence of DDHE in the cells was weakened ( Figure 14 a), and the decrease in fluorescence intensity is positively correlated with the concentration of acetylsalicylic acid ( Figure 14 b) This phenomenon indicates that acetylsalicylic acid inhibits the pseudoesterase activity of HSA. This reveals that the intracellular fluorescence signal of DDHE is activated by HSA. Therefore, the intracellular fluorescence intensity of DDHE can be used as a sensitive indicator of HSA concentration.

Claims

1. A type of HSA fluorescent probe, characterized in that: The HSA fluorescent probe contains a total of 8 molecules, abbreviated as DDHB, DDHC, DDHD, DDHE, DDHF, DDHG, DDHH, and DDHI, and their structural formulas are shown in formula (I): 。 2. The method for preparing the HSA fluorescent probe according to claim 1, wherein The following steps are involved: Step 1: Add 3-hydroxy-1,8-naphthalene anhydride and 4-(2-aminoethyl)morpholine to an organic solvent, heat under reflux, and after the reaction is complete, separate and obtain the intermediate product DDHE-OH, the structural formula of which is shown below: ； Step 2: Dissolve the intermediate product DDHE-OH obtained in step 1 and the base in an organic solvent, stir in an ice-water bath until the mixture is uniform, and then add , continue stirring until the reaction is terminated, and separate the HSA 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 4-(2-aminoethyl)morpholine 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 used 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 intermediate product DDHE-OH, The molar ratio of the base used 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 or 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 or 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, stir in an ice-water bath for 30 to 120 minutes, then add , continue stirring for 1-8 hours to terminate the reaction.

10. Use of the HSA fluorescent probe according to claim 1 in preparing an HSA detection reagent.

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