HSA fluorescent probe as well as synthesis and application thereof

Abstract

The invention provides an HSA fluorescent probe as well as synthesis and application thereof. The fluorescent probe is an enzymatic HSA fluorescent probe and has a structure as shown in a formula (I). The fluorescent probe has high selectivity and high sensitivity, can distinguish HSA from other enzymes (such as chymotrypsin, lipase, protease, beta-galactosidase, pepsin, acetylcholin esterase, butyrylcholine esterase, carboxylesterase, trypsin, DNA enzyme, alkaline phosphatase, lysozyme and the like), and has the detection limit as low as 0.27 mu g / mL, so that the fluorescent probe has the advantages of high selectivity, high sensitivity, high sensitivity and the like. The fluorescent probe provided by the invention can realize qualitative and quantitative detection of HSA in serum, urine or cells.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a type of HSA fluorescent probe and the synthesis and application thereof. Background Art

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

[0003] In recent years, the detection methods of HSA mainly include dye binding method, immunochemical method, chromatography, nanoparticle detection method, electrophoresis and fluorescent probe method. Dye binding method, such as bromocresol green method (BCG) and bromocresol purple method (BCP), can detect low concentrations of HSA, but it is easily interfered by globulin, heparin, etc., and has poor selectivity and stability. Although the immunochemical method has good selectivity, it is costly, time-consuming and has a high background value. The chromatography method has high precision, but the operation steps are complicated, time-consuming and costly. The nanoparticle detection method has good specificity and high sensitivity, but the sample preparation requirements are high, and the sample processing steps and conditions need to be precisely controlled. It is also susceptible to interference from various factors, such as impurities in the sample, environmental noise, etc. The electrophoresis method can distinguish HSA from other plasma proteins, but the operation is complicated and may cause protein inactivation, and requires high professional skills of the operator. Compared with the above methods, the fluorescent probe method has the advantages of high sensitivity, good selectivity, response rate, simple operation and low cost, and shows broad application prospects in the detection of HSA in biological matrices such as serum and urine.

[0004] The existing HSA fluorescent probes mainly perform qualitative and quantitative analysis by changes in fluorescence signals (such as intensity, wavelength, lifetime, polarity, etc.) after binding to HSA. However, since HSA may bind non-specifically to other biological molecules (such as globulin, heparin, etc.), affecting the fluorescence signal, this method is difficult to achieve accurate quantification of HSA in complex biological matrices.

[0005] In order to improve the selectivity, researchers have developed a new type of enzymatic reaction fluorescent probe based on the pseudoesterase activity of HSA. Under the action of HSA, this type of probe produces a strong fluorescent product by hydrolyzing the ester bond, thereby realizing the detection of HSA. However, although the enzymatic reaction has substrate specificity, the existing HSA enzymatic probes lack suitable ester recognition groups and are easily hydrolyzed by other enzymes such as acetylcholinesterase, butyrylcholinesterase, carboxylesterase, lipase, etc., resulting in low selectivity for HSA and easy interference from other enzymes. In addition, the existing HSA enzymatic probes have low sensitivity, which limits their application in the precise qualitative and quantitative determination of HSA. For example, Jin's research group developed an enzymatic fluorescent probe DDAP based on the pseudoesterase activity and substrate selection of HSA. It has a 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 defects of low selectivity and sensitivity of HSA fluorescent probes in the prior art, the purpose of the present invention is to provide a class of HSA fluorescent probes and their synthesis and application, wherein the fluorescent probes have high selectivity and high sensitivity and can realize 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, which are abbreviated as DDHB, DDHC, DDHD, DDHE, DDHF, DDHG, DDHH, and DDHI, and their structures are 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 to reflux, and after the reaction is completed, 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 a 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, And the molar amount 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, the mixture is stirred in an ice water bath for 30 to 120 minutes, and then added 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, which 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 applied to 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] Fig. 9 For the fluorescent probe DDHI 1 H NMR spectrum (Figure a) and 13 C NMR spectrum (Figure b).

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

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

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

[0036] Fig.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] Fig.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 average fluorescence intensity in A549 cells and the concentration of acetylsalicylic acid (Figure b) (FL field: λex=488 nm, λem=520-650 nm; scale bar: 20 μm). DETAILED DESCRIPTION

[0038] 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.

[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 introduced 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, 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 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 Cs 2 CO 3 (1.30 g, 4 mM), dissolved in anhydrous dichloromethane (15 mL), stirred in an ice bath for 30 min, then added benzothiophene-2-carbonyl chloride (1.04 g, 4.0 mM), and continued stirring for 8 hours. 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 fluorescent probe DDHE for detecting HSA, and 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] The 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 corresponds to the chemical structure of DDHE-OH, indicating its successful synthesis.

[0049] 1 H NMR (500MHz, 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, 1 H NMR spectra and 13 The C NMR spectrum corresponds 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 corresponds 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 corresponds to the chemical structure of the fluorescent probe DDHD, indicating its successful synthesis.

[0057] 1 H NMR (500 MHz, CDCl 3 )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] 13 C NMR (126 MHz, CDCl 3)δ163.95,163.35,159.31,148.83,139.61,135.08,133.67,132.48,131.10,129.98,127.90 ,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 (500 MHz, CDCl 3 )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=14.3,8.1 Hz,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 (126 MHz, CDCl 3 )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 and13 The 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 (500 MHz, CDCl 3 )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),7.6 1–7.55(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 (126 MHz, CDCl 3 )δ164.99,164.00,163.43,158.11,149.55,133.61,132.54,130.86,130.28,127.73,126.35 ,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 corresponds to the chemical structure of the fluorescent probe DDHH, indicating its successful synthesis.

[0067] 1 H NMR (500 MHz, CDCl 3 )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, CDCl 3 )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 Fig. 9 a and Fig. 9 As shown in b, 1 H NMR spectra and 13The C NMR spectrum corresponds to the chemical structure of the fluorescent probe DDHI, indicating its successful synthesis.

[0070] 1 H NMR (500 MHz, CDCl 3 )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),3.68(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=1 2.9,3.7Hz,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 (126 MHz, CDCl 3 )δ174.38,164.01,163.48,149.42,133.54,132.52,130.82,127.70,126.27,126.1 7,124.93,124.27,122.74,67.06,56.15,53.84,43.19,37.33,28.95,25.67,25.33.

[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 (1mM), added to the EP tube, and then primary water, PBS buffer (200mM, pH 7.4) and HSA stock solution (1mg / mL) were added to the tube in sequence. After being fully mixed, it was placed in a 37°C constant temperature incubator for 70 minutes, and then the absorption spectrum and fluorescence spectrum were measured. Among them, the final concentration of PBS was 10mM, the final concentration of DDHE was 10μM, the final concentration of HSA was 200μg / mL, and the excitation wavelength λex=460nm.

[0075] like Fig.10As 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 Fig.10 As shown in b, under the excitation of 460nm, the probe has no fluorescence signal in the ≥500nm light region. After incubation with HSA, the fluorescence of the probe is significantly enhanced, forming a fluorescence emission peak at 585nm. Under the test conditions, HSA has no absorption and fluorescence signal in the ≥500nm light region.

[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] Take a series of EP tubes, add DMSO stock solution (1mM) of fluorescent probe DDHE, primary water, PBS buffer (200mM, pH 7.4) and different volumes of HSA stock solution (1mg / mL) respectively. Mix thoroughly, place in a constant temperature incubator at 37℃ for 70min, and then measure its fluorescence spectrum. Among them, the final concentration of PBS is 10mM, the final concentration of DDHE is 10μM, the final concentration of HSA is 0-900μg / mL, and λex=460nm.

[0080] like Fig.11 As shown in Figure 2, the fluorescence of DDHE is weak, and its fluorescence gradually increases with the increase of HSA concentration ( Fig.11 a). When 900 μg / mL HSA was added, the fluorescence intensity of the probe at 585 nm increased by about 340 times, indicating that it has a high sensitivity to HSA.

[0081] like Fig.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), 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) prepared from DDHE in Example 1, primary 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 test substances 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), 18γ-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), 56NH 4 + (2mM), 57Ca 2+ (2mM), 58Mg 2+ (2mM), 59Zn 2+ (2mM), 60Mn 2+ (2mM), 61Cu 2+ (2mM), 62PO 4 3- (2mM), 63S 2 O 3 2- (2mM), 64Br - (2mM), 65SO 4 2- (2mM), 66Cl - (2mM), 67I - (2mM), 68NO 3 - (2mM), 69CO 3 2- (2mM).

[0086] like Fig.12 As shown in the figure, the fluorescence of the fluorescent probe DDHE is significantly enhanced after adding HSA, but the change is relatively small after adding 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: Fig.13 shown.

[0088] Depend on Fig.13 a As can be seen, the fluorescence of the fluorescent probe DDHB 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.

[0089] Depend on Fig.13 b It can be seen that the fluorescence of the fluorescent probe DDHC 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.

[0090] Depend on Fig.13 c 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 Fig.13 d It can be seen that the fluorescence of the fluorescent probe DDHF 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.

[0092] Depend on Fig.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 Fig.13 f It can be seen that the fluorescence of the fluorescent probe DDHH 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.

[0094] Depend on Fig.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 show that the eight fluorescent probes described in the present invention 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] Add the DMSO stock solution (1 mM) prepared by DDHE in Example 1, primary water, PBS buffer (200 mM, pH 7.4) and serum samples pre-added with different concentrations of HSA into the EP tube, mix thoroughly, incubate at 37°C for 70 min, and then measure the fluorescence spectrum. The final concentration of DDHE is 10 μM, the final concentration of PBS is 1 mM, the final volume fraction of serum is 2%, the final concentration of HSA is 0, 10, 20, 40 μg / mL, respectively, and λex=460 nm.

[0098] As shown in Table 1, using the fluorescence analysis method based on the fluorescent probe DDHE, the measured value of HSA concentration in the serum sample was 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 by fluorescent probe DDHE

[0100]

[0101]

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

[0103] Add the DMSO stock solution (1 mM) prepared by DDHE in Example 1, primary water, PBS buffer (200 mM, pH 7.4), and urine samples pre-added with different concentrations of HSA into the EP tube, mix thoroughly, incubate at 37°C for 70 min, and measure the fluorescence spectrum. The total volume of the solution is 1 mL, the final concentration of DDHE is 10 μM, the final concentration of PBS is 10 mM, the concentration of HSA is 0 μg / mL, 35 μg / mL, 70 μg / mL, 100 μg / mL, the final volume fraction of urine is 10%, and λex=460 nm.

[0104] As shown in Table 2, using the fluorescence analysis method based on the fluorescent probe DDHE, the measured value of HSA concentration in the urine sample was 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 The cells were inoculated into a confocal dish at a density of 10 cells / dish and cultured for 24 hours to allow them to adhere to the wall. Then, 1 mL of cell culture medium was aspirated, 1 mL of cell culture medium containing acetylsalicylic acid was added, and cultured at 37°C for 2 hours. Then, 1 mL of cell culture medium was aspirated, 1 mL of cell culture medium containing DDHE was added, and after culturing for 20 minutes, the cells were washed twice with PBS, and then the cell fluorescence imaging was observed using a confocal fluorescence microscope. The final concentrations of acetylsalicylic acid were 0mM, 0.5mM, 1mM, and 2mM, and the final concentration of DDHE was 10μM.

[0109] like Fig.14 As shown, when A549 cells were co-incubated with acetylsalicylic acid, the fluorescence of DDHE in the cells was weakened ( Fig.14 a), and the decrease in fluorescence intensity is positively correlated with the concentration of acetylsalicylic acid ( Fig.14 b) This phenomenon indicates that acetylsalicylic acid can inhibit the pseudoesterase activity of HSA. It reveals that the fluorescence signal of DDHE in cells is activated by HSA. Therefore, the fluorescence intensity of DDHE in cells 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 comprises a total of 8 molecules, which are referred to 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, characterized in that: The following steps are involved: Step 1: Add 3-hydroxy-1,8-naphthalene anhydride and 4-(2-aminoethyl)morpholine to an organic solvent, heat to reflux, and after the reaction is completed, 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 a 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 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 to 8 hours.

6. The preparation method according to claim 2, characterized in that: In step 2, the intermediate product DDHE-OH, And the molar amount 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, add Continue stirring for 1-8 hours to terminate the reaction.

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

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