Environment-sensitive fluorescent probe for detecting human serum albumin as well as preparation and application of environment-sensitive fluorescent probe

Abstract

The invention discloses an environment-sensitive fluorescent probe for detecting human serum albumin (HSA) as well as a preparation method and an application of the environment-sensitive fluorescent probe. According to the technical scheme adopted by the invention, a fluorescent probe formula 1 is designed and synthesized based on a dinitrile isophorone electron withdrawing group, the probe molecule is extremely sensitive to a microenvironment, and the fluorescence spectrum is obviously changed along with the change of the polarity and the viscosity of a solvent. In a mixed system of Tris-HCl and water, a probe and HSA are added, fluorescence is enhanced, and the concentration of HSA can be detected by using the change of fluorescence intensity. The fluorescent probe disclosed by the invention is simple to synthesize, large in Stokes shift, long in emission wavelength, sensitive to microenvironment, good in HSA detection selectivity, high in sensitivity and wide in linear range.

Description

Technical Field

[0001] The invention relates to a microenvironment-sensitive fluorescent probe and a preparation method thereof and application to human serum albumin detection, belonging to the technical field of fluorescent probes. Background Art

[0002] Human serum albumin (HSA) is the most abundant and important protein in the human circulatory system. It participates in the catalytic activity of enzymes, maintains the osmotic pressure of plasma, regulates immune function, and transports drug molecules. Abnormal HSA content can serve as an early warning of renal impairment and related kidney diseases. The plasma concentration of healthy adults is usually 35-55g / L. Low plasma HSA concentration, i.e. hypoalbuminemia, is closely related to the symptoms of many diseases such as malnutrition, rheumatoid arthritis, liver failure, cirrhosis, chronic hepatitis and sepsis. In addition, preoperative hypoalbuminemia can also be used to predict postoperative mortality and morbidity. In contrast to the albumin concentration in plasma, the normal albumin concentration in urine is less than 30 mg / L. This is because the kidneys can reduce the penetration of various proteins, including albumin, into the urine. However, under the influence of diseases such as chronic kidney disease or acute nephritis, the normal filtration capacity of the kidneys is impaired, resulting in a large amount of unfiltered protein penetrating into the urine, thus causing proteinuria. In clinical practice, proteinuria has been identified as an early and sensitive marker for the diagnosis, monitoring, treatment planning, and response assessment of a variety of renal diseases.

[0003] Among the many HSA detection methods, fluorescent probes have attracted widespread attention due to their fast response speed, high sensitivity and simple operation. At present, many studies on HSA molecular fluorescent probes mainly divide them into three types: environmentally sensitive fluorescent probes, aggregation-decomposition fluorescent probes and enzyme reaction fluorescent probes. However, most of the reported fluorescent probes for detecting HSA in aqueous phases have problems such as complex preparation, short emission wavelength, large background interference and poor specificity. Therefore, it is particularly important to design a simple, efficient and accurate fluorescent probe for detecting HSA. Summary of the invention

[0004] In view of the problems of existing HSA detection probes such as short emission wavelength, large background interference, and poor specificity, the present invention aims to provide a microenvironment-sensitive fluorescent probe for detecting HSA. The fluorescent probe is a new type of organic small molecule fluorescent probe with the characteristics of long emission wavelength, high specificity and high sensitivity.

[0005] Another object of the present invention is to provide a method for preparing a microenvironment-sensitive fluorescent probe for detecting HSA, which has simple synthesis steps, high yield and low cost.

[0006] Another object of the present invention is to provide a microenvironment-sensitive fluorescent probe for detecting HSA, which can be used to prepare a reagent for detecting the HSA content in the cell tissue environment and urine.

[0007] The present invention is achieved by the following technical solution: a microenvironment-sensitive fluorescent probe, whose structural formula is shown in Formula 1.

[0008] .

[0009] The method for preparing the microenvironment-sensitive fluorescent probe comprises the following steps.

[0010] Step 1: isophorone, malononitrile, sodium acetate and anhydrous ethanol are mixed, and the mixture is heated to react. After the reaction is completed, it is recrystallized with cold ethanol and filtered to obtain a solid product QN-1.

[0011] Step 2: dissolve p-acetaminobenzaldehyde and QN-1 in anhydrous acetonitrile, add piperidine to the mixture under the protection of inert gas, heat the mixture to react, and after the reaction is completed, filter to obtain a solid product QN-2.

[0012] Step 3: QN-2 is mixed with concentrated hydrochloric acid and anhydrous ethanol, and the mixture is heated to react. After the reaction is completed, the mixture is neutralized with sodium hydroxide solution, and then the mixture is extracted, water is removed, and the solvent is removed under reduced pressure to obtain a crude product; the crude product is subjected to column chromatography to obtain a solid product QN-3.

[0013] Step 4: Under low temperature, QN-3 is dissolved with concentrated hydrochloric acid, and sodium nitrite is added. After a period of reaction, stannous chloride solution is added to the mixture, and the reaction is continued with stirring. After the reaction is completed, the mixture is neutralized with sodium hydroxide solution, and then the mixture is extracted, water is removed, and the solvent is removed under reduced pressure to obtain a crude product; the crude product is subjected to column chromatography to obtain a solid product QN-4.

[0014] Step 5: QN-4, salicylaldehyde and anhydrous ethanol are mixed, and the mixture is heated to react. After the reaction is completed, a crude product is obtained; the crude product is subjected to column chromatography to obtain a fluorescent probe formula 1.

[0015] The synthetic steps included in this route are as follows.

[0016] .

[0017] The application of the microenvironment-sensitive HSA fluorescent probe formula 1 is applied to the detection of HSA in a mixed system of Tris-HCl and water and in normal human urine.

[0018] In a mixture of Tris-HCl and water, the fluorescent probe formula 1 reacts with HSA, and the concentration of HSA is detected by the change in fluorescence intensity: before the addition of HSA, the fluorescence signal of the probe is weak, and after the addition of HSA, the fluorescence signal of the probe is enhanced.

[0019] Detection of HSA in actual healthy human urine: By adding HSA to healthy human urine and fluorescent probe formula 1, the concentration of HSA is detected by the change in fluorescence intensity: before the addition of HSA, the fluorescence signal is weak because healthy human urine contains a small amount of HSA. After adding HSA, the fluorescence signal is significantly enhanced.

[0020] The fluorescence characteristics of the microenvironment-sensitive fluorescent probe formula 1 prepared by the present invention are that in a mixed system of glycerol and water, as the proportion of glycerol increases, the fluorescence emission wavelength red-shifts and the fluorescence intensity increases; in a mixed system of 1,4-dioxane and water, as the proportion of 1,4-dioxane increases, the fluorescence emission wavelength blue-shifts and the fluorescence intensity increases.

[0021] The fluorescent probe formula 1 prepared by the present invention has a dinitrile isophorone electron-withdrawing group, has obvious intramolecular charge transfer characteristics, has a long emission wavelength, and has a large Stokes shift. The recognition mechanism of probe formula 1 for HSA includes two aspects. First, the probe molecule has a D-π-A structure that is highly sensitive to the environment, which is conducive to the intramolecular charge transfer (ICT) process. In a highly polar solvent, the free rotation of the double bond is conducive to the formation of a twisted intramolecular charge transfer (TICT), which leads to fluorescence quenching. After binding to HSA, the molecular rotation of probe formula 1 is restricted and TICT is inhibited, resulting in a sharp increase in fluorescence. Second, the probe molecule can enter the low-polarity hydrophobic pocket of HSA, where hydrogen bonds, hydrophobic interactions, etc. exist, and the microenvironment in which probe formula 1 is located changes. Probe formula 1 will show weak fluorescence in an aqueous medium (high polarity environment), and once it enters the binding site in the HSA cavity (low polarity microenvironment), it will show significant fluorescence enhancement, accompanied by a wavelength shift.

[0022] The beneficial effects of the present invention are: The present invention provides a microenvironment-sensitive fluorescent probe, a preparation method thereof, and an application to HSA detection. The fluorescent probe has a simple preparation method, a large probe Stokes shift, a long emission wavelength, high sensitivity, and a strong anti-interference ability. The fluorescence emission wavelength and intensity change with changes in solvent polarity and viscosity. At the same time, HSA in urine can be detected, and the probe has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the reaction mechanism of probe formula 1.

[0024] Figure 2This is the high-resolution mass spectrum of probe type 1.

[0025] Figure 3 The fluorescence spectrum of probe formula 1 varies with viscosity (a) and polarity (b).

[0026] Figure 4 This is a probe type 1 stability test chart.

[0027] Figure 5 The graphs are the fluorescence spectrum of probe type 1 changing with HSA concentration (a) and the linear relationship between fluorescence intensity and HSA concentration (b).

[0028] Figure 6 This is a graph showing the change in fluorescence intensity of the reaction between probe formula 1 and HSA over time.

[0029] Figure 7 This is a schematic diagram of the fluorescence intensity changes of probe type 1 before and after the reaction with different interfering analytes. The concentration of probe type 1 is 10 μM, and the metal ions (Na + , K + Mg 2+ , Mn 2+ , Cu 2+ , Fe 2+ , Fe 3+ 、Zn 2+ , Hg 2+ 、Al 3+ , Pb 2+ 、Ni 2+ )、Anions(SO3 2- 、SO4 2- , ClO - , Cl - 、NO3 - 、CH3COO - ), amino acids (GSH, Cys, Ser, Gly) concentrations were all 1 mM.

[0030] Figure 8 This is a schematic diagram of the fluorescence spectrum of probe type 1 in the urine of healthy people as the HSA concentration changes. Implementation

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. The described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1: Synthesis of probe molecule 1.

[0033] Isophorone (2210 μL, 14.48 mmol), malononitrile (0.9566 g, 14.48 mmol) and sodium acetate (0.8004 g, 10 mmol) were placed in a 50 mL round-bottom flask and 18 mL of anhydrous ethanol was added. The mixture was stirred and refluxed at 78 °C for 12 h. After cooling to room temperature, the precipitate was removed by filtration. The liquid was concentrated under reduced pressure to remove half of the solvent. The mixture was placed at 4 °C for 0.5 h, filtered, and then recrystallized from ethanol to obtain compound QN-1, which was a yellow powdery solid.

[0034]

[0035] At room temperature and N2 atmosphere, QN-1 (0.503 g, 2.7 mmol) and p-acetaminobenzaldehyde (0.449 g, 2.7 mmol) were added with 0.1 mL of piperidine in a 50 mL round-bottom flask and 10 mL of anhydrous acetonitrile was added. The mixture was heated at 82 o C, stirred and refluxed for 1 h. After cooling to room temperature, the precipitate was filtered and washed with cold acetonitrile to obtain the product QN-2, which was a red solid.

[0036]

[0037] QN-2 (0.25 g, 0.75 mmol) and concentrated hydrochloric acid (10 mL) were placed in a 50 mL round-bottom flask and 20 mL of ethanol was added. The mixture was refluxed and stirred at 78 ºC for 5.5 h. After cooling to room temperature, the mixture was neutralized with sodium hydroxide solution, and then the aqueous solution was extracted with ethyl acetate and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure to obtain a crude product, which was then separated and purified by column chromatography (eluent ratio: dichloromethane: petroleum ether = 9:1). After removing the solvent, a red solid QN-3 was obtained.

[0038]

[0039] QN-3 (0.1 g, 0.346 mmol) and an aqueous solution of sodium nitrate (0.2630 g, 0.93 mmol) were placed in a 50 mL round-bottom flask and 10 mL of concentrated hydrochloric acid solution was added. oAfter stirring at 40 °C for 1.5 h, a hydrochloric acid solution (5 mL) of stannous chloride (0.0642 g, 1.387 mmol) was slowly added dropwise to the mixture, and stirring was continued for 1.5 h. After the reaction was completed, the mixture was neutralized with sodium hydroxide solution, and then the aqueous solution was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product, which was then separated and purified by column chromatography (eluent ratio: dichloromethane: methanol = 100:1). After removing the solvent, a red-black solid QN-4 was obtained.

[0040]

[0041] QN-4 (0.06 g, 0.20 mmol) and salicylaldehyde (0.037 g, 0.30 mmol) were placed in a 50 mL round-bottom flask, and 12 mL of anhydrous ethanol was added. o C, stirred and refluxed for 5 h, cooled to room temperature, and then the solvent was removed under reduced pressure to obtain a crude product, which was then separated and purified by column chromatography (eluent ratio: dichloromethane: methanol = 40:1). After removing the solvent, a red-black solid formula 1 was obtained. The high-resolution mass spectrum is shown in FIG. Figure 2 shown.

[0042]

[0043] Example 2: Study on the sensitivity of probe molecule 1 to microenvironment

[0044] The probe molecule 1 was prepared into a 1 mM stock solution with dimethyl sulfoxide, and HSA was prepared into 100 μM and 10 μM stock solutions with Tris-HCl.

[0045] In order to test the sensitivity of the probe molecule to the microenvironment, the spectral properties of the probe in systems with different viscosities and polarities were studied. Figure 3 As can be seen in a, in a mixed system of glycerol and water, with the increase of viscosity (increase of glycerol ratio), the maximum emission wavelength of fluorescence slightly red-shifts and the fluorescence intensity gradually increases; Figure 3 As can be seen in b, in the mixed system of 1,4-dioxane and water, as the polarity decreases (the proportion of 1,4-dioxane increases), the maximum emission wavelength of fluorescence changes from 725 nm to 680 nm, a blue shift occurs, and the fluorescence intensity gradually increases. Figure 4 It can be seen that within 60 min, the fluorescence intensity of the probe molecule remained basically unchanged, indicating that the probe has good photostability.

[0046] Example 3: Study on the change of fluorescence intensity of probe formula 1 under different HSA concentration conditions

[0047] In order to test the response of the probe molecule to different concentrations of HSA, the changes in the fluorescence intensity of the probe molecule under different concentrations of HSA (0, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, 10.0 μM) were studied. Figure 5 As can be seen from a and b, as the concentration of HSA increases, the fluorescence intensity also increases. And through linear fitting, there is a good linear relationship between the increase in the fluorescence intensity of the probe molecule and the concentration of HSA (R 2 = 0.9928), the linear range is 0.5-10 μM, and the linear regression equation is Y=10.183X+11.688, where X is the concentration of HSA and Y is the fluorescence intensity of the probe molecule. The detection limit is calculated to be 0.106 μM by LOD = 3r / K, indicating that the fluorescent probe molecule of the present invention shows high sensitivity to different concentrations of HSA.

[0048] Example 4: Study on the time response of probe formula 1 to HSA

[0049] In order to test the time response of the probe molecule to HSA, the change of the fluorescence intensity of the probe molecule reacting with HSA over time (0-70 min) was studied. Figure 6 As can be seen from a and 6b, the reaction rate between the probe molecule and HSA is relatively fast in the first 10 min, and the reaction is basically completed in about 30 min.

[0050] Example 5: Study on the Selectivity of Probe Formula 1 to HSA

[0051] To test the selectivity of the probe molecule for HSA, the probe molecule was subjected to the reaction with different probe metal ions (Na + , K + Mg 2+ , Mn 2+ , Cu 2+ , Fe 2+ , Fe 3+ 、Zn 2+ , Hg 2+ 、Al 3+ , Pb 2+ 、Ni 2+ )、Anions(SO3 2- 、SO4 2- , ClO - , Cl - 、NO3 - 、CH3COO - The changes in fluorescence intensity before and after the reaction of ), amino acids (GSH, Cys, Ser, Gly) were experimentally studied. The concentration of HSA was 10 μM, and the concentrations of the other interfering substances were 1 mM. Figure 7As can be seen from 7a and 7b, the detection of HSA by the probe molecule is not interfered by other analytes and is highly selective.

[0052] Example 6: Detection of HSA in actual healthy urine using probe molecule formula 1

[0053] In order to test the detection of HSA by the probe molecule in the urine of actual healthy people, the changes in the fluorescence intensity of the probe molecule under different HSA concentrations (0, 1, 3, 5, 8, 10.0 μM) were studied. Figure 8 It can be seen from Figures 8a and 8b that as the concentration of HSA increases, the fluorescence intensity also increases. And through linear fitting, there is a good linear relationship between the increase in the fluorescence intensity of the probe molecule and the concentration of HSA (R 2 = 0.9908), the linear range is 1-10 μM, and the linear regression equation is Y=10.411X+68.353, where X is the concentration of HSA and Y is the fluorescence intensity of the probe molecule. This shows that the probe molecule can be applied to the detection of actual urine samples.

Claims

1. A microenvironment-sensitive fluorescent probe, It is characterized in that The fluorescent probe structural formula is shown in Formula 1: 。 2. The method for preparing the microenvironment-sensitive fluorescent probe according to claim 1, It is characterized in that The synthesis steps are as follows: (1) isophorone, malononitrile, sodium acetate and anhydrous ethanol are mixed, the mixture is heated to react, and after the reaction is completed, it is recrystallized with cold ethanol and filtered to obtain a solid product QN-1; ； (2) dissolving p-acetaminobenzaldehyde and QN-1 in anhydrous acetonitrile, adding piperidine to the mixture under the protection of an inert gas, heating the mixture to react, and filtering to obtain a solid product QN-2 after the reaction is completed; ； (3) QN-2 is mixed with concentrated hydrochloric acid and anhydrous ethanol, and the mixture is heated to react. After the reaction is completed, the mixture is neutralized with sodium hydroxide solution, and then the mixture is extracted, water is removed, and the solvent is removed under reduced pressure to obtain a crude product; the crude product is subjected to column chromatography to obtain a solid product QN-3; ； (4) Dissolve QN-3 with concentrated hydrochloric acid at low temperature, and add sodium nitrite. After a period of reaction, add stannous chloride solution to the mixture, continue stirring and reacting. After the reaction is completed, neutralize the mixture with sodium hydroxide solution, and then extract the mixture, remove water, and remove the solvent under reduced pressure to obtain a crude product; the crude product is subjected to column chromatography to obtain a solid product QN-4; ； (5) QN-4, salicylaldehyde and anhydrous ethanol are mixed, and the mixture is heated to react. After the reaction is completed, a crude product is obtained; the crude product is subjected to column chromatography to obtain a fluorescent probe formula 1; 。 3. The preparation method according to claim 2, Features: In step (1), the molar ratio of isophorone, malononitrile and sodium acetate is (5.1-7.2): (5.4-7.1): (3.5-5.5); the heating reaction temperature is 60-80 °C, and the reaction time is 9-15 h; In step (2), the molar ratio of the product QN-1, 4-acetaminobenzaldehyde, and piperidine is (2.4-3.1): (2.2-3.5): (0.8-1.2); The heating reaction temperature is 70-90 ° C, and the reaction time is 0.5-2.0 h; In step (3), the molar ratio of the product QN-2 to concentrated hydrochloric acid is (24-26.5): (3.2-5.5); The heating temperature is 60-80 ° C, and the reaction time is 3.0-7.0 h; The eluent is dichloromethane:petroleum ether, with a volume ratio of (5:1)-(12:1); In step (4), the molar ratio of the product QN-3, sodium nitrite and stannous chloride is (0.5-1.4): (2.0-3.0): (3.2-4.4); The cryogenic temperature is -3-5 °C, and the reaction time is 2.0-6.0 h; The eluent is dichloromethane:methanol, with a volume ratio of (75:1)-(110:1); In step (5), the molar ratio of the product QN-4 to salicylaldehyde is (0.7-2.4):(1.8-3.8); The heating temperature is 60-80 ° C, and the reaction time is 3.5-7.0 h; The eluent was dichloromethane:methanol in a volume ratio of (30:1)-(60:1).

4. Use of the microenvironment-sensitive fluorescent probe as claimed in claim 1, It is characterized by: The fluorescent probe is applied to the detection of HSA in a mixed system of glycerol and water, a mixed system of 1,4-dioxane and water, a mixed system of Tris-HCl and water, and actual healthy urine.

5. Use of the microenvironment-sensitive fluorescent probe according to claim 4, It is characterized by: In the mixed system of glycerol and water: as the proportion of glycerol increases, the fluorescence emission wavelength red-shifts and the fluorescence intensity increases; in the mixed system of 1,4-dioxane and water, as the proportion of 1,4-dioxane increases, the fluorescence emission wavelength blue-shifts and the fluorescence intensity increases.

6. Use of the microenvironment-sensitive fluorescent probe according to claim 4, It is characterized by: In a mixed system of Tris-HCl and water, the fluorescent probe reacts with HSA, and the concentration of HSA is detected by the change in fluorescence intensity: before the addition of HSA, the fluorescence signal of the probe is weak, and after the addition of HSA, the fluorescence signal of the probe is enhanced.

7. Use of the microenvironment-sensitive fluorescent probe according to claim 4, It is characterized by: By adding HSA to healthy urine and fluorescent probes, the concentration of HSA is detected by using changes in fluorescence intensity: before adding HSA, the fluorescence signal is weak because healthy urine contains a small amount of HSA. After adding HSA, the fluorescence signal is significantly enhanced.

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