Fluorescent probe containing pyrene group, preparation method and application

Abstract

The invention discloses a fluorescent probe containing a pyrene group as well as a preparation method and application of the fluorescent probe. The fluorescent probe disclosed by the invention is a ratio type fluorescent molecule, and is combined with protamine through electrostatic interaction to induce emission of excimer. Protamine can be hydrolyzed by adding trypsin, and fluorescence recovery monomer emission is realized, so that trypsin detection is realized. In addition, the probe and gamma-cyclodextrin form a host-guest compound system, and excimer fluorescence emission is shown. Based on the hydrolysis effect of alpha-amylase on cyclodextrin, fluorescence recovers monomer emission, and quantitative detection of alpha-amylase is realized. The invention provides a multifunctional fluorescence detection probe which has good selectivity and sensitivity and can be applied to a serum dilution environment.

Description

Technical Field

[0001] The invention belongs to the technical field of functional materials, relates to fluorescent probe materials, and specifically relates to a fluorescent probe containing a pyrene group, a preparation method and an application thereof. Background Art

[0002] With the improvement of people's living standards and the popularization of medical knowledge, people pay more and more attention to health issues. In 2024, the average life expectancy in my country has reached 78.8 years, and it is increasing year by year. This is largely due to the development of medical standards, which allows people to achieve early detection of diseases and more accurate treatment. Biomacromolecules play an important role in physiological processes and disease detection and treatment. Protamine has high nutritional and functional properties. It can be used as a heparin detoxifier, and can also be used to lower blood pressure and promote digestion; trypsin is an important digestive enzyme, an enzyme drug, and one of the detection indicators for pancreatic diseases; α-amylase plays an important role in the digestion of carbohydrates and is related to diseases such as acute pancreatitis. Too high or too low levels of these biomacromolecules will have some side effects on the human body, so appropriate methods need to be used for quantitative detection.

[0003] Fluorescence sensing methods have the advantages of simplicity, high sensitivity, and rapid response, and have made great progress over the past few decades. The fluorescent probe binds specifically to the analyte, and the interaction between them leads to a significant change in the fluorescence properties, thereby realizing the detection of the analyte. Fluorescence probe methods have been widely used in the detection of various substances. In the field of biomolecular sensing, fluorescence probe methods have been successfully used for the detection of many biological small molecules and macromolecules, such as ATP, amino acids, metal ions, protamine, serum albumin, biogenic amines, heparin, etc. To this day, the design and development of multifunctional fluorescent probe molecules with excellent performance is still a very attractive and challenging topic. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a fluorescent probe containing a pyrene group, a preparation method and an application. The chemical structure of the pyrene-containing fluorescent probe provided by the present invention is shown in Formula I. The probe binds to protamine through electrostatic interaction to achieve quantitative detection of protamine. The host-guest complex formed by the probe and γ-cyclodextrin can achieve the detection of α-amylase. Using the probe, high-selectivity and high-sensitivity detection of protamine, trypsin and α-amylase can be achieved.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a fluorescent probe containing a pyrene group, and its chemical structure is shown in Formula I:

[0007]

[0008] The present invention also provides a method for preparing the fluorescent probe containing a pyrene group, comprising the following steps:

[0009] Step 1: Dissolve compound 1 and triethylamine in dichloromethane, stir at room temperature, then add DMAP, EDCI, succinic acid or adipic acid or suberic acid or sebacic acid, stir to react. Add water for extraction, take the organic phase, rotary evaporate, remove the solvent, and obtain white solid compound 2.

[0010] Step 2: Dissolve compound 2, DMAP and EDCI in dichloromethane, stir at room temperature, then add 1-aminopyrene and triethylamine, stir and react at low temperature and room temperature in turn. Add water for extraction, take the organic phase, rotary evaporate and remove the solvent to obtain compound 3.

[0011] Step 3: dissolving compound 3 and sodium carbonate in a water-ethanol mixed solvent and subjecting the mixture to reflux reaction. After removing the solvent, the fluorescent probe is obtained.

[0012] The chemical structural formula of the compound 1 is shown in Formula II:

[0013]

[0014] The chemical structural formula of the compound 2 is shown in Formula III:

[0015]

[0016] The chemical structural formula of the compound 3 is shown in Formula IV:

[0017]

[0018] The molar ratio of compound 1 and succinic acid or adipic acid or suberic acid or sebacic acid in step 1 is 1:2-1:3; the molar ratio of compound 1 and EDCI is 1:1.05-1:1.15; the ratio of compound 1 and DMAP is 1:0.2-1:0.4; the ratio of compound 1 and triethylamine is 1:1.1-1:1.3. Step 1 is the reaction of ethyl glutamate hydrochloride and diacid, the purpose of which is to obtain a single-end amidated diacid derivative, while retaining a carboxyl group as a functional group to participate in the next amidation reaction.

[0019] The stirring method in step 1 is stirring at room temperature, wherein the first stirring time at room temperature is 0.5 to 2 hours, and the second stirring time is 5 to 8 hours; the number of water extractions is 2 to 4 times.

[0020] The molar ratio of compound 2 and 1-aminopyrene in step 2 is 1:1.05-1:1.15; the molar ratio of compound 2 and EDCI is 1:1.3-1:1.6; the ratio of compound 2 and DMAP is 1:0.2-1:0.4; the ratio of compound 2 and triethylamine is 1:1.1-1:1.3. Step 2 is to carry out an amidation reaction between 1-aminopyrene and the carboxyl group retained in compound 2, so as to obtain a diacid derivative whose two ends are respectively subjected to an amidation reaction with glutamic acid and 1-aminopyrene.

[0021] The stirring method in step 2 is first stirring at room temperature, then stirring at low temperature, and finally stirring at room temperature again, wherein the first stirring time at room temperature is 0.5 to 2 hours, the low temperature stirring temperature is -8 to -12°C, the stirring time is 0.5 to 2 hours, and the third stirring time is 20 to 30 hours; the number of water extractions is 4 to 6 times.

[0022] The volume ratio of the water-ethanol mixed solvent in step 3 is 1:0.8-1:1.2; the molar ratio of compound 3 to sodium carbonate is 1:1-1:1.1. Step 3 is a deprotection reaction of the carboxyl group, that is, a hydrolysis reaction of the ester to obtain a fluorescent probe in the form of a sodium salt.

[0023] The stirring method in step 3 is reflux stirring, wherein the first stirring time is 8 to 12 hours, and the second stirring time is 1 to 3 hours.

[0024] The invention also provides the application of the fluorescent probe containing the pyrene group in the process of detecting protamine and trypsin.

[0025] The present invention also provides the application of the host-guest composite system of the fluorescent probe containing the pyrene group and gamma-cyclodextrin in detecting alpha-amylase.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides a fluorescent probe molecule containing a pyrene group, which is synthesized from glutamic acid, 1-aminopyrene, succinic acid or adipic acid or suberic acid or sebacic acid as raw materials. The probe is combined with protamine through electrostatic binding, and the emission peak of excimer appears, thereby realizing the quantitative detection of protamine. Furthermore, trypsin has the effect of hydrolyzing protamine, causing the fluorescence emission to return to monomer emission, thereby realizing the quantitative detection of trypsin. In addition, the probe forms a host-guest complex with γ-cyclodextrin, showing excimer emission. α-amylase hydrolyzes γ-cyclodextrin, and the fluorescence returns to monomer emission, thereby realizing the quantitative detection of α-amylase.

[0028] The probe exhibits multiple detection functions, has good selectivity and sensitivity, and has the potential to be applied in complex media. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1a is a schematic diagram of the chemical structure of the fluorescent probe molecule containing a pyrene group in Example 1 of the present invention;

[0030] Figure 1b is the H NMR spectrum (600 MHz, D2O) of the fluorescent probe molecule in Example 1 of the present invention;

[0031] Figure 2 is the fluorescence spectrum of the fluorescent probe molecule in Example 1 of the present invention;

[0032] Figure 3 The fluorescence change of the fluorescent probe solution before and after the addition of protamine in Example 2 of the present invention;

[0033] Figure 4 is the ratio of the emission intensity of the fluorescent probe excimer to the peak emission intensity of the monomer in Example 2 of the present invention (I 489 / I 385 ) and the linear relationship between the concentration of protamine;

[0034] Figure 5 The effect of the fluorescent probe in Example 2 of the present invention in detecting protamine at different pH values;

[0035] Figure 6 is the selectivity of the fluorescent probe to protamine in Example 3 of the present invention;

[0036] Figure 7 is the linear response interval of the fluorescent probe in Example 4 of the present invention to protamine in a diluted fetal bovine serum (FBS) solution;

[0037] Figure 8 The fluorescence of the complex system of the fluorescent probe and protamine in Example 5 of the present invention changes with the addition of trypsin;

[0038] Fig. 9 is a linear fitting graph of the fluorescence intensity ratio of the fluorescent probe and protamine complex system and the trypsin concentration in Example 5 of the present invention;

[0039] Fig.10 The selectivity of the fluorescent probe and protamine complex in detecting trypsin in Example 5 of the present invention;

[0040] Fig.11 The fluorescence spectra and photos of the fluorescent probe in Example 6 of the present invention after adding α-, β-, and γ-cyclodextrin respectively;

[0041] Fig.12 The change of fluorescence over time after adding α-amylase to the composite system of the fluorescent probe and γ-cyclodextrin in Example 7 of the present invention;

[0042] Fig.13 The fluorescence changes over time after adding different concentrations of α-amylase to the composite system of the fluorescent probe and γ-cyclodextrin in Example 7 of the present invention;

[0043] Fig.14 The selectivity of the composite system of the fluorescent probe and γ-cyclodextrin in Example 8 of the present invention for α-amylase in the presence of an interfering substance;

[0044] Fig.15 The fluorescence changes over time after adding different concentrations of α-amylase into the composite system of the fluorescent probe and γ-cyclodextrin in Example 9 of the present invention. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the present invention clearer, the following examples will further illustrate the specific steps and features of the invention, which are only used for illustration and are not intended to limit the present invention. The methods used in the present invention are conventional methods in the art unless otherwise specified. The reagents and materials involved in the present invention are commercially available unless otherwise specified.

[0046] Example 1 Synthesis of fluorescent probe (n=1)

[0047]

[0048] See also Figure 1a , which is a schematic diagram of the chemical structure of the fluorescent probe containing a pyrene group in an embodiment of the present invention. The preparation process includes the following steps:

[0049] Compound 1 (1.2 g, 5 mmol) and triethylamine (0.6 g, 6 mmol) were dissolved in dichloromethane (50 mL). After stirring at room temperature for 30 min, EDCI (5.5 mmol, 1.05 g), DMAP (0.15 mmol, 0.018 g), and succinic acid (10 mmol, 1.18 g) were added and stirred at room temperature for 6 h. After the reaction was completed, the reaction solution was washed with acidic water (pH 1-2) (2×50 mL) and water (2×50 mL) in sequence. The organic phase was collected and the solvent was removed to obtain compound 2. The yield was 78%. Characterization by mass spectrometry. ESI-MS: m / z 301.7[(M-H) － ].

[0050] Compound 2 (5mmol, 1.5g), EDCI (7.5mmol, 1.4g), DMAP (0.15mmol, 0.018g) were dissolved in dichloromethane (50mL). After stirring at room temperature for 30min, 1-aminopyrene (5.5mmol, 1.2g) and triethylamine (6mmol, 0.6g) were added. Stirred for 30min under ice bath conditions, and stirred for 24h after returning to room temperature. The solution was washed with acidic water (pH 1-2) (2×50mL), alkaline water (pH 8-9) (2×50mL) and water (2×50mL) in sequence. The organic phase was collected and the solvent was removed to obtain compound 3. The yield was 50%.

[0051] Compound 3 (0.5 g, 1 mmol) was dissolved in ethanol (20 mL), and an aqueous solution (20 mL) of sodium carbonate (0.12 g, 1.1 mmol) was added, and the mixture was refluxed overnight. The solvent was removed to obtain a fluorescent probe molecule in the form of a sodium salt (n = 1). The fluorescence probe molecule was characterized by mass spectrometry. ESI-MS: m / z 221.9 [(M-2Na) 2－ ]. The sodium salt product was characterized by H NMR spectroscopy, such as Figure 1b shown.

[0052] Fluorescence spectra of aqueous solutions with different concentrations are shown in Figure 2. Figure 2 As shown in Figure 2, the maximum emission wavelengths of the fluorescent probe molecule aqueous solution are at 385nm and 405nm (the excitation wavelength is 340nm).

[0053] Example 2 Fluorescence response of fluorescent probe (n=1) to protamine

[0054] Prepare 5 mL of HEPES buffer solution (10 mmol / L, pH 7.4) of the probe, with a probe concentration of 10 μmol / L, and record the changes in the probe's fluorescence spectrum after adding different concentrations of protamine (excitation wavelength is 340 nm). With the addition of protamine (0-14 μg / mL), an excimer emission peak appears at 489 nm, and the sample's luminescent color changes from blue to blue-green under a 360 nm ultraviolet lamp. Figure 3 The ratio of the emission peak intensity at 489 nm to the emission peak intensity at 385 nm (I 489 / I 385 ) showed a linear relationship with the concentration of protamine in the range of 1-14 μg / mL, such as Figure 4 As shown in Figure 2, the detection limit of the fluorescent probe molecule for protamine in HEPES buffer solution is calculated to be 13.8 ng / mL.

[0055] To further determine the applicable pH range of the fluorescent probe, pure water was used to prepare the solution and concentrated hydrochloric acid or sodium hydroxide was used to adjust the pH value. Figure 5As shown, the probe achieves fluorescence response to protamine in the pH range of 4-10.

[0056] Example 3 Selectivity of fluorescent probe (n=1) for protamine

[0057] By comparing the probes under the same conditions for various proteins with different pI values, including hemoglobin, bovine serum albumin, pepsin, ovalbumin, lysozyme (14 μg / mL), two amino acids (lysine and arginine) (14 μg / mL), glucose, and two common ions (Zn 2+ and Ca 2+ The selectivity of the probe for protamine determination was investigated by measuring the fluorescence response of )(140 μmol / L). Figure 6 As shown, except for protamine, the other analytes mentioned above have little effect on the fluorescence emission of the probe, indicating that the probe has good selectivity for protamine.

[0058] Example 4 Fluorescence detection of protamine by fluorescent probe (n=1) in serum solution

[0059] The detection ability of the probe for protamine in 0.1% fetal bovine serum (FBS) solution was further investigated. 489 / I 385 ) was linearly correlated with protamine concentration in the range of 7-16 μg / mL (y=0.00369x-0.01109, R 2 =0.9972), such as Figure 7 The detection limit was calculated to be 0.64 μg / mL. Different amounts of protamine (8, 11, 12, 13 μg / mL) were added to the probe solution for spike addition and recovery experiments. According to the results shown in Table 1, the probe showed good stability and recovery in 0.1% FBS, indicating that it has the potential to detect protamine in complex biological systems.

[0060] Table 1 Detection of protamine by fluorescent probe (n=1) in 0.1% FBS solution

[0061]

[0062] Example 5 Response of the fluorescent probe (n=1)-protamine system (10 μmol / L-14 μg / mL) to trypsin

[0063] Trypsin can catalyze the hydrolysis of protamine, the complex between the probe and protamine will dissociate, and the fluorescence emission will return to monomer emission. As the concentration of trypsin increases (0-1μg / mL), the initial hydrolysis rate increases, and I 489 / I 385 The rate of descent increases accordingly, such as Figure 8 As shown. Recorded 12 minutes later 489 / I 385 The relationship between the concentration of trypsin and the concentration of trypsin was found to be linear in the range of 0-1 μg / mL, such as Fig. 9 This shows that the system can achieve quantitative detection of trypsin.

[0064] In addition, the selectivity of the fluorescent probe molecule-protamine complex in detecting trypsin was investigated. Fig.10 As shown, except for trypsin, other biological molecules such as lysozyme, bovine serum albumin, ovalbumin, pepsin (1 μg / mL) and glucose (10 μmol / L) have little effect on the fluorescence of the fluorescent probe molecule-protamine complex, which can be ignored. This shows that the system has a high selectivity for trypsin.

[0065] Example 6 Fluorescence emission of the host-guest complex of fluorescent probe (n=4) and γ-cyclodextrin

[0066] Since the pyrene group can interact with cyclodextrin as a host and guest, and the formation of the host-guest complex affects the fluorescence of the pyrene group, when α-, β-, and γ-cyclodextrin (2mmol / L) were added to the fluorescent probe (n=4) solution (0.2mmol / L), it was found that only γ-cyclodextrin could make the excimer peak (excitation wavelength of 356nm) observed in the fluorescence spectrum, such as Fig.11 As shown, under 360nm ultraviolet light, the solution fluorescence color changes from blue to green, as shown in Fig.11 This indicates that the addition of γ-cyclodextrin induces the formation of excimers, and the change of fluorescence color is observed.

[0067] Example 7 Fluorescence response of the host-guest complex of fluorescent probe (n=4) and γ-cyclodextrin to α-amylase

[0068] The addition of γ-cyclodextrin can induce a change in fluorescence color, so removing cyclodextrin can restore green light to blue light. α-amylase can cut the 1,4-glycosidic bond and destroy the ring structure of cyclodextrin, thereby dissociating its host-guest complex. Fig.12 As shown in the figure, α-amylase (10U / mL) was added to the fluorescent probe (n=4)-γ-cyclodextrin (0.2mmol / L-0.8mmol / L) system in HEPES buffer solution. As time goes by, γ-cyclodextrin is gradually hydrolyzed, and the emission intensity of the excimer gradually decreases, and finally returns to the initial state.

[0069] Different concentrations of α-amylase were added to the HEPES solution of fluorescent probe (n=4)-γ-cyclodextrin (0.2mmol / L-0.8mmol / L), and the change of fluorescence over time was monitored, such as Fig.13 As shown in Figure 2, the higher the concentration of α-amylase, the higher the fluorescence intensity ratio (I 485 / I 385 ) decreases faster. Take 30min later I 485 / I 385 The value of is linearly related to the concentration of α-amylase (y = 0.1726-0.01944x, R 2 =0.9929), the linear range is 1-5μg / mL, and the detection limit is 0.055U / mL. This shows that the system has the ability to quantitatively detect α-amylase.

[0070] Example 8 Selectivity of the host-guest complex of fluorescent probe (n=4) and γ-cyclodextrin in detecting α-amylase

[0071] The selectivity and anti-interference of α-amylase detection based on the host-guest complex of pyrene-containing fluorescent probe (n=4) and γ-cyclodextrin were further investigated, and trypsin, β-amylase, α-glucosidase, alkaline protease, glucose oxidase, laccase, lysozyme, saccharifying enzyme, pepsin, and lipase were selected for anti-interference experiments. α-Amylase (5U / mL) was added to the host-guest complex system of fluorescent probe (n=4) and γ-cyclodextrin (0.2mmol / L-0.8mmol / L), and then the above proteins (5U / mL) were added respectively, and the fluorescence emission before and after the addition was recorded. Fig.14 As shown, except for α-amylase, the presence of other enzymes has no obvious effect on the detection of α-amylase, indicating that the system has good selectivity and anti-interference ability for α-amylase.

[0072] Example 9 Application of the host-guest complex of fluorescent probe (n=4) and γ-cyclodextrin to detect α-amylase in serum solution

[0073] The author investigated whether the host-guest complex system of fluorescent probe (n=4) and γ-cyclodextrin in 0.1% FBS solution can detect α-amylase. In 0.1% FBS solution, a complex system of fluorescent probe (n=4) and γ-cyclodextrin (0.2mmol / L-0.8mmol / L) was prepared, and different concentrations of α-amylase were added to monitor the changes of fluorescence emission over time. Fig.15 As shown, the fluorescence intensity ratio (I 485 / I 385 ) showed an accelerating downward trend with the increase of α-amylase concentration. The fluorescence intensity ratio (I 485 / I 385) was plotted against enzyme concentration and found to be linear between 0.6 and 5 μg / mL (y = 0.1752 - 0.02248x, R 2 =0.9953). The detection limit was 0.062U / mL. The recovery test results are shown in Table 2. The experiments with the four selected concentrations all showed good recovery and stability. This shows that in low-concentration serum solutions, the composite system of fluorescent probe (n=4) and γ-cyclodextrin has the ability to quantitatively detect α-amylase.

[0074] Table 2 Detection of α-amylase by the complex of fluorescent probe (n=4) and γ-cyclodextrin in 0.1% FBS solution

[0075]

[0076] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A fluorescent probe containing a pyrene group, characterized in that: The structural formula of the fluorescent probe is shown in formula (I): Here, n is any integer from 1 to 4.

2. The method for preparing a fluorescent probe containing a pyrene group according to claim 1, characterized in that: The following steps are involved: Step 1, dissolve compound 1 and triethylamine in dichloromethane, stir at room temperature, then add 4-dimethylaminopyridine (DMAP), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), succinic acid or adipic acid or suberic acid or sebacic acid, stir to react; add water for extraction, take the organic phase, and rotary evaporate to obtain compound 2; Step 2: dissolving the compound 2, DMAP and EDCI in dichloromethane, stirring at room temperature, then adding 1-aminopyrene and triethylamine, stirring and reacting at low temperature and room temperature in turn; extracting with water, taking the organic phase, rotary evaporating, and removing the solvent to obtain compound 3; Step 3, dissolving the compound 3 and sodium carbonate in a water-ethanol mixed solvent, reflux reaction, and removing the solvent to obtain the fluorescent probe; The chemical structural formula of the compound 1 is shown in Formula II: The chemical structural formula of the compound 2 is shown in Formula III: The chemical structural formula of the compound 3 is shown in Formula IV:

3. The preparation method according to claim 2, characterized in that: The molar ratio of compound 1 in step 1 to succinic acid, adipic acid, suberic acid or sebacic acid is 1:2 to 1:3; the molar ratio of compound 1 to EDCI is 1:1.05 to 1:1.15; the ratio of compound 1 to DMAP is 1:0.2 to 1:0.4; the ratio of compound 1 to triethylamine is 1:1.1 to 1:1.

3.

4. The preparation method according to claim 2, characterized in that: The stirring method in step 1 is stirring at room temperature, wherein the first stirring time at room temperature is 0.5 to 2 hours, and the second stirring time is 5 to 8 hours; the number of water extractions is 2 to 4 times.

5. The preparation method according to claim 2, characterized in that: The molar ratio of compound 2 and 1-aminopyrene in step 2 is 1:1.05 to 1:1.15; the molar ratio of compound 2 to EDCI is 1:1.3 to 1:1.6; the ratio of compound 2 to DMAP is 1:0.2 to 1:0.4; the ratio of compound 2 to triethylamine is 1:1.1 to 1:1.

3.

6. The preparation method according to claim 2, characterized in that: The stirring method in step 2 is first stirring at room temperature, then stirring at low temperature, and finally stirring at room temperature again, wherein the first stirring time at room temperature is 0.5 to 2 hours, the low temperature stirring temperature is -8 to -12°C, the stirring time is 0.5 to 2 hours, and the third stirring time is 20 to 30 hours; the number of water extractions is 4 to 6 times.

7. The preparation method according to claim 2, characterized in that: The volume ratio of the water-ethanol mixed solvent in step 3 is 1:0.8 to 1:1.2; the molar ratio of compound 3 to sodium carbonate is 1:1 to 1:1.

1.

8. The preparation method according to claim 2, characterized in that: The stirring method in step three is reflux stirring, wherein the first stirring time is 8 to 12 hours, and the second stirring time is 1 to 3 hours.

9. Use of the fluorescent probe containing a pyrene group as claimed in any one of claims 1 to 8 in the detection of protamine and trypsin.

10. Use of a host-guest complex system of a fluorescent probe containing a pyrene group as claimed in any one of claims 1 to 8 and γ-cyclodextrin in detecting α-amylase.

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