Fluorescent probe for detecting glucosidase as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for detecting glucosidase as well as a preparation method and application of the fluorescent probe, relates to the technical field of analysis and detection, and comprises the fluorescent probe for detecting glucosidase, which is # imgabs0. The fluorescent probe for detecting glucosidase as well as the preparation method and application of the fluorescent probe have the advantages that berberrubine is selected as a fluorophore; according to the present invention, the alpha-glucosidase probe is synthesized and is combined with the monosaccharide to form the glucosidic bond, such that the probe can be specifically recognized and catalyzed by the alpha-glucosidase so as to produce the fluorescence signal change, such that the high selectivity of the probe on the alpha-glucosidase is provided so as to reduce the interference caused by the non-specific reaction; the catalytic combination of 2-chloro-1, 3-dimethyl imidazoline chloride and triethylamine can efficiently promote the reaction between berberrubine and monosaccharide, thereby ensuring the efficient synthesis of the probe. The fluorescent probe can be used for rapidly screening candidate compounds of hypoglycemic drugs for inhibiting the activity of alpha-glucosidase and substances influencing the activity of alpha-glucosidase in food.

Description

Technical Field

[0001] The present invention relates to analysis and detection technology, and in particular to a fluorescent probe for detecting glucosidase, a preparation method thereof and an application thereof. Background Art

[0002] Glucosidases are enzymes that hydrolyze glucosidic bonds and are widely found in plants, animals, and microorganisms. Their primary physiological function is to participate in the metabolism of carbohydrates in organisms, hydrolyzing complex glycosides into simple sugar molecules such as glucose, providing energy and carbon sources for the organism. α-Glucosidase is a type of glucosidase that primarily acts on α-glucosidic bonds.

[0003] Accurate detection of α-glucosidase is crucial in many fields, including biomedicine and drug development. Traditional detection methods have numerous limitations. For example, some colorimetric methods have relatively low sensitivity and are susceptible to interference from factors such as sample color and turbidity. Other methods, such as enzyme-linked immunosorbent assays (ELISAs), while offering improved sensitivity, are cumbersome and time-consuming.

[0004] Compared with traditional detection methods, fluorescent probe technology offers many advantages, including high sensitivity, good selectivity, and real-time monitoring. A variety of fluorescent probes targeting glucosidase have been developed, but these probes often have shortcomings. Traditional probes are mostly endpoint assays, unable to observe dynamic changes in α-glucosidase activity and inhibitor effects in real time. Furthermore, some probes lack ideal stability during long-term use or storage, prone to signal attenuation or structural changes, affecting detection accuracy. Summary of the Invention

[0005] The purpose of the present invention is to provide a fluorescent probe for detecting glucosidase and a preparation method and application thereof, so as to solve the above-mentioned deficiencies in the prior art.

[0006] In order to achieve the above object, the present invention provides the following technical solution: a fluorescent probe for detecting glucosidase, which is

[0007] A fluorescent probe for detecting glucosidase, which is The derivatives of The derivatives of

[0008]

[0009] A method for preparing a fluorescent probe for detecting glucosidase comprises the following steps:

[0010] S1. Dissolve D-glucose in 20 mL of anhydrous dichloromethane, then add triethylamine, and stir under argon for 10 minutes to obtain a mixed solution;

[0011] S2. Slowly add berberine to the mixed solution, then add 2-chloro-1,3-dimethylimidazoline chloride, raise the temperature to 40° C., continue stirring to react, and monitor the reaction by TLC until berberine is completely converted to obtain an intermediate;

[0012] S3. Cool the intermediate to 0-5°C in an ice-water bath, then add 10 mL of pre-cooled deionized water dropwise and continue stirring for 10 minutes. Then add dichloromethane and transfer the solution to a separatory funnel. Let it stand for 5-10 minutes. After the two phases are clearly separated, collect the lower organic phase.

[0013] S4. Add anhydrous sodium sulfate to the organic phase, shake until the organic phase solution is clear, and let it stand to dry for 30 minutes to obtain a dried organic phase;

[0014] S5. Purify the dried organic phase by silica gel column chromatography. The amount of silica gel used in the column is 20 times the mass of the dried organic phase. The eluent is a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:3. The target component is collected and evaporated using a rotary evaporator to obtain a fluorescent probe.

[0015] Furthermore, the amount of D-glucose in S1 is 1.2 mmol; the amount of triethylamine in S1 is 0.4 mmol.

[0016] Furthermore, the amount of berberine described in S2 is 1.0 mmol; the amount of 2-chloro-1,3-dimethylimidazoline chloride described in S2 is 0.2 mmol.

[0017] Furthermore, the developing solvent for TLC monitoring in S2 is a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:3.

[0018] Furthermore, the dropping speed of the 10 mL pre-cooled deionized water in S3 is 1 mL / min.

[0019] Furthermore, the dichloromethane is added in S3 in two portions, with each addition amount being 10 mL.

[0020] Furthermore, the amount of anhydrous sodium sulfate in S4 is 10% of the mass of the organic phase; the rotary evaporator temperature in S5 is 35° C., and the vacuum degree is -0.09 MPa.

[0021] The invention discloses an application of a fluorescent probe for detecting glucosidase in detecting α-glucosidase.

[0022] Compared with the prior art, the present invention provides a fluorescent probe for detecting α-glucosidase, its preparation method, and application. The fluorescent probe uses berberine as a fluorophore, and its emission wavelength is in the red channel (615 nm), which can effectively avoid fluorescence interference from other biological molecules. It has high sensitivity and high selectivity for α-glucosidase and can accurately detect the activity of α-glucosidase. By detecting changes in the fluorescence signal in the red channel, the activity of α-glucosidase and the inhibitory effect of hypoglycemic bioactive substances on it can be monitored in real time, providing a dynamic monitoring method for biomedical research and drug screening.

[0023] This method for preparing a fluorescent probe for detecting glucosidase uses berberine as a fluorophore and combines it with a monosaccharide to form a glycosidic bond. This allows the probe to be specifically recognized and catalyzed by α-glucosidase, thereby generating a change in the fluorescent signal. This makes the probe highly selective for α-glucosidase and reduces interference caused by nonspecific reactions. The catalytic combination of 2-chloro-1,3-dimethylimidazoline chloride and triethylamine can efficiently promote the reaction between berberine and the monosaccharide, ensuring efficient synthesis of the probe.

[0024] This fluorescent probe can be used to quickly screen candidate compounds for hypoglycemic drugs that inhibit α-glucosidase activity. In the field of food safety testing, it can be used to detect substances in food that affect α-glucosidase activity to ensure food quality and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments described in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0026] Figure 1 A schematic diagram of the overall synthesis route of the fluorescent probe provided in an embodiment of the present invention;

[0027] Figure 2 A schematic diagram of the overall preparation method of the fluorescent probe provided in an embodiment of the present invention;

[0028] Figure 3 Schematic diagram of sensitivity detection of fluorescent probes provided in an embodiment of the present invention;

[0029] Figure 4 Schematic diagram of selective detection of fluorescent probes provided by an embodiment of the present invention;

[0030] Figure 5 Schematic diagram of fluorescent probe stability detection provided by an embodiment of the present invention;

[0031] Figure 6Schematic diagram of the detection of hypoglycemic bioactive substances by the fluorescent probe provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0033] Example 1:

[0034] A fluorescent probe for detecting glucosidase, which is

[0035]

[0036] The specific implementation method is that berberine is a fluorophore with special luminescence properties, and its emission wavelength is 615nm, which means that when it is irradiated with specific excitation light, it will emit light with a wavelength of 615nm, which corresponds to the signal of the red channel.

[0037] The monomeric fluorescent probe "Sugar Red" constructed based on berberine is mainly targeted at α-glucosidase. In the detection system, when α-glucosidase and the probe "Sugar Red" are present at the same time, a series of key chemical reactions and signal changes will occur. α-glucosidase has enzymatic catalytic activity, which can recognize and act on the glycosidic bond-like structure on the probe "Sugar Red" to cut it off. As this cleavage reaction proceeds, the luminescence environment or binding state of the berberine fluorophore changes, so that the fluorescent signal can be collected in the red channel (that is, the detection channel corresponding to the berberine emission wavelength range), which indicates that the enzyme is catalytically functioning normally at this time and the probe has responded accordingly.

[0038] However, when there are substances with hypoglycemic biological activity, the situation will be different. This type of hypoglycemic active substance will inhibit α-glucosidase, preventing or weakening the enzyme's catalytic activity on its substrate (here is the part of the probe "Sugar Red" that resembles a glycosidic bond). Because the catalytic action of the enzyme is blocked, the glycosidic bond that should have been cut off is retained, and the luminescence of the berberine fluorophore is also changed, which is directly reflected in the intensity of the fluorescence signal detected in the red channel. At this time, the fluorescence signal in the red channel will be weakened. By detecting the change in the intensity of this fluorescence signal, it is possible to determine whether there is a hypoglycemic biological active substance and its degree of inhibition on α-glucosidase, thereby providing an effective detection method for the screening and research of hypoglycemic active substances.

[0039] In summary, this detection system of the monomeric fluorescent probe "Sugar Red" based on the berberine fluorophore cleverly utilizes the correlation between enzyme-catalyzed reactions and fluorescence signal changes to achieve the detection of α-glucosidase activity and hypoglycemic active substances.

[0040] Example 2:

[0041] A fluorescent probe for detecting glucosidase, which is derivatives of The derivatives of

[0042]

[0043] Example 3:

[0044] See also Figures 1 to 2 This embodiment provides a technical solution based on the first embodiment, a method for preparing a fluorescent probe for detecting glucosidase, which is used to prepare a fluorescent probe for detecting glucosidase, comprising the following steps:

[0045] S1. Dissolve D-glucose in 20 mL of anhydrous dichloromethane, then add triethylamine, and stir under argon for 10 minutes to obtain a mixed solution; the amount of D-glucose used is 1.2 mmol; the amount of triethylamine used is 0.4 mmol;

[0046] The specific implementation method is as follows: in a fume hood, 1.2 mmol of D-glucose is accurately weighed and placed in a dry three-necked flask, 20 mL of anhydrous dichloromethane is added to the flask, and the D-glucose is fully dissolved under stirring to obtain a dichloromethane solution of D-glucose;

[0047] Then, 0.4 mmol of triethylamine was weighed and slowly added to the above-mentioned D-glucose dichloromethane solution. The flask was transferred to an argon protection device, and argon was introduced to eliminate oxygen in the system to prevent the oxidation reaction from interfering with the experiment. The stirring device was turned on and stirring was continued for 10 minutes under argon protection to fully mix the D-glucose, triethylamine and dichloromethane to obtain a uniform mixed solution, which provided a good reaction medium for subsequent reactions.

[0048] S2. Berberrubine was slowly added to the mixed solution, followed by the addition of 2-chloro-1,3-dimethylimidazoline chloride, and the temperature was raised to 40° C. and the reaction was continued with stirring. The reaction was monitored by TLC until berberrubine was completely converted to obtain an intermediate. The amount of berberrubine used was 1.0 mmol; the amount of 2-chloro-1,3-dimethylimidazoline chloride used was 0.2 mmol; the developing solvent for TLC monitoring was a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:3;

[0049] A specific implementation method is as follows: 1.0 mmol of berberrubine is weighed and slowly added to the mixed solution obtained in step S1. During the addition process, the solution is continuously stirred to ensure that berberrubine is fully in contact with the mixed solution to obtain a berberrubine mixed solution; then 0.2 mmol of 2-chloro-1,3-dimethylimidazoline chloride is weighed and added to the berberrubine mixed solution, the heating device is turned on, and the temperature is slowly raised to 40°C, and the reaction system is continuously stirred at this temperature.

[0050] During the reaction, samples were taken regularly and monitored by thin layer chromatography (TLC). The developing solvent used for TLC monitoring was a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:3. When TLC showed that berberine was completely converted, the reaction reached the end point and the intermediate was obtained.

[0051] S3. Cool the intermediate to 0-5°C in an ice-water bath, then add 10 mL of pre-cooled deionized water dropwise at a rate of 1 mL / min, continue stirring for 10 minutes, then add dichloromethane twice, each time adding 10 mL, and transfer the solution to a separatory funnel. Let it stand for 5-10 minutes, and after the two phases are clearly separated, collect the lower organic phase;

[0052] A specific implementation method is to quickly place the intermediate obtained in step S2 in an ice water bath and rapidly cool it to 0-5°C by continuous stirring to lower the temperature of the intermediate, reduce the occurrence of side reactions, and stabilize the components in the intermediate.

[0053] Slowly add 10 mL of 4°C pre-cooled deionized water to the cooled intermediate at a rate of 1 mL / min, maintaining the solution at a low temperature and continuing to stir for 10 minutes to facilitate the subsequent extraction and separation process. After the deionized water is added, add 10 mL of dichloromethane to the current solution twice, stirring thoroughly after each addition to fully dissolve the organic components in the solution in the dichloromethane to obtain the final solution. Transfer the final solution to a separatory funnel and let it stand for 5-10 minutes. Once the final solution is clearly separated, open the stopcock of the separatory funnel and collect the lower organic phase to prepare for subsequent drying.

[0054] S4. Add anhydrous sodium sulfate to the organic phase, shake until the organic phase solution is clear, and let it stand and dry for 30 minutes to obtain a dried organic phase; the amount of anhydrous sodium sulfate is 10% of the mass of the organic phase;

[0055] The specific implementation method is to weigh an appropriate amount of anhydrous sodium sulfate, which is 10% of the mass of the organic phase, and slowly add the anhydrous sodium sulfate to the organic phase obtained in step S3 while gently shaking the flask to allow the anhydrous sodium sulfate to fully contact the organic phase and absorb moisture in the organic phase.

[0056] Shake until the organic phase solution becomes clear, indicating that the water has been fully absorbed. Then, let the mixture of anhydrous sodium sulfate and the organic phase stand and dry for 30 minutes to ensure that the water in the organic phase is completely removed to obtain a dried organic phase, which provides a dry reactant for the subsequent purification step.

[0057] S5. Purify the dried organic phase by silica gel column chromatography. The amount of silica gel used for the column is 20 times the mass of the dried organic phase. The eluent is a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:3. Collect the target component and use a rotary evaporator to evaporate the target component to obtain a fluorescent probe; the rotary evaporation temperature of the rotary evaporator is 35°C, and the vacuum degree is -0.09 MPa.

[0058] The specific embodiment is to purify the dried organic phase obtained in step S4 by silica gel column chromatography. Weigh silica gel in an amount 20 times the mass of the dried organic phase and load the silica gel into the chromatography column, paying attention to the uniformity and tightness of the column to ensure a good separation effect.

[0059] Prepare an eluent consisting of a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:3. Slowly add the dried organic phase to a chromatography column packed with silica gel and elute with the eluent to collect the target component during the chromatography process.

[0060] The collected target component was transferred to a flask on a rotary evaporator. The rotary evaporation temperature was set to 35°C and the vacuum was set to -0.09 MPa. The solvent was removed by rotary evaporation under reduced pressure to obtain a fluorescent probe for detecting glucosidase. The prepared fluorescent probe was sealed and stored in a dry, light-proof environment for subsequent detection applications.

[0061] Example 4:

[0062] See also Figure 3 This embodiment provides a technical solution based on the first embodiment: a sensitivity detection experiment of a fluorescent probe for detecting glucosidase.

[0063] Experimental steps: Prepare a series of α-glucosidase solutions with different concentrations (0 U / mL, 0.1 U / mL, 0.5 U / mL, 1 U / mL, 2 U / mL and 5 U / mL).

[0064] Add the same concentration of "Sugar Red" fluorescent probe solution to each test tube containing different concentrations of α-glucosidase solution, allowing the probe to fully contact the enzyme and react (reaction system volume 1 mL, reaction time 1 hour). Use a fluorescence spectrometer to excite the reacted solution at the excitation wavelength of berberine, and record the fluorescence intensity in the red channel (emission wavelength 615 nm).

[0065] See also Figure 3 , a standard curve between the α-glucosidase concentration and the red channel fluorescence intensity was drawn. It can be seen that with the increase of the α-glucosidase concentration, the fluorescence intensity of the red channel gradually increased, showing a good linear relationship, thereby determining the sensitivity range of the fluorescent probe for α-glucosidase detection.

[0066] Through linear regression analysis, the linear equation was calculated as follows: y = 152.94x + 137.46, where y is the red channel fluorescence intensity and x is the α-glucosidase concentration; the correlation coefficient R 2 It is 0.9941, indicating that there is a good linear relationship between fluorescence intensity and enzyme concentration within the experimental concentration range.

[0067] Experimental data demonstrated that the fluorescent probe exhibited good linear response across a wide dynamic range of α-glucosidase concentrations from 0 to 5 U / mL. Sensitivity experiments confirmed that the "Sugar Red" fluorescent probe exhibited high sensitivity and good linearity for α-glucosidase detection. The experimental data demonstrated that the probe can accurately detect low concentrations of α-glucosidase, making it suitable for enzyme activity analysis in practical applications.

[0068] Embodiment 5:

[0069] See also Figure 4 This embodiment provides a technical solution based on the first embodiment: a selective detection experiment of a fluorescent probe for detecting glucosidase.

[0070] Experimental steps: Prepare a series of solutions of other substances that may interact with the "Sugar Red" fluorescent probe, and set up blank group, positive control group and interference group.

[0071] Blank group: containing only reaction buffer.

[0072] Positive control group: contained α-glucosidase (1 U / mL) and fluorescent probe "Sugar Red".

[0073] Interference group: β-glucosidase (1 U / mL), α-amylase (1 U / mL), β-amylase (1 U / mL), sucrase (1 U / mL), lactase (1 U / mL), glucose (1 mM) or sucrose (1 mM), and the fluorescent probe "Sugar Red" were added respectively.

[0074] Similar to Experiment 1, these substance solutions were mixed with the same concentration of "sugar red" fluorescent probe solution, reacted under the same reaction conditions, and the fluorescence intensity of the red channel was recorded using a fluorescence spectrometer.

[0075] See also Figure 4Compared with the blank group, the fluorescence intensity of the positive control group was significantly enhanced, indicating that in the absence of interference, the "Sugar Red" fluorescent probe had a significant response to α-glucosidase; the fluorescence intensity of each interference group was similar to that of the blank group, and compared with the positive control group, the fluorescence intensity change was less than 15%, which was much lower than the 620% increase in the positive control group, indicating that other substances had little interference with the "Sugar Red" fluorescent probe, and the probe had good selectivity for α-glucosidase.

[0076] When detecting α-glucosidase, the "Sugar Red" fluorescent probe can effectively distinguish the target enzyme from other potential interfering substances, has the ability to specifically detect α-glucosidase, and can provide accurate enzyme activity assessment in complex component detection, indicating that the fluorescent probe has good selectivity and can specifically detect α-glucosidase.

[0077] Example 6:

[0078] See also Figure 5 This embodiment provides a technical solution based on the first embodiment: a stability detection experiment of a fluorescent probe for detecting glucosidase.

[0079] A certain concentration of "Sugar Red" fluorescent probe solution was prepared and stored under different storage conditions (4°C, 25°C, 37°C).

[0080] At different time points after storage (1 day, 3 days, 1 week, 2 weeks, and 4 weeks), a portion of the probe solution was taken out and mixed with the α-glucosidase solution of the same concentration for reaction. The fluorescence intensity of the red channel was excited and recorded according to the above method. The fluorescence intensity at the initial storage was used as the benchmark to calculate the fluorescence intensity under different storage conditions and time, and the stability index was calculated. The calculation formula of the stability index is as follows:

[0081]

[0082] See also Figure 5 Under 4°C storage conditions, the stability of the "Sugar Red" fluorescent probe is good. After 4 weeks, the stability index can still reach 90.3%, indicating that low temperature helps maintain the performance of the probe; when stored at room temperature (25°C), the stability index gradually decreases over time, and drops to 80.6% after 4 weeks, indicating that the stability of the probe at room temperature is relatively poor, but it still has certain practicality in the short term; under 37°C storage conditions, the stability index decreases the fastest, and is only 66.7% after 4 weeks, indicating that high temperature has a greater impact on the stability of the probe, and long-term storage in a high temperature environment is not recommended.

[0083] Regardless of the storage conditions, the stability index of the fluorescent probe shows a downward trend with the extension of storage time, indicating that long-term storage will lead to the gradual weakening of the probe performance.

[0084] According to stability test data, the "Sugar Red" fluorescent probe is most stable when stored at 4°C, maintaining high performance for four weeks. Storage at room temperature allows for short-term use, but long-term stability is poor. High-temperature storage (37°C) accelerates the degradation of probe performance and should be avoided. To ensure the accuracy and reliability of test results, it is recommended to store the fluorescent probe at 4°C and use it within two weeks for optimal detection results.

[0085] Embodiment seven:

[0086] See also Figure 6 This embodiment provides a technical solution based on the first embodiment: a fluorescent probe for detecting hypoglycemic bioactive substances.

[0087] The experiment was divided into the following groups: blank group: containing only reaction buffer and the fluorescent probe "Tanghong"; positive control group: containing α-glucosidase and the fluorescent probe "Tanghong"; inhibition group: containing α-glucosidase, the fluorescent probe "Tanghong" and the hypoglycemic bioactive substance acarbose.

[0088] The inhibition groups were divided into inhibition group 1, inhibition group 2, inhibition group 3 and inhibition group 4. The concentration of acarbose added to inhibition group 1 was 50 μg / mL, the concentration of acarbose added to inhibition group 2 was 100 μg / mL, the concentration of acarbose added to inhibition group 3 was 200 μg / mL, and the concentration of acarbose added to inhibition group 4 was 400 μg / mL.

[0089] After the reaction under the same conditions, the fluorescence intensity of the red channel was recorded using a fluorescence spectrometer, and the inhibition rate was calculated. The calculation formula for the inhibition rate is as follows:

[0090]

[0091] Compared with the blank group, the fluorescence intensity of the positive control group was significantly enhanced, indicating that in the absence of inhibitors, the "Sugar Red" fluorescent probe has a significant response to α-glucosidase; please refer to Figure 6 As the concentration of acarbose increased, the fluorescence intensity of the red channel gradually weakened and the inhibition rate gradually increased, indicating that acarbose has a significant inhibitory effect on α-glucosidase. Through this experiment, it was verified that the "Sugar Red" fluorescent probe can effectively detect the inhibitory effect of hypoglycemic bioactive substances on α-glucosidase. As the concentration of the inhibitor increased, the fluorescence signal of the red channel gradually weakened and the inhibition rate gradually increased, indicating that this fluorescent probe has good responsiveness and accuracy for the detection of hypoglycemic bioactive substances. This provides a reliable method for screening and evaluating hypoglycemic drugs.

[0092] The above description is merely illustrative of certain exemplary embodiments of the present invention. It goes without saying that those skilled in the art will be able to modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the above drawings and description are illustrative in nature and should not be construed as limiting the scope of protection of the claims.

Claims

1. A fluorescent probe for detecting glucosidase, characterized in that Its 2. A fluorescent probe for detecting glucosidase, characterized in that Its The derivatives of The derivatives of 3. A method for preparing a fluorescent probe for detecting glucosidase, characterized in that: The method is used to prepare a fluorescent probe for detecting glucosidase according to claim 1, comprising the following steps: S1. Dissolve D-glucose in 20 mL of anhydrous dichloromethane, then add triethylamine, and stir under argon for 10 minutes to obtain a mixed solution; S2. Slowly add berberine to the mixed solution, then add 2-chloro-1,3-dimethylimidazoline chloride, raise the temperature to 40° C., continue stirring to react, and monitor the reaction by TLC until berberine is completely converted to obtain an intermediate; S3. Cool the intermediate to 0-5°C in an ice-water bath, then add 10 mL of pre-cooled deionized water dropwise and continue stirring for 10 minutes. Then add dichloromethane and transfer the solution to a separatory funnel. Let it stand for 5-10 minutes. After the two phases are clearly separated, collect the lower organic phase. S4. Add anhydrous sodium sulfate to the organic phase, shake until the organic phase solution is clear, and let it stand to dry for 30 minutes to obtain a dried organic phase; S5. Purify the dried organic phase by silica gel column chromatography. The amount of silica gel used in the column is 20 times the mass of the dried organic phase. The eluent is a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:

3. The target component is collected and evaporated using a rotary evaporator to obtain a fluorescent probe.

4. The method for preparing a fluorescent probe for detecting glucosidase according to claim 3, wherein The amount of D-glucose in S1 is 1.2 mmol; the amount of triethylamine in S1 is 0.4 mmol.

5. The method for preparing a fluorescent probe for detecting glucosidase according to claim 3, wherein The amount of berberine in S2 is 1.0 mmol; the amount of 2-chloro-1,3-dimethylimidazoline chloride in S2 is 0.2 mmol.

6. The method for preparing a fluorescent probe for detecting glucosidase according to claim 3, wherein The developing solvent for TLC monitoring described in S2 is a mixture of ethyl acetate and petroleum ether in a volume ratio of 1:

3.

7. The method for preparing a fluorescent probe for detecting glucosidase according to claim 3, wherein The dropping speed of the 10 mL pre-cooled deionized water in S3 is 1 mL / min.

8. The method for preparing a fluorescent probe for detecting glucosidase according to claim 3, wherein The dichloromethane in S3 is added in two steps, with each step adding 10 mL.

9. The method for preparing a fluorescent probe for detecting glucosidase according to claim 3, wherein The amount of anhydrous sodium sulfate in S4 is 10% of the mass of the organic phase; the rotary evaporator temperature in S5 is 35° C. and the vacuum degree is -0.09 MPa.

10. Use of the fluorescent probe for detecting glucosidase according to any one of claims 1 to 2 in detecting α-glucosidase.

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