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

Abstract

The invention belongs to the field of chemical detection, and discloses a fluorescent probe for detecting butyrylcholine esterase as well as a preparation method and application of the fluorescent probe. The structural formula of the fluorescent probe is # imgabs0. The fluorescent probe provided by the invention has weak fluorescence in a PBS solution system, and can be hydrolyzed by butyrylcholine esterase to generate a product with strong fluorescence. The fluorescent probe has high sensitivity in detection of butyrylcholine esterase, and is not interfered by coexisting substances such as metal ions, amino acids and anions. Based on the specific inhibition effect of pesticides on the activity of butyrylcholine esterase, the fluorescent probe is applied to detect the inhibition degree of butyrylcholine esterase, a rapid detection method for various pesticides is developed, and the fluorescent probe has great market application and popularization value in the field of pesticide residue detection.

Description

Technical Field

[0001] The invention relates to the field of chemical detection, and relates to a pesticide residue detection reagent, in particular to a fluorescent probe for butyrylcholinesterase detection, and a preparation method and application thereof. Background Art

[0002] Pesticide residues are one of the most sensitive topics in the quality and safety of agricultural products. Pesticide residues accumulate in the human body through the food chain and are an important cause of diseases such as cancer, arteriosclerosis, cardiovascular disease, fetal malformation, stillbirth, premature death, premature aging, etc., which seriously threaten human health. Taking organophosphorus pesticides as an example, they can enter the human body and spread through channels such as food, respiratory system, and skin. Once organophosphorus pesticides enter the human body, they will inhibit the activity of the original active substances in the body, causing a decrease in the content of cholinesterase in the human body, causing irreversible damage to human functions. When the organophosphorus content in the human body exceeds the tolerable range, symptoms such as muscle cramps, sweating, and accelerated heartbeat will appear. If the disease is not controlled in time, it will cause partial loss of function of the human nervous system, and in severe cases, disorders will occur. The newly revised "National Food Safety Standard Maximum Residue Limits of Pesticides in Food" (GB 2763-2021) stipulates 10,092 maximum residue limits for 564 pesticides in 376 types (categories) of food. The number of standards has exceeded 10,000 for the first time, nearly twice that of the Codex Alimentarius Commission (CAC), ensuring the quality and safety of agricultural products on a larger scale and ensuring the "safety on the tip of the tongue" of the people.

[0003] Traditional methods for detecting organophosphorus pesticides mainly include chromatography, electrochemical method, capillary electrophoresis, flow injection spectrophotometry, ion mobility spectrometry, etc. Although the above methods have their own advantages in sensitivity, stability and detection efficiency, they have disadvantages such as high instrument operation requirements, complex sample preparation process and expensive instruments and equipment. Therefore, it is necessary to develop fast, efficient and low-cost pesticide detection technology, which is of great significance in life sciences, environmental sciences, medical sciences and agricultural production. Rapid detection technology is conducive to the rapid screening of samples, but the rapid detection technology of pesticide residues in food and agricultural products is still in the qualitative or semi-quantitative stage, and the accuracy in quantitative aspects is not high. Therefore, the development of rapid quantitative detection technology for trace pesticide residues has important scientific significance and application prospects.

[0004] Fluorescent probe detection methods based on organic small molecules have the advantages of good selectivity, high sensitivity, simple and fast operation, and real-time online detection, and have attracted widespread attention. Fluorescent probes are essentially a tool that converts microscopic interactions between molecules into optical signals that are easily recognized by analytical instruments. When fluorescent probes interact with specific target analytes, the fluorescent signal of the system changes significantly, thereby achieving accurate detection of specific target analytes. Fluorescent probe methods have been widely used in the detection and analysis of analytes in the environment and organisms.

[0005] Since the mechanism of action of many pesticides is through inhibiting the activity of various enzymes in harmful organisms, carboxylesterases (CESs), acetylcholinesterase (AchE), butyrylcholinesterase (BchE), etc. are used as identification elements of pesticides to detect pesticide residues in food and environmental water samples. The enzyme inhibition method based on the inhibitory effect of organophosphorus pesticides on cholinesterase has the advantages of simple operation and rapid response, and has become a common method for rapid detection of pesticide residues. However, most of the reported fluorescent probes based on the enzyme inhibition method have the disadvantages of being severely interfered by thiols, low sensitivity, and cumbersome operation. Patent CN111592504A discloses a fluorescent probe for detecting butyrylcholinesterase activity, its synthesis method and application, and the structural formula of the probe is The specific reaction site cyclopropylbutyrate is introduced into the non-emissive skeleton to produce the BchE target receptor P1, thereby eliminating the intrinsic fluorescence of the probe itself. At the same time, after the introduction of BchE, cyclopropylbutyrate can be specifically removed and undergo a spontaneous cyclization reaction. The generated fluorescent product can indicate the activity of BchE; the detection limit of the probe is 0.092μg / mL. Therefore, it is an urgent problem for technicians in this field to develop a fluorescent probe with strong anti-interference and high sensitivity based on the enzyme inhibitor method and apply it to the rapid and accurate detection of pesticide residues. Summary of the invention

[0006] In order to solve the above technical problems, the present invention proposes a fluorescent probe for butyrylcholinesterase detection and its preparation method and application. The present invention provides a fluorescent probe molecule for efficient detection of butyrylcholinesterase, and integrates fluorescent probe technology with enzyme inhibition method for accurate detection of pesticide residues.

[0007] To achieve the above object, the technical solution of the present invention is implemented as follows:

[0008] A fluorescent probe for butyrylcholinesterase detection, the structural formula of which is as follows:

[0009]

[0010] The preparation method of the fluorescent probe for butyrylcholinesterase detection is as follows:

[0011]

[0012] The preparation method of the fluorescent probe for butyrylcholinesterase detection is as follows:

[0013] (1) dissolving 1-ethyl-2,3,3-trimethylindolium iodide and p-hydroxybenzaldehyde in ethanol, subjecting the mixture to reflux reaction, and concentrating the reaction solution to obtain an orange compound, namely, the intermediate CYN-OH;

[0014] (2) The intermediate CYN-OH and triethylamine are dissolved in anhydrous dichloromethane solvent, and an anhydrous dichloromethane solution containing cyclopropylcarbonyl chloride is added dropwise at 0°C. The mixture is reacted at room temperature and washed three times with saturated brine. The organic phase is dried, filtered, concentrated, and purified by column chromatography to obtain a yellow solid product, which is a fluorescent probe for butyrylcholinesterase detection.

[0015] In the above step (1), the molar ratio of 1-ethyl-2,3,3-trimethylindolium iodide to p-hydroxybenzaldehyde is 1:(0.8-4).

[0016] In the above step (1), the reflux reaction temperature is 80-95° C. and the reaction time is 12-36 h. The yield of the intermediate CYN-OH is 65-85%.

[0017] The molar ratio of the intermediate CYN-OH, triethylamine and cyclopropylcarbonyl chloride in the above step (2) is 1:(1-10):(1-8).

[0018] The reaction time at room temperature in the above step (2) is 8-24 hours.

[0019] In the above step (2), the eluent used for column chromatography purification is a mixture of dichloromethane and methanol in a volume ratio of (25-60):1; the yield of the fluorescent probe CYN is 55-75%.

[0020] Application of the above fluorescent probe in detecting butyrylcholinesterase activity.

[0021] Furthermore, the application in detecting the activity of butyrylcholinesterase comprises the following steps: adding the fluorescent probe to a sample to be tested, incubating the fluorescent probe to react with the butyrylcholinesterase in the sample to be tested and removing the cyclopropanecarboxyl group to generate an intermediate CYN-OH; then, performing fluorescence detection, the maximum fluorescence intensity of the emission peak of the fluorescent probe is proportional to the butyrylcholinesterase activity concentration, and the butyrylcholinesterase activity can be quantified.

[0022] The above-mentioned incubation conditions include: temperature of 30-40°C, pH value of 6.5-9, and time of 15-60min. For example, in the incubation conditions, the temperature is 30-35°C, the pH value is 6.5-7, and the time is 15-30min; in the incubation conditions, the temperature is 35-40°C, the pH value is 7-9, and the time is 30-60min; in the incubation conditions, the temperature is 32-40°C, the pH value is 7-9, and the time is 30-60min; in the incubation conditions, the temperature is 30, the pH value is 6.7, and the time is 40min; in the incubation conditions, the temperature is 34, the pH value is 7, and the time is 30min; in the incubation conditions, the temperature is 37, the pH value is 7.4, and the time is 20min; in the incubation conditions, the temperature is 40, the pH value is 9, and the time is 60min.

[0023] In the above fluorescence detection, the excitation wavelength is 450-515nm, and the emission wavelength is 520-700nm. For example, the excitation wavelength is 450-500nm, and the emission wavelength is 520-600nm; the excitation wavelength is 500-515nm, and the emission wavelength is 600-700nm; the excitation wavelength is 510nm, and the emission wavelength is 550nm.

[0024] The application of the above fluorescent probe in the detection of organophosphorus pesticide residues.

[0025] Further, the application in pesticide residue detection, the specific steps are:

[0026] (1) According to the conventional method for extracting pesticides from agricultural products, sample extraction is performed to prepare a sample extract to be tested; a certain volume of the sample extract to be tested is taken as a sample solution to be tested, an enzyme reagent is added, and then a fluorescent probe solution is added, and after sufficient mixing, fluorescence detection is performed; the reaction conditions include: temperature of 37° C., pH value of 7.42, and time of 20 min; the excitation wavelength of fluorescence detection is 510 nm, and the detection wavelength is 555 nm; the enzyme reagent is butyrylcholinesterase.

[0027] (2) The inhibition rate was calculated using the detected fluorescence signal (the enzyme activity inhibition rate calculation formula is shown in formula ①), and then compared with the pesticide standard curve (y = 37.2x-41.4, R 2 =0.999), and the pesticide concentration of the sample solution to be tested was calculated.

[0028]

[0029] Wherein: R is the inhibition rate (%), F0 is the relative fluorescence intensity of the control group, and F1 is the relative fluorescence intensity of the experimental group.

[0030] The beneficial effects produced by the present invention are:

[0031] (1) The preparation method of the fluorescent probe for butyrylcholinesterase detection of the present invention is simple and has high yield. The analysis and test results show that the probe CYN has high efficiency and specific selectivity for butyrylcholinesterase, has strong anti-interference ability, has a minimum detection limit of 0.0125 μg / mL, has a wide pH range, and has strong practical application value.

[0032] (2) The recognition mechanism of the fluorescent probe CYN of the present invention for butyrylcholinesterase is as follows:

[0033]

[0034] It was confirmed by high-resolution mass spectrometry. The probe recognized butyrylcholinesterase and converted it into an intermediate CYN-OH with strong fluorescence. The theoretical molecular weight of the intermediate CYN-OH is [C 20 H 22 N O ] + :292.1696 (positive ion mode hydrogenation peak), high resolution test data is 292.1696 (positive ion mode). High resolution mass spectrometry data verified the recognition mechanism of the fluorescent probe of the present invention for recognizing butyrylcholinesterase, and provided theoretical support for further developing more related fluorescent probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe CYN of the present invention (the solvent is CD3Cl).

[0037] Figure 2 This is the carbon nuclear magnetic resonance spectrum of the fluorescent probe CYN of the present invention (the solvent is CD3Cl).

[0038] Figure 3 This is the high-resolution mass spectrum of the fluorescent probe CYN of the present invention (positive ion mode, solvent is CH3OH).

[0039] Figure 4 The figure is a selectivity diagram of ultraviolet absorption spectrum of the fluorescent probe CYN of the present invention in recognizing butyrylcholinesterase.

[0040] Figure 5This is a fluorescence emission spectrum selectivity diagram of the fluorescent probe CYN of the present invention for recognizing butyrylcholinesterase (a common enzyme), with an excitation wavelength of 510 nm.

[0041] Figure 6 This is the fluorescence emission spectrum selectivity diagram (common metal ions, anions and amino acids) of the fluorescent probe CYN of the present invention for recognizing butyrylcholinesterase, with an excitation wavelength of 510 nm.

[0042] Figure 7 This is a fluorescence titration diagram of the fluorescent probe CYN of the present invention recognizing butyrylcholinesterase, with an excitation wavelength of 510 nm and an emission wavelength of 550 nm.

[0043] Figure 8 This is the minimum detection limit diagram of the fluorescent probe CYN of the present invention for identifying butyrylcholinesterase, with an excitation wavelength of 510 nm and an emission wavelength of 550 nm.

[0044] Fig. 9 This is a diagram showing the pH applicable range of the fluorescent probe CYN of the present invention for identifying butyrylcholinesterase, with an excitation wavelength of 510 nm and an emission wavelength of 550 nm.

[0045] Fig.10 This is a high-resolution mass spectrum (positive ion mode, solvent is CH3OH) for mechanism verification of the fluorescent probe CYN of the present invention after recognizing butyrylcholinesterase.

[0046] Fig.11 The graph is a working curve of the fluorescence emission spectrum intensity and inhibition efficiency of the fluorescent probe CYN of the present invention and butyrylcholinesterase after the addition of carbofuran, with an excitation wavelength of 510 nm and an emission wavelength of 550 nm. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0048] Example 1

[0049] The preparation method of a fluorescent probe for butyrylcholinesterase detection in this embodiment comprises the following steps:

[0050] (1) 1-Ethyl-2,3,3-trimethylindolium iodide (315 mg, 1 mmol) and p-hydroxybenzaldehyde (122 mg, 1 mmol) were dissolved in ethanol (15 mL). After reflux at 80° C. for 12 h, the reaction solution was concentrated to obtain 272 mg of an orange solid product, which was the intermediate CYN-OH, with a yield of 65%.

[0051] (2) The intermediate CYN-OH (419 mg, 1 mmol) and triethylamine (101 mg, 1 mmol) were dissolved in 15 mL of anhydrous dichloromethane solution, and anhydrous dichloromethane solution (5 mL) containing cyclopropylcarbonyl chloride (105 mg, 1 mmol) was added dropwise at 0°C. After the addition was completed, the mixture was reacted at room temperature for 8 h, and then washed three times with saturated brine. The organic phase was dried, filtered, concentrated, and purified by column chromatography (volume ratio of dichloromethane: methanol = 25 / 1) to obtain 268 mg of a yellow solid product, which was the fluorescent probe CYN, with a yield of 55%.

[0052] H NMR spectroscopy: 1 H NMR (CDCl3, 400MHz) δ1.08 (t, J = 3.8Hz, 2H), 1.18 (s, 2H), 1.62 (q, J = 7.0Hz, 3H), 1.71 (s, 1H), 1.87 (s, 6H), 5.04 (d, J = 7.2Hz, 2H), 7.30 (s, 2H), 7.60 (s, 3H), 7.69 (s, 1H), 7.83 (d, J = 16.4Hz, 1H), 8.32 (m, 3H). The hydrogen nuclear magnetic resonance spectrum is shown in Figure 1 shown.

[0053] Carbon NMR spectroscopy determination: 13 C NMR (CDCl3, 100MHz) δ9.6, 13.1, 14.57, 27.0, 44.6, 52.6, 112.7, 115.0, 122.8, 122.9, 129.8, 130.1, 131.1, 133.1, 140.2, 143.5, 154.0, 155.3, 172.8, 181.5. The carbon NMR spectrum is shown in Figure 2 shown.

[0054] High resolution mass spectrometry: HR-ESI-MS calculation for C 24 H 26 NO2 + :360.1958[MI - ] + ,found360.1958[MI - ] + . High-resolution mass spectra such as Figure 3 shown.

[0055] Example 2

[0056] The preparation method of a fluorescent probe for butyrylcholinesterase detection in this embodiment comprises the following steps:

[0057] (1) 1-Ethyl-2,3,3-trimethylindolium iodide (315 mg, 1 mmol) and p-hydroxybenzaldehyde (244 mg, 2 mmol) were dissolved in ethanol (15 mL). After reflux at 85° C. for 18 h, the reaction solution was concentrated to obtain 293 mg of an orange solid product, which was the intermediate CYN-OH, with a yield of 70%.

[0058] (2) The intermediate CYN-OH (419 mg, 1 mmol) and triethylamine (202 mg, 2 mmol) were dissolved in 15 mL of anhydrous dichloromethane solution, and anhydrous dichloromethane solution (5 mL) containing cyclopropylcarbonyl chloride (210 mg, 2 mmol) was added dropwise at 0°C. After the addition was completed, the mixture was reacted at room temperature for 12 h, and then washed three times with saturated brine. The organic phase was dried, filtered, concentrated, and purified by column chromatography (volume ratio of dichloromethane: methanol = 40 / 1) to obtain 317 mg of a yellow solid product, which was the fluorescent probe CYN, with a yield of 65%.

[0059] H NMR spectroscopy: 1 H NMR (CDCl3, 400MHz) δ1.08 (t, J = 3.8Hz, 2H), 1.18 (s, 2H), 1.62 (q, J = 7.0Hz, 3H), 1.71 (s, 1H), 1.87 (s, 6H), 5.04 (d, J = 7.2Hz, 2H), 7.30 (s, 2H), 7.60 (s, 3H), 7.69 (s, 1H), 7.83 (d, J = 16.4Hz, 1H), 8.32 (m, 3H). The hydrogen nuclear magnetic resonance spectrum is as follows Figure 1 shown.

[0060] Carbon NMR spectroscopy determination: 13 C NMR (CDCl3, 100MHz) δ9.6, 13.1, 14.57, 27.0, 44.6, 52.6, 112.7, 115.0, 122.8, 122.9, 129.8, 130.1, 131.1, 133.1, 140.2, 143.5, 154.0, 155.3, 172.8, 181.5. The carbon NMR spectrum is as attached. Figure 2 shown.

[0061] High resolution mass spectrometry: HR-ESI-MS calculation for C 24 H 26 NO2+ :360.1958[MI - ] + ,found360.1958[MI - ] + The high-resolution mass spectrum is shown in the attached Figure 3 shown.

[0062] Example 3

[0063] (1) 1-Ethyl-2,3,3-trimethylindolium iodide (315 mg, 1 mmol) and p-hydroxybenzaldehyde (98 mg, 0.8 mmol) were dissolved in ethanol (15 mL). After reflux at 90° C. for 36 h, the reaction solution was concentrated to obtain 273 mg of an orange solid product, which was the intermediate CYN-OH, with a yield of 65%.

[0064] (2) The intermediate CYN-OH (210 mg, 0.5 mmol) and triethylamine (505 mg, 5 mmol) were dissolved in 15 mL of anhydrous dichloromethane solution, and an anhydrous dichloromethane solution (5 mL) containing cyclopropylcarbonyl chloride (53 mg, 0.5 mmol) was added dropwise at 0°C. After the addition was completed, the mixture was reacted at room temperature for 16 h, and then washed three times with saturated brine. The organic phase was dried, filtered, concentrated, and purified by column chromatography (volume ratio of dichloromethane: methanol = 50 / 1) to obtain 134 mg of a yellow solid product, which was the fluorescent probe CYN, with a yield of 55%.

[0065] H NMR spectroscopy: 1 H NMR (CDCl3, 400MHz) δ1.08 (t, J = 3.8Hz, 2H), 1.18 (s, 2H), 1.62 (q, J = 7.0Hz, 3H), 1.71 (s, 1H), 1.87 (s, 6H), 5.04 (d, J = 7.2Hz, 2H), 7.30 (s, 2H), 7.60 (s, 3H), 7.69 (s, 1H), 7.83 (d, J = 16.4Hz, 1H), 8.32 (m, 3H). The hydrogen nuclear magnetic resonance spectrum is shown in Figure 1 shown.

[0066] Carbon NMR spectroscopy determination: 13 C NMR (CDCl3, 100MHz) δ9.6, 13.1, 14.57, 27.0, 44.6, 52.6, 112.7, 115.0, 122.8, 122.9, 129.8, 130.1, 131.1, 133.1, 140.2, 143.5, 154.0, 155.3, 172.8, 181.5. The carbon NMR spectrum is shown in Figure 2 shown.

[0067] High resolution mass spectrometry: HR-ESI-MS calculation for C 24 H 26 NO2 + :360.1958[MI - ] + ,found360.1958[MI - ] + . High-resolution mass spectra such as Figure 3 shown.

[0068] Example 4

[0069] (1) 1-Ethyl-2,3,3-trimethylindolium iodide (315 mg, 1 mmol) and p-hydroxybenzaldehyde (490 mg, 4 mmol) were dissolved in ethanol (15 mL). After reflux at 95° C. for 24 h, the reaction solution was concentrated to obtain 293 mg of an orange solid product, which was the intermediate CYN-OH, with a yield of 70%.

[0070] (2) The intermediate CYN-OH (210 mg, 0.5 mmol) and triethylamine (505 mg, 5 mmol) were dissolved in 15 mL of anhydrous dichloromethane solution, and anhydrous dichloromethane solution (5 mL) containing cyclopropylcarbonyl chloride (525 mg, 4 mmol) was added dropwise at 0°C. After the addition was completed, the mixture was reacted at room temperature for 24 h, and then washed three times with saturated brine. The organic phase was dried, filtered, concentrated, and purified by column chromatography (volume ratio of dichloromethane: methanol = 40 / 1) to obtain 171 mg of a yellow solid product, which was the fluorescent probe CYN, with a yield of 70%.

[0071] H NMR spectroscopy: 1 H NMR (CDCl3, 400MHz) δ1.08 (t, J = 3.8Hz, 2H), 1.18 (s, 2H), 1.62 (q, J = 7.0Hz, 3H), 1.71 (s, 1H), 1.87 (s, 6H), 5.04 (d, J = 7.2Hz, 2H), 7.30 (s, 2H), 7.60 (s, 3H), 7.69 (s, 1H), 7.83 (d, J = 16.4Hz, 1H), 8.32 (m, 3H). The hydrogen nuclear magnetic resonance spectrum is shown in Figure 1 shown.

[0072] Carbon NMR spectroscopy determination: 13C NMR (CDCl3, 100MHz) δ9.6, 13.1, 14.57, 27.0, 44.6, 52.6, 112.7, 115.0, 122.8, 122.9, 129.8, 130.1, 131.1, 133.1, 140.2, 143.5, 154.0, 155.3, 172.8, 181.5. The carbon NMR spectrum is shown in Figure 2 shown.

[0073] High resolution mass spectrometry: HR-ESI-MS calculation for C 24 H 26 NO2 + :360.1958[MI - ] + ,found360.1958[MI - ] + . High-resolution mass spectra such as Figure 3 shown.

[0074] Example 5

[0075] (1) 1-Ethyl-2,3,3-trimethylindolium iodide (315 mg, 1 mmol) and p-hydroxybenzaldehyde (366 mg, 3 mmol) were dissolved in ethanol (15 mL). After reflux at 90° C. for 36 h, the reaction solution was concentrated to obtain 335 mg of an orange solid product, which was the intermediate CYN-OH, with a yield of 80%.

[0076] (2) The intermediate CYN-OH (419 mg, 1 mmol) and triethylamine (505 mg, 5 mmol) were dissolved in 15 mL of anhydrous dichloromethane solution, and anhydrous dichloromethane solution (5 mL) containing cyclopropylcarbonyl chloride (336 mg, 4 mmol) was added dropwise at 0°C. After the addition was completed, the mixture was reacted at room temperature for 16 h, and then washed three times with saturated brine. The organic phase was dried, filtered, concentrated, and purified by column chromatography (volume ratio of dichloromethane: methanol = 60 / 1) to obtain 365 mg of a yellow solid product, which was the fluorescent probe CYN, with a yield of 75%.

[0077] H NMR spectroscopy: 1 H NMR (CDCl3, 400MHz) δ1.08 (t, J = 3.8Hz, 2H), 1.18 (s, 2H), 1.62 (q, J = 7.0Hz, 3H), 1.71 (s, 1H), 1.87 (s, 6H), 5.04 (d, J = 7.2Hz, 2H), 7.30 (s, 2H), 7.60 (s, 3H), 7.69 (s, 1H), 7.83 (d, J = 16.4Hz, 1H), 8.32 (m, 3H). The hydrogen nuclear magnetic resonance spectrum is shown in Figure 1shown.

[0078] Carbon NMR spectroscopy determination: 13 C NMR (CDCl3, 100MHz) δ9.6, 13.1, 14.57, 27.0, 44.6, 52.6, 112.7, 115.0, 122.8, 122.9, 129.8, 130.1, 131.1, 133.1, 140.2, 143.5, 154.0, 155.3, 172.8, 181.5. The carbon NMR spectrum is shown in Figure 2 shown.

[0079] High resolution mass spectrometry: HR-ESI-MS calculation for C 24 H 26 NO2 + :360.1958[MI - ] + ,found360.1958[MI - ] + . High-resolution mass spectra such as Figure 3 shown.

[0080] Implementation effect example

[0081] (1) UV selectivity

[0082] The fluorescent probe CYN was dissolved in dimethyl sulfoxide (DMSO) to prepare a probe stock solution with a concentration of 1mM for use, and a PBS buffer solution with a pH of 7.4 and a concentration of 20mM was prepared. In the DMSO-PBS (1 / 99, v / v) buffer system, the UV selectivity of the probe CYN for butyrylcholinesterase was investigated using a UV-visible spectrophotometer under the condition of 37°C water bath for 20 minutes. The results are shown in Figure 4As shown in the figure, the single probe CYN (10 μM) has a large UV absorption intensity at 360 nm in the buffer system; when butyrylcholinesterase (20 μg / mL) is added to the probe buffer system (DMSO-PBS buffer system), the UV absorption intensity of the solution system at 525 nm is significantly increased, but when other active substances (20 μg / mL) are added, such as bovine serum albumin (BSA), acetylcholinesterase (AchE), recombinant lipase-A (Lipase A), α-chymotrypsin, human serum albumin (HAS), phosphorylase (PPE), alkaline phosphatase (ALP), bilirubin oxidase (BOD), triglyceride (TG), glucose oxidase (GOD), porcine pancreatic lipase (PPL), trypsin pepsin, tyrosinase, lysozyme (Lysozyme human), carboxypeptidase A (Carboxypeptidase After the addition of the probe to butyrylcholinesterase A, carboxypeptidase B, α-galactosidase and β-galactosidase, the UV absorption intensity (525nm) of the solution system did not change significantly compared with that of the single probe. The results showed that the probe has a specific UV selectivity for butyrylcholinesterase.

[0083] (2) Fluorescence selectivity

[0084] The fluorescent probe CYN was dissolved in dimethyl sulfoxide (DMSO) to prepare a probe stock solution with a concentration of 1mM for use, and a PBS buffer solution with a pH of 7.4 and a concentration of 20mM was prepared. In the DMSO-PBS (1 / 99, v / v) buffer system, the selectivity of the probe CYN for butyrylcholinesterase was investigated using a fluorescence spectrometer under the condition of 37℃ water bath insulation for 20 minutes. The results are shown in Figure 5 and Figure 6 Under the condition of excitation at 510nm, the single probe CYN (10μM) has a weak fluorescence emission intensity at 550nm in the buffer system. When butyrylcholinesterase (20μg / mL) is added, the fluorescence emission intensity of the solution system at 550nm is significantly enhanced. However, when other common enzymes (20μg / mL) and other small molecules (metal ions, common anions, active small molecules and amino acids, all at a concentration of 100μM) are added, the fluorescence emission spectrum of the solution system does not change significantly compared with the fluorescence emission spectrum of the single probe system. The probe CYN has a specific fluorescence selectivity for butyrylcholinesterase.

[0085] (3) Minimum detection limit

[0086] A good detection limit is one of the criteria for testing whether a probe molecule has application value. The probe CYN was dissolved in DMSO to prepare a probe stock solution with a concentration of 1mM for standby use, and a PBS buffer solution with a pH of 7.4 and a concentration of 20mM was prepared. In the DMSO-PBS (1 / 99, v / v) buffer system, the concentration of the fixed probe CYN was 10μM under the condition of 37°C water bath for 20 minutes, and its response intensity to different concentrations of butyrylcholinesterase was measured. With the increase of butyrylcholinesterase concentration (0-23μg / mL), the fluorescence emission intensity of the system at 550nm was significantly enhanced (such as Figure 7 ). The study found that the fluorescence emission intensity of the solution system (F 550 ) was linear in the range of butyrylcholinesterase concentration from 0 to 0.5 μg / mL (R 2 =0.9995), such as Figure 8 According to the rules of the International Union of Pure and Applied Chemistry, the detection limit of the probe molecule for butyrylcholinesterase was calculated to be 0.0125 μg / mL (3σ / k).

[0087] (4) Effect of pH on probe recognition ability

[0088] In order to test the recognition ability of the probe for butyrylcholinesterase under different pH conditions, the effect of different pH on its recognition of butyrylcholinesterase was investigated. PBS buffer solutions with pH values ​​of 4.6, 5.4, 6.5, 7.4, 8.5, and 9.2 (all at a concentration of 20 mM) were prepared, and the probe CYN was dissolved in DMSO to prepare a probe stock solution with a concentration of 1 mM for standby use. In the DMSO-PBS (1 / 99, v / v) buffer system, the concentration of the fixed probe CYN was 10 μM under the condition of 37°C water bath incubation for 20 minutes. The pH value of the buffer system was changed (4.6-8.5), and the fluorescence emission intensity (F 550 ) remained basically unchanged, but in the presence of butyrylcholinesterase (concentration of 20 μg / mL), the fluorescence emission intensity (F 550 ) has a more obvious enhancement between pH 6.5-8.5, such as Fig. 9 The results showed that the probe can be used to identify butyrylcholinesterase under physiological conditions.

[0089] The recognition mechanism of the fluorescent probe CYN of the present invention for butyrylcholinesterase is as follows:

[0090]

[0091] It was confirmed by high-resolution mass spectrometry. Using the activity of butyrylcholinesterase, the probe recognized butyrylcholinesterase and turned into an intermediate CYN-OH with strong fluorescence. The theoretical molecular weight of the intermediate CYN-OH is [C 20 H 22 N O ] + :292.1696 (positive ion mode hydrogenation peak), high resolution test data is 292.1696 (positive ion mode, Fig.10 ). The high-resolution mass spectrometry data verified the recognition mechanism of the fluorescent probe of the present invention for recognizing butyrylcholinesterase, and provided theoretical support for the further development of more related fluorescent probes.

[0092] Application Examples

[0093] The application of fluorescent probes in the detection of organophosphorus pesticide residues is as follows:

[0094] (1) Specific preparation process of standard curve: First, prepare the standard solution of carbofuran pesticide with different concentration gradients (20-1000μg / L); then add 961μL of PBS buffer (pH=7.4, 20mM), 13μL of carbofuran (different concentrations: 20-1000μg / L), butyrylcholinesterase solution (13μL, final concentration is 20μg / mL), and fluorescent probe CYN (13μL, final concentration is 10μmol / L) into the sample bottle. React at 37℃ for 20min, and add 300μL of acetonitrile to terminate the reaction. After mixing and reacting fully, perform fluorescence detection to obtain the relative fluorescence intensity of the experimental group. Control group: add 974μL of PBS buffer (pH=7.4, 20mM), butyrylcholinesterase solution (13μL, final concentration is 20μg / mL), and fluorescent probe CYN (13μL, final concentration is 10μmol / L) into the sample bottle. After the mixed reaction, fluorescence detection was performed to obtain the relative fluorescence intensity of the control group. The reaction conditions included: temperature of 37°C, pH of 7.4, and time of 20 min; the excitation wavelength of fluorescence detection was 510 nm, the detection wavelength was 550 nm, and the inhibition rate was calculated using the detected fluorescence signal (the enzyme activity inhibition rate calculation formula is shown in formula ①).

[0095]

[0096] Wherein: R is the inhibition rate (%), F0 is the relative fluorescence intensity of the control group, and F1 is the relative fluorescence intensity of the experimental group.

[0097] A standard curve was obtained with the logarithmic value of the concentration of carbofuran as the abscissa and the inhibition rate of the corresponding concentration of carbofuran as the ordinate.

[0098] (2) Dilute the standard substance carbofuran with methanol to 50 mg / L to obtain a methanol solution of carbofuran. Weigh 6 portions of peanuts, corn and wheat of 10 g each, of which 3 portions are used as blank control groups (no pesticide spraying) and 3 portions are used as experimental groups. Then, evenly spray 40 μL of the methanol solution of carbofuran on the surface of the experimental group samples, place them in a cool place at room temperature to evaporate (20-30 minutes), rinse them repeatedly with 40 mL of PBS buffer with pH = 7.4, and then ultrasonicate for about 20-30 minutes, let them stand, take the supernatant, filter it with a microfilter, and use it as the extract. Take 2.94 mL of the sample extract as the sample solution to be tested, add butyrylcholinesterase (30 μL, final concentration of 20 μg / mL), then add the fluorescent probe CYN (30 μL, final concentration of 10 μmol / L) solution, mix and react fully, and then perform fluorescence detection; the reaction conditions include: temperature of 37° C., pH value of 7.42, time of 20 min; the excitation wavelength of fluorescence detection is 510 nm, and the detection wavelength is 550 nm; the enzyme reagent is butyrylcholinesterase.

[0099] (3) The inhibition rate is calculated using the detected fluorescence signal (the enzyme activity inhibition rate calculation formula is shown in formula ①), and then the inhibition rate is substituted into the pesticide standard curve y = 37.2x-41.4, R 2 =0.999( Fig.11 ), and the pesticide concentration in the sample extract to be tested is calculated, as shown in Table 1.

[0100] Table 1

[0101]

[0102] * Mean ± standard deviation

[0103] It can be seen from Table 1 that the spiked recovery results of the obtained test results all meet the requirements of the “Laboratory Quality Control Standards for Physical and Chemical Testing of Food”.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A fluorescent probe for detecting butyrylcholinesterase, characterized in that: The structural formula of the fluorescent probe is as follows: 。 2. The method for preparing a fluorescent probe for butyrylcholinesterase detection according to claim 1, characterized in that: Here are the steps: (1) Dissolve 1-ethyl-2,3,3-trimethylindolium iodide and p-hydroxybenzaldehyde in ethanol, reflux for reaction, and then concentrate the reaction solution to obtain the intermediate CYN-OH; (2) The intermediate CYN-OH and triethylamine were dissolved in anhydrous dichloromethane solvent, and an anhydrous dichloromethane solution containing cyclopropylcarbonyl chloride was added dropwise at 0°C. The mixture was reacted at room temperature and washed with saturated brine. The organic phase was dried, filtered, concentrated, and purified by column chromatography to obtain a fluorescent probe for butyrylcholinesterase detection.

3. The preparation method according to claim 2, characterized in that: In the step (1), the molar ratio of 1-ethyl-2,3,3-trimethylindolium iodide to p-hydroxybenzaldehyde is 1:(0.8-4); the reflux reaction temperature is 80-95° C. and the reaction time is 12-36 hours.

4. The preparation method according to claim 3, characterized in that: In the step (2), the molar ratio of the intermediate CYN-OH, triethylamine and cyclopropylcarbonyl chloride is 1:(1-10):(1-8); and the reaction time at room temperature is 8-24 hours.

5. The preparation method according to claim 4, characterized in that: The eluent used for column chromatography purification in step (2) is a mixture of dichloromethane and methanol in a volume ratio of (25-60):

1.

6. Use of the fluorescent probe according to claim 1 in detecting butyrylcholinesterase activity.

7. The use according to claim 6, characterized in that: The method comprises the following steps: adding the fluorescent probe to a sample to be tested, incubating the sample, and then performing fluorescence detection; the maximum fluorescence intensity of the emission peak of the fluorescent probe is proportional to the butyrylcholinesterase activity concentration, and the butyrylcholinesterase activity can be quantified.

8. The use according to claim 7, characterized in that: The incubation conditions include: temperature of 30-40° C., pH value of 6.5-9, and time of 15-60 min.

9. The use according to claim 7, characterized in that: In the fluorescence detection, the excitation wavelength is 450-515 nm, and the emission wavelength is 520-700 nm.

10. Use of the fluorescent probe according to claim 1 in detecting organophosphorus pesticide residues.

Images