Compound based on hemicyanine fluorescent dye as well as preparation method and application thereof

Abstract

The invention discloses a hemicyanine fluorescent dye-based compound as well as a preparation method and application thereof. The invention provides a compound MCYA based on hemicyanine fluorescent dye targeting AChE, the compound has high sensitivity to AChE, the detection limit can be as low as 0.0225 U / mL, and high-sensitivity detection on AChE can be realized; and the MCYA has good selectivity and strong anti-interference capability on common ions and amino acids in a biological system. A fluorescence sensor platform built by combining MCYA and AChE can realize qualitative and quantitative analysis of organophosphorus and carbamate pesticide residues, and has the advantages of high sensitivity, simplicity in operation, low cost, short detection time, suitability for rapid detection in the market and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of pesticide residue detection. More specifically, it relates to a compound based on a semi-cyanine fluorescent dye, its preparation method and application. Background Art

[0002] In modern agricultural production, pesticides are widely used to prevent various agricultural pests, diseases and weeds that may damage crop products, and play a crucial role in improving agricultural productivity and product quality. Due to the short residual effect under natural conditions and high effectiveness in resisting pest infestation, organophosphates (OPs) and carbamates (CBs) are two major types of pesticides currently widely used in agriculture. However, unreasonable and continuous overuse of pesticides may cause serious pollution to the environment and crop products, ultimately endangering human health and the stable development of the ecosystem. Therefore, developing a sensitive, efficient, reliable and convenient pesticide residue detection method is a very promising task.

[0003] Acetylcholinesterase (AChE) is a key enzyme in biological nerve conduction and belongs to the serine hydrolase family. Acetylcholinesterase can degrade acetylcholine into acetic acid and choline, terminating the continuous excitatory effect of neurotransmitters on the postsynaptic membrane, thus ensuring the normal transmission of nerve signals in the body. Research shows that organophosphorus (OPs) and carbamate (CBs) pesticides have a toxic effect of inhibiting the enzyme activity of acetylcholinesterase; based on the enzyme inhibition principle, the inhibition rate of organophosphorus or carbamate pesticides on AChE has a linear relationship with the content of pesticides. In recent years, the enzyme inhibition method developed based on this toxicological effect has become one of the mainstream methods for rapid detection of pesticide residues in the market.

[0004] The rapid detection method for pesticide residues based on the inhibition of acetylcholinesterase activity (enzyme inhibition method) has advantages such as portable operation and fast detection speed, and has become a common screening method for the quality and safety of agricultural products. However, the traditional enzyme inhibition method is restricted in terms of detection sensitivity, which limits its further development. To improve this defect, fluorescence methods have been introduced. By using characteristic excitation / emission spectra, non-specific interference can be effectively reduced and detection sensitivity can be improved. Currently, a variety of fluorescent probes have been developed for targeting the response of acetylcholinesterase activity, and then realizing the rapid detection of pesticide residues. These fluorescent probes usually have high specificity and sensitivity, and can accurately identify target pesticide residues in complex samples.

[0005] Although significant progress has been made in the detection of pesticide residues using fluorescent probes, there are still some deficiencies and challenges: (1) Stability issues: Some fluorescent probes may be interfered with in complex environments, leading to a decrease in stability, thereby affecting the accuracy of detection results. (2) Cost issues: The preparation cost of certain fluorescent probes is relatively high, limiting their application in large-scale detection. (3) Detection range limitations: Although fluorescent probes have high specificity, their detection range may be limited to some extent and they cannot detect multiple pesticide residues simultaneously. (4) Operational complexity: Although fluorescence detection methods are relatively portable and fast, the preparation and use processes of some probes may be relatively complex and require professional operation.

[0006] In summary, fluorescent probes that can respond to or target acetylcholinesterase and are used for detecting pesticide residues have important application value in pesticide residue detection. To further improve their detection performance and practicality, it is necessary to continue to develop more compounds or fluorescent probes with simple operation, fast speed, high sensitivity, low cost, broad-spectrum efficiency, and easy promotion and use to address the above-mentioned deficiencies and challenges. Currently, there are relatively few fluorescent probes for detecting acetylcholinesterase, and their response speed is still not satisfactory. Therefore, based on the enzyme inhibition principle, it is very necessary to develop a fluorescent probe that can achieve a rapid response and efficiently detect pesticide residues. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the deficiencies of existing compounds or products for detecting organophosphorus and carbamate pesticide residues, and to provide a compound based on a semi-cyanine fluorescent dye, its preparation method, and application.

[0008] The first object of the present invention is to provide a compound MCYA based on a semi-cyanine fluorescent dye.

[0009] The second object of the present invention is to provide a preparation method of the compound MCYA.

[0010] The third object of the present invention is to provide the application of the compound MCYA.

[0011] The fourth object of the present invention is to provide a product.

[0012] The fifth object of the present invention is to provide a fluorescence sensor.

[0013] The sixth object of the present invention is to provide a method for detecting organophosphorus and carbamate pesticide residues.

[0014] The above objects of the present invention are achieved by the following technical solutions:

[0015] The present invention provides a compound based on a semi-cyanine fluorescent dye, and the structure of the compound is shown as follows:

[0016] In the formula, X is selected from halogens.

[0017] In the present invention, 1-ethyl-2-[2-(4-hydroxyphenyl)vinyl]-3,3-dimethyl-3H-indole and acetyl chloride are used as raw materials to synthesize a compound MCYA that can target acetylcholinesterase (AChE). After being catalytically hydrolyzed by AChE, the hydrolysis product of MCYA has fluorescence properties. Therefore, the compound MCYA combined with the enzyme inhibition method can achieve rapid and sensitive detection of the product and the substrate. And the detection process of MCYA and AChE activity is not affected by the biological system matrix and impurities, has good selectivity for AChE in complex biological matrices, strong anti-interference ability, can be used to detect acetylcholinesterase, and the detection limit can be as low as 0.0225 U / mL, enabling highly sensitive detection of acetylcholinesterase activity. Combining the compound MCYA with AChE for detecting organophosphorus and carbamate pesticides has the advantages of high sensitivity, good selectivity, and high stability. By recording the changes in the fluorescence intensity of the detection system before and after drug inhibition, qualitative and quantitative analysis of pesticide residues in the sample solution to be detected can be achieved, providing more detection products and methods with simple synthesis, high sensitivity, and easy operation for pesticide residue detection.

[0018] The present invention provides a preparation method of the compound MCYA. Dissolve 1-ethyl-2-[2-(4-hydroxyphenyl)vinyl]-3,3-dimethyl-3H-indole (MCYO) and triethylamine in an organic solvent, then slowly dropwise add acetyl chloride, and ice-bath for 5 - 20 minutes. After the reaction is completed, remove the solvent and purify the crude product by silica gel column chromatography. The obtained yellow solid powder is the compound MCYA based on the semi-cyanine fluorescent dye, that is, 1-ethyl-2-[2-(4-acetylphenyl)vinyl]-3,3-dimethyl-3H-indole;

[0019] Among them, the structural formula of 1-ethyl-2-[2-(4-hydroxyphenyl)vinyl]-3,3-dimethyl-3H-indole is:

[0020] In the formula, X is selected from halogens.

[0021] The synthesis route of the compound MCYA based on the semi-cyanine fluorescent dye is:

[0022]

[0023] In the formula, X is selected from halogens, preferably I - , Br - , Cl - , F - .

[0024] Preferably, the molar ratio of 1-ethyl-2-[2-(4-hydroxyphenyl)vinyl]-3,3-dimethyl-3H-indole to triethylamine is 1:(1-2).

[0025] More preferably, the molar ratio of 1-ethyl-2-[2-(4-hydroxyphenyl)vinyl]-3,3-dimethyl-3H-indole to triethylamine is 1:1.

[0026] Preferably, the eluent for silica gel column chromatography purification is a mixed solution of methanol and dichloromethane, and the volume ratio thereof is 1:(15-25).

[0027] More preferably, the volume ratio of methanol to dichloromethane is 1:20.

[0028] The present invention provides the application of compound MCYA in the aspect of being used as a fluorescence probe detection material, and compound MCYA is used in combination with acetylcholinesterase.

[0029] The response mechanism of compound MCYA to AChE: After the reaction of compound MCYA with AChE, the enzymatic hydrolysis product MCYO has the highest fluorescence intensity at 560 nm under the optimal excitation wavelength of 520 nm, indicating that MCYA can achieve a signal-on response to acetylcholinesterase. MCYA can perform fluorescence titration measurement on the enzyme activity of acetylcholinesterase, and the detection limit is as low as 0.0225 U / mL, and high-sensitivity detection of the enzyme activity of acetylcholinesterase can be achieved.

[0030] Therefore, the present invention provides the application of compound MCYA in the detection of acetylcholinesterase.

[0031] The present invention provides the application of compound MCYA in the aspect of being used as a fluorescence probe targeting acetylcholinesterase.

[0032] The present invention provides the application of compound MCYA in the detection of organophosphorus and carbamate pesticide residues, or in the preparation of products for detecting organophosphorus and carbamate pesticide residues.

[0033] The present invention provides a product containing the above compound MCYA.

[0034] The present invention provides a fluorescence sensor containing compound MCYA and acetylcholinesterase.

[0035] Furthermore, the fluorescent compound MCYO, which is the product of the hydrolysis of compound MCYA by AChE, is excited under the excitation condition of 520 nm and emits a wavelength of 560 nm. According to the change in the fluorescence signal intensity before and after the detection system is inhibited by the drug, the pesticide residues in the sample solution to be detected can be detected, so as to achieve qualitative and quantitative analysis.

[0036] The present invention also provides a method for detecting organophosphorus and carbamate pesticide residues, which uses the compound MCYA or a fluorescence sensor for detection.

[0037] The present invention has the following beneficial effects:

[0038] The present invention provides a new compound MCYA that can target acetylcholinesterase. The compound MCYA itself has no fluorescence, while its hydrolysis product has fluorescence properties. After being catalytically hydrolyzed by AChE, the decomposition product of MCYA is the fluorescent compound MCYO, thus turning on the fluorescence signal, and a microplate reader can be used to achieve rapid and sensitive detection of the product and the substrate. During the activity detection of MCYA and AChE, it will not be affected by the biological system matrix and impurities, has good selectivity for AChE in complex biological matrices, strong anti-interference ability, and the detection limit can be as low as 0.0225 U / mL, enabling highly sensitive detection of the activity of acetylcholinesterase.

[0039] Furthermore, by combining the compound MCYA and acetylcholinesterase to construct a fluorescence sensor, it can be used to detect organophosphorus and carbamate pesticides, and has the advantages of high sensitivity, simple operation, low cost, short detection time, and being suitable for rapid detection in the market. In the detection of the residues of organophosphorus and carbamate pesticides by this fluorescence sensor, 4 organophosphorus pesticides and 13 carbamate pesticides that meet the maximum residue limit standard of GB2763-2021 have been detected, and the detection time only takes 10 minutes. Among them, dichlorvos has the highest sensitivity, and the detection limit can reach 0.1 ng / mL. Therefore, the compound MCYA provided by the present invention and the fluorescence sensor platform built by it for detecting organophosphorus and carbamate pesticide residues have the characteristics of simple operation, rapidity, high sensitivity, low cost, broad-spectrum high efficiency, easy popularization and use, etc., are suitable for screening a large number of samples in the market and realizing on-site real-time monitoring, and can be used to efficiently screen products with excessive organophosphorus and carbamate pesticide residues in the market, and have good practical application value. Description of the Drawings

[0040] Figure 1 1H NMR spectrum of MCYA in deuterated methanol 1 1H NMR spectrum.

[0041] Figure 2 13C NMR spectrum of MCYA in deuterated methanol 13 13C NMR spectrum.

[0042] Figure 3 Spectral response diagram of MCYA to AChE (in the figure, a is the absorption spectrum of MCYA (30 μM) before and after adding AChE (2.5 U / mL); b is the fluorescence spectrum of MCYA (30 μM) before and after adding AChE (2.5 U / mL), λex = 470 nm).

[0043] Figure 4 UV absorption and fluorescence spectral properties of the fluorescent compound MCYO (in the figure, a is the absorption spectrum of the fluorescent compound MCYO (30 μM) (λex = 470 nm); b is the fluorescence spectrum of the fluorescent compound MCYO (30 μM) (λex = 470 nm); c is the luminescence stability of the fluorescent compound MCYO (30 μM) over a certain period of time (λex = 520 nm)).

[0044] Figure 5 HPLC-MS verification results of the reaction mechanism of MCYA with AChE (in the figure, a is the HPLC chromatograms of MCYA (30 μM), MCYO (30 μM), and MCYA (30 μM) + AChE (2.5 U / mL). Separation conditions: chromatographic column (Athena C18, 120A, 4.6×200 mm, 5 μm); mobile phase: 0.2% formic acid in water and acetonitrile; elution conditions (gradient elution): 5% - 5% (0 - 10 min), 5% - 90% (10 - 25 min), 90% - 90% (25 - 35 min); column temperature: 30 °C; flow rate: 0.8 mL / min; detection wavelength: 430 nm; b is the mass spectrum of the reaction solution of MCYA and AChE).

[0045] Figure 6 Results of the effect of temperature on the MCYA + AChE detection system (at different temperatures (0 °C, 10 °C, 25 °C, 37 °C, 55 °C), a is the fluorescence spectrum of MCYA (30 μM); b is the fluorescence intensity of MCYA (30 μM) at 560 nm; c is the fluorescence spectrum of MCYA (30 μM) + AChE (2.5 U / mL); d is the fluorescence intensity of MCYA (30 μM) and MCYA (30 μM) + AChE (2.5 U / mL) at 560 nm; all experiments were carried out in PB (50 mM, pH = 7.4), λex = 470 nm).

[0046] Figure 7 Results of the effect of pH on the MCYA + AChE detection system (under the conditions of pH 3.0 - 9.0, a is the fluorescence spectrum of MCYA (30 μM), b is the fluorescence intensity of MCYA (30 μM) at 560 nm; c is the fluorescence spectrum of MCYA (30 μM) + AChE (2.5 U / mL); d is the fluorescence intensity of MCYA (30 μM) and MCYA (30 μM) + AChE (2.5 U / mL) at 560 nm; all experiments were carried out in PB (50 mM), λex = 470 nm).

[0047] Figure 8Fluorescence titration experimental results of MCYA on AChE enzyme activity (in the figure, a is the fluorescence spectrum of MCYA (30 μM) after adding AChE (0 - 66 U / mL); b is the change diagram of the linear relationship between the fluorescence intensity and enzyme concentration at 560 nm in the MCYA (30 μM) + AChE reaction system, λex = 470 nm).

[0048] Figure 9 It is the exploration of the reaction time between MCYA and AChE.

[0049] Figure 10 It is the figure of the best enzyme activity determination results of the MCYA + AChE detection system (the corresponding inhibition rates of detecting drugs in the MCYA + AChE system at different enzyme activities: a is dichlorvos (0.0006 μg / mL); b is malathion (0.8 μg / mL); c is carbofuran (0.002 μg / mL); d is benfuracarb (0.007 μg / mL)).

[0050] Figure 11 It is the figure of the best reaction time determination results of the MCYA + AChE detection system (the corresponding inhibition rates of detecting drugs in the MCYA + rBmAChE system when the probe MCYA and rBmAChE are incubated for different times: a is dichlorvos (0.0006 μg / mL); b is malathion (0.8 μg / mL); c is carbofuran (0.002 μg / mL); d is benfuracarb (0.007 μg / mL)).

[0051] Figure 12 It is the standard curve drawn for detecting drugs by MCYA + AChE (in the figure, a is 4 kinds of organophosphorus pesticides; b is 13 kinds of carbamate pesticides).

[0052] Figure 13 It is the selectivity of the MCYA probe and the anti - interference experiment of the system (after adding various analytes, a is the fluorescence intensity of MCYA (30 μM) at 560 nm; b is the fluorescence intensity of MCYA (30 μM) + rBmAChE (2.5 U / mL) at 560 nm; analytes and their concentrations include: 1. Na + (1 mM), 2. K + (1 mM), 3. Ca 2+ (1 mM), 4. Mg 2+ (1 mM), 5. Fe 3+ (1 mM), 6. Cl - (1 mM), 7. NO 3- (1 mM), 8. CO3 2- (1 mM), 9. SO4 2-(1 mM), 10. EDTA (1 mM), 11. Lys (1 mM), 12. Gln (1 mM), 13. Ala (1 mM), 14. His (1 mM), 15. Gly (1 mM), 16. Leu (1 mM), 17. Arg (1 mM), 18. rBmAChE (2.5 U / mL)).

[0053] Figure 14 These are the experimental results of the enzymatic reaction kinetics of MCYA on AChE. Specific implementation manners

[0054] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0055] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0056] Example 1 Preparation of compound MCYA

[0057] According to the synthesis route recorded in the prior art (Wang X, Li P, Ding Q, et al.. Observation of acetylcholinesterase in stress-induced depression phenotypes by two-photon fluorescence imaging in the mouse brain[J]. Journal of the American Chemical Society, 2019, 141(5):2061-2068.), the fluorescent compound 1-ethyl-2-[2-(4-hydroxyphenyl)vinyl]-3,3-dimethyl-3H-indole (MCYO) was prepared from compound a.

[0058] Preparation method of compound MCYA: Under ice bath conditions, add the red fluorescent compound MCYO (0.058 g, 0.2 mmol) and triethylamine (45 μL, 0.2 mmol) to dichloromethane, and then slowly dropwise add acetyl chloride (30 μL, 0.4 mmol). Continue the reaction for 10 minutes under ice bath. After the reaction is completed, remove the solvent and purify the crude product by silica gel column chromatography (the eluent is dichloromethane:methanol = 20:1). The obtained yellow solid powder is compound MCYA, that is, 1-ethyl-2-[2-(4-acetylphenyl)vinyl]-3,3-dimethyl-3H-indole.

[0059] The synthesis route is:

[0060]

[0061] Among them, the paired ion of compound MCYA is I - , and similarly, halogens can be used for pairing.

[0062] The H spectrum and C spectrum of compound MCYA in deuterated methanol are as shown in Figure 1 and Figure 2 shown, and its 1 H NMR and 13 the characterization results of C NMR are as follows:

[0063] 1 H NMR (600 MHz, methanol-d4) δ (ppm): 8.48 (d, J = 16.3 Hz, 1H), 8.16 (d, J = 8.7 Hz, 2H), 7.91 - 7.86 (m, 1H q, J = 7.4 Hz, 3H), 2.33 (s, 3H), 1.87 (s, 6H), 1.61 (t, J = 7.4 Hz, 3H).

[0064] 13 C NMR (151 MHz, methanol-d4) δ (ppm): 183.48, 170.44, 156.46, 155.19, 145.42, 141.80, 133.27, 132.94, 131.24, 130.68, 124.22, 124.05, 116.19, 113.16, 54.15, 43.81, 26.40, 20.97, 14.13.

[0065] Example 2 Spectral Responsiveness of Compound MCYA to AChE

[0066] 1. Spectral Responsiveness of Compound MCYA to AChE

[0067] In a 300 μL black microplate, the following were set respectively: (1) MCYO group: 225 μL of 50 mM PB (pH = 7.4) + 75 μL of MCYO (30 μM); (2) MCYA + AChE group: 150 μL of 50 mM PB (pH = 7.4) + 75 μL of MCYA (30 μM) + 75 μL of AChE (2.5 U / mL); (3) MCYA group: 225 μL of 50 mM PB (pH = 7.4) + 75 μL of MCYA (30 μM); Incubate the reaction at room temperature for 10 minutes, and measure the ultraviolet absorption spectrum (ABS) and fluorescence spectrum (FL) of the three reaction systems.

[0068] The measurement results are as shown in Figure 3As shown, after adding AChE, the ABS and FL peak graphs of compound MCYA before and after reacting with AChE are the same as those of MCYO, indicating that both can undergo enzymatic hydrolysis response, thereby turning on the fluorescence signal.

[0069] The schematic diagram of compound MCYA for AChE detection is as follows. After MCYA is catalytically hydrolyzed by AChE, the acetyl group is cleaved off to expose the hydroxyl group, becoming the fluorescent compound MCYO, thus emitting fluorescence.

[0070]

[0071] 2. Optimal excitation and emission wavelengths of compound MCYA

[0072] Add 75 μL of MCYO (30 μM) to 300 μL of transparent and black microplates containing 225 μL of 50 mM PB (pH = 7.4) respectively, and measure its ultraviolet absorption spectrum and fluorescence spectrum to determine the optimal excitation and emission wavelengths.

[0073] The measurement results are as Figure 4 shown. It can be seen that the optimal excitation and emission wavelengths of the fluorescent compound MCYO, the decomposition product of compound MCYA, are 520 nm and 560 nm respectively ( Figure 4 a - b). At the optimal excitation wavelength of 520 nm, the fluorescence intensity of compound MCYO at 560 nm was measured within 60 min) ( Figure 4 c), showing good time stability.

[0074] Example 3 Verification of the reaction mechanism of compound MCYA with AChE

[0075] Perform HPLC - MS detection on compound MCYA, fluorescent compound MCYO, and AChE. The liquid - phase detection conditions for the reaction solution are as follows: C18 reverse - phase column (250×4.6 mm, 5 μm), mobile phase is 0.2% formic acid in water (phase A) and acetonitrile (phase B), flow rate is 0.8 mL / min, column temperature is 30 °C, and detection wavelength is 430 nm. Among them, the reaction solution of MCYA and AChE is detected by mass spectrometry in positive ion mode.

[0076] The detection results are as Figure 5 shown, showing that the elution times of the main peaks of the reaction solution of MCYA and AChE are 2.559 min and the elution time of MCYO (2.523 min) are basically the same ( Figure 5 a), and this result further proves that MCYA is converted into MCYO under the catalytic action of AChE, and the mass spectrometry diagram of this reaction solution also shows the mass spectrometry data of MCYO ( Figure 5 b).

[0077] Example 4 Influence Factors of the Reaction between Compound MCYA and AChE

[0078] 1. Influence of pH on the MCYA + AChE Detection System

[0079] In a 300 μL microplate, 150 μL of 50 mM PB buffer with different pH values (pH 3.0 - 9.0), 75 μL of rBmAChE working solution (2.5 U / mL), and 75 μL of MCYA working solution (30 μM) were added in sequence. Meanwhile, a blank control group containing only MCYA was set. After incubation at 25 °C for 10 min, the fluorescence spectrum was measured.

[0080] The measurement results are as Figure 6 shown. It can be seen that MCYA exhibits weak fluorescence and remains at a stable level at pH 3 - 7. When pH > 7, the fluorescence increases, indicating an increase in the self-hydrolysis rate of the probe; the MCYA + AChE system has better responsiveness at 6.0 - 9.0. Considering that the pH of the vast majority of biological matrices is 7.0 - 7.4, the detection pH was selected as 7.4, and subsequent detections were carried out under this condition.

[0081] 2. Influence of Temperature on the MCYA + AChE Detection System

[0082] In a 300 μL microplate, 150 μL of 50 mM PB buffer (pH = 7.4), 75 μL of rBmAChE working solution (2.5 U / mL), and 75 μL of MCYA working solution (30 μM) were added in sequence. Meanwhile, a blank control group containing only MCYA was set. After incubation at different temperatures (0 °C, 10 °C, 25 °C, 37 °C, 55 °C) for 10 min, the fluorescence spectrum was measured.

[0083] The measurement results are as Figure 7 shown. It can be seen that MCYA exhibits weak fluorescence and remains at a stable level at 0 - 37 °C. When the temperature > 37 °C, the fluorescence increases, indicating an increase in the self-hydrolysis rate of the probe; for the MCYA + AChE detection system, as the temperature increases, the fluorescence enhancement amplitude is not large. Finally, room temperature (25 °C) was selected as the detection temperature for this detection system, and subsequent detections were carried out under this condition.

[0084] Example 5 Fluorescence Titration Experiment of Compound MCYA on AChE Enzyme Activity

[0085] 1. Detection Limit

[0086] In a 300 μL microplate, 150 μL of 50 mM PB buffer (pH = 7.4), 75 μL of rBmAChE working solution (0 - 66 U / mL), and 75 μL of MCYA working solution (30 μM) were added in sequence. Incubate at 25 °C for 10 minutes, and record the fluorescence intensity at 560 nm (excitation wavelength 520 nm) using a continuous wavelength multifunctional microplate reader. Then, with acetylcholinesterase activity as the abscissa and fluorescence intensity as the ordinate, plot the working curve of MCYA against a series of active acetylcholinesterases. And according to the formula: detection limit = 3σ / k (σ is the standard deviation of blank measurement, k is the slope between fluorescence intensity and AChE concentration), calculate the detection limit of compound MCYA for AChE. The fluorescence intensity of the MCYA blank solution at 520 nm was measured ten times to obtain the standard deviation of blank measurement.

[0087] The determination results are as Figure 8 shown. It can be seen that MCYA shows good analytical performance for acetylcholinesterase activity, and the detection limit can be as low as 0.0225 U / mL, enabling highly sensitive detection of acetylcholinesterase activity.

[0088] 2. Reaction time between MCYA and AChE

[0089] In a 300 μL microplate, 150 μL of 50 mM PB buffer (pH = 7.4), 75 μL of rBmAChE working solution (2.5 U / mL), and 75 μL of MCYA working solution (30 μM) were added in sequence. Incubate at 25 °C for 35 minutes, and record the fluorescence intensity at 560 nm (excitation wavelength 520 nm) every 5 minutes using a continuous wavelength multifunctional microplate reader. Then, with the reaction incubation time as the abscissa and fluorescence intensity as the ordinate, plot the enzymatic hydrolysis curve of MCYA over time.

[0090] The determination results are as Figure 9 shown. It can be seen that within 25 minutes, the fluorescence intensity shows a linear relationship with time over time.

[0091] Example 6 Optimization of the Conditions of the MCYA + AChE Detection System

[0092] 1. Determination of the Optimal Enzyme Activity

[0093] At room temperature (25 °C), in a 300 μL microplate, the following were set up respectively: (1) Inhibition group: 75 μL of 50 mM PB buffer (pH = 7.4) + 75 μL of AChE working solution with different enzyme activities + 75 μL of the drug. After incubating for 5 minutes, 75 μL of the MCYA working solution (30 μM) was added and reacted for 5 minutes. The fluorescence intensity at 560 nm (excitation wavelength 520 nm) was recorded and denoted as FL; (2) Blank group: 150 μL of 50 mM PB buffer (pH = 7.4) + 75 μL of AChE working solution with different enzyme activities + 75 μL of the MCYA working solution (30 μM) reacted for 5 minutes. The fluorescence intensity at 560 nm (excitation wavelength 520 nm) was recorded and denoted as FL0.

[0094] The above drug selections and their concentrations were as follows: Dichlorvos (0.0006 μg / mL); Malathion (0.8 μg / mL); Carbofuran (0.002 μg / mL); Propoxur (0.007 μg / mL). Based on the enzyme inhibition principle, the enzyme activity inhibition rate was calculated. The inhibition rate calculation formula was as follows:

[0095] Inhibition rate = (FL0 - FL) / FL0

[0096] The measurement results were as Figure 10 shown. It can be seen that when the AChE working enzyme activity was 2.5 U / mL, the inhibition rates of the four drugs were the highest.

[0097] 2. Determination of the optimal reaction time

[0098] At room temperature (25 °C), in a 300 μL microplate, the following were set up respectively: (1) Inhibition group: 75 μL of 50 mM PB buffer (pH = 7.4) + 75 μL of AChE working solution (2.5 U / mL) + 75 μL of the drug. After incubating for 5 minutes, 75 μL of the MCYA working solution (30 μM) was added and reacted for 30 min. The fluorescence intensity at 560 nm (excitation wavelength 520 nm) was recorded every 5 minutes and denoted as FL; (2) Blank group: 150 μL of 50 mM PB buffer (pH = 7.4) + 75 μL of AChE working solution (2.5 U / mL) + 75 μL of the MCYA working solution (30 μM) reacted for 30 minutes. The fluorescence intensity at 560 nm (excitation wavelength 520 nm) was recorded every 5 minutes and denoted as FL0. And the calculation was carried out according to the above inhibition rate formula. The drug selections and their concentrations were as follows: Dichlorvos (0.0006 μg / mL); Malathion (0.8 μg / mL); Carbofuran (0.002 μg / mL); Propoxur (0.007 μg / mL).

[0099] The measurement results were as Figure 11As shown, when the incubation reaction time of compound MCYA and AChE is 5 minutes, the inhibition rates of the four drugs are the highest.

[0100] Example 7 Selectivity of Compound MCYA and Anti-Interference Experiment of the System

[0101] 1. Drawing and Determination of Standard Curves of Different Detection Substances

[0102] At room temperature (25 °C), in a 300 μL microplate, set up: (1) Drug inhibition group: 75 μL of 50 mM PB buffer (pH = 7.4) + 75 μL of AChE working solution (2.5 U / mL) + 75 μL of the drug pre-incubated for 5 minutes, then add 75 μL of MCYA working solution (30 μM) and react for 5 minutes, record the fluorescence intensity at 560 nm (excitation wavelength 520 nm), denoted as FL; (2) Blank group: 150 μL of 50 mM PB buffer (pH = 7.4) + 75 μL of AChE working solution (2.5 U / mL) + 75 μL of MCYA working solution (30 μM) react for 10 minutes, record the fluorescence intensity at 560 nm (excitation wavelength 520 nm), denoted as FL0; Use the semi-logarithmic concentration of the drug as the abscissa and FL / FL0 as the ordinate.

[0103] According to the above settings, experiments were carried out using different pesticides respectively, and the standard curves of 4 organophosphorus pesticides and 13 carbamate pesticides (profenofos, malathion, dichlorvos, trichlorfon, fenobucarb, methiocarb, propoxur, benfuracarb, carbaryl, 3-hydroxycarbofuran, methomyl, carbofuran, isoprocarb, aldicarb sulfoxide, metolcarb, pirimicarb, butocarboxim) were drawn.

[0104] The standard curves drawn by detecting different pesticides with MCYA + AChE are as Figure 12 shown. The detection statistical results of 4 organophosphorus pesticides and 13 carbamate pesticides are shown in Table 1 below, indicating that MCYA + AChE can be used to detect the 16 pesticides in the following table.

[0105] Table 1 Detection Performance of MCYA-AChE Detection System for Different Pesticides

[0106]

[0107]

[0108] 2. Selectivity of MCYA and Anti-Interference Experiment of the System

[0109] (1) Selectivity experiment: Add 150 μL of 50 mM PB buffer (pH = 7.4) + 75 μL of analyte + 75 μL of MCYA (30 μM) to a 300 μL microplate and incubate at room temperature for 10 minutes. Record the fluorescence intensity at 560 nm (excitation wavelength 520 nm) using a continuous wavelength multifunctional microplate reader. Types and concentrations of analytes used: 1. Na + (1 mM), 2. K + (1 mM), 3. Ca 2+ (1 mM), 4. Mg 2+ (1 mM), 5. Fe 3+ (1 mM), 6. Cl - (1 mM), 7. NO 3- (1 mM), 8. CO3 2- (1 mM), 9. SO4 2- (1 mM), 10. EDTA (1 mM), 11. Lys (1 mM), 12. Gln (1 mM), 13. Ala (1 mM), 14. His (1 mM), 15. Gly (1 mM), 16. Leu (1 mM), 17. Arg (1 mM), 18. rBmAChE (2.5 U / mL).

[0110] The measurement results are as Figure 13 shown in a. It can be seen that the fluorescence intensity of the reaction solution of compound MCYA and AChE is significantly higher than that of other analytes, indicating that it has good selectivity for AChE in complex biological matrices.

[0111] (2) Anti-interference experiment: Add 75 μL of 50 mM PB buffer (pH = 7.4) + 75 μL of analyte + 75 μL of MCYA (30 μM) + 75 μL of AChE (2.5 U / mL) to a 300 μL microplate and incubate at room temperature for 10 minutes. Record the fluorescence intensity at 560 nm (excitation wavelength 520 nm) using a continuous wavelength multifunctional microplate reader. Types and concentrations of analytes used: 1. Na + (1 mM), 2. K + (1 mM), 3. Ca 2+ (1 mM), 4. Mg 2+ (1 mM), 5. Fe 3+ (1 mM), 6. Cl - (1 mM), 7. NO 3- (1 mM), 8. CO3 2- (1 mM), 9. SO4 2-(1 mM), 10. EDTA (1 mM), 11. Lys (1 mM), 12. Gln (1 mM), 13. Ala (1 mM), 14. His (1 mM), 15. Gly (1 mM), 16. Leu (1 mM), 17. Arg (1 mM), 18. rBmAChE (2.5 U / mL).

[0112] The measurement results are as Figure 13 shown in b. It can be seen that the fluorescence signals of the reaction solution of compound MCYA and AChE are not very different among the above common amino acids and ions, indicating that the reaction system has strong anti-interference ability.

[0113] Example 8 Enzymatic reaction kinetics experiment of compound MCYA on AChE

[0114] Add 150 μL of 50 mM PB buffer (pH = 7.4) + 75 μL of MCYA (0 - 0.2 mM) + 75 μL of AChE (2.5 U / mL) into a 300 μL microplate and incubate at room temperature. Use the Michaelis Menten template in the SoftMax Pro software of the continuous wavelength multi-functional microplate reader platform to directly calculate the Michaelis constant Km to determine the affinity between compound MCYA and AChE.

[0115] The results are as Figure 14 shown. The calculated Michaelis constant K of AChE is 0.134 mM. It shows that compound MCYA has a strong affinity for binding to AChE.

[0116] In summary, the present invention provides a new compound MCYA that can target acetylcholinesterase. After being catalytically hydrolyzed by AChE, the decomposition product is a fluorescent compound MCYO, thus turning on the fluorescence signal. Compound MCYA itself has no fluorescence, while its hydrolysis product has fluorescence properties, and a microplate reader can be used to achieve rapid and sensitive detection of the product and substrate; and during the activity detection process of MCYA and AChE, it will not be affected by the biological system matrix and impurities, has strong anti-interference ability, has good selectivity for AChE in complex biological matrices, and the detection limit can be as low as 0.0225 U / mL, enabling highly sensitive detection of the activity of acetylcholinesterase.

[0117] Furthermore, the fluorescence sensor constructed by combining compound MCYA and acetylcholinesterase can be used to detect organophosphorus and carbamate pesticides. It has the advantages of high sensitivity, good selectivity, high stability, simple operation, low cost, short detection time, and is suitable for rapid detection in the market. In the residual detection of organophosphorus and carbamate pesticides, this fluorescence sensor has detected 4 organophosphorus pesticides and 13 carbamate pesticides that meet the maximum residue limit standard of GB2763-2021. The detection time only takes 10 minutes, and dichlorvos has the highest sensitivity, with a detection limit of up to 0.1 ng / mL. Therefore, the compound MCYA and the fluorescence sensor platform provided by the present invention for detecting organophosphorus and carbamate pesticide residues have the characteristics of simple operation, rapidity, high sensitivity, low cost, broad-spectrum high efficiency, and easy popularization and use, and are suitable for screening a large number of samples in the market and realizing on-site real-time monitoring.

[0118] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A compound MCYA based on semi-cyanine fluorescent dye, characterized in that, The structure of the said compound is as follows: In the formula, X is selected from halogen.

2. The preparation method of the compound according to claim 1, characterized in that, 1-Ethyl-2-[2-(4-hydroxyphenyl)vinyl]-3,3-dimethyl-3H-indole and triethylamine are dissolved in an organic solvent, and then acetyl chloride is slowly added dropwise. After ice-bathing for 5 to 20 minutes, the solvent is removed and the crude product is purified by silica gel column chromatography. The obtained yellow solid powder is the compound MCYA based on hemicyanine fluorescent dye, that is, 1-ethyl-2-[2-(4-acetylphenyl)vinyl]-3,3-dimethyl-3H-indole; Among them, the structural formula of 1-ethyl-2-[2-(4-hydroxyphenyl)vinyl]-3,3-dimethyl-3H-indole is: In the formula, X is selected from halogens.

3. The preparation method according to claim 2, wherein The molar ratio of 1-ethyl-2-[2-(4-hydroxyphenyl)vinyl]-3,3-dimethyl-3H-indole to triethylamine is 1:(1 - 2).

4. The preparation method according to claim 2, characterized in that, The eluent for silica gel column chromatography purification is a mixed solution of methanol and dichloromethane, and their volume ratio is 1:(15 - 25).

5. Use of the compound according to claim 1 as a material for detecting a fluorescent probe, characterized in that, The compound described in claim 1 is used in combination with acetylcholinesterase.

6. The application of the compound described in claim 1 as a fluorescent probe targeting acetylcholinesterase.

7. The application of the compound described in claim 1 in detecting organophosphorus and carbamate pesticide residues, or in preparing products for detecting organophosphorus and carbamate pesticide residues.

8. A product, characterized in that, Containing the compound described in claim 1.

9. A fluorescence sensor, characterized in that, Containing the compound described in claim 1 and acetylcholinesterase.

10. A method for detecting organophosphorus and carbamate pesticide residues, characterized in that, Detection is carried out using the compound described in claim 1 or the fluorescent sensor described in claim 9.

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