Paclobutrazol hapten as well as synthesis method and application thereof

Abstract

The invention discloses a paclobutrazol hapten as well as synthesis and application thereof. The structure of the paclobutrazol hapten is shown as a formula I. Compared with 3-6 carbon chains, the synthetic method of the paclobutrazol hapten provided by the invention has the characteristics of mild reaction conditions, high yield, few reaction steps and capability of reducing the cost of raw materials. The paclobutrazol colloidal gold detection product provided by the invention has the characteristics of strong specificity, high sensitivity and the like, can realize accurate detection of paclobutrazol in a sample, and has no cross reaction with other drugs. # imgabs0 #

Description

Technical Field

[0001] The invention relates to the technical field of biochemical engineering, and in particular to a paclobutrazol hapten and a synthesis method and application thereof. Background Art

[0002] Paclobutrazol is a plant growth regulator that has the effects of delaying plant growth, inhibiting stem elongation, shortening internodes, promoting plant tillering, increasing plant stress resistance, and improving yield. Paclobutrazol is suitable for crops such as rice, wheat, peanuts, fruit trees, tobacco, rapeseed, and soybeans, and has significant effects. Although the use of paclobutrazol has many benefits, it has been found in practice that excessive use of paclobutrazol will affect the quality of fruits and vegetables and even cause fruit deformities. In addition, excessive paclobutrazol residues will cause vegetable plants to stagnate, shrink, or even die, causing serious economic losses.

[0003] Paclobutrazol can enter the human body through various pathways and may cause chronic toxicity in the human body. Countries around the world pay particular attention to this. Paclobutrazol is widely used in my country. Although it has not been found to have obvious harm to human health, its widespread use in vegetables has led to long-term dietary exposure to human body. Therefore, the impact of paclobutrazol on human health still needs attention and research. Regarding the residual amount of paclobutrazol, all countries have strict regulations. The paclobutrazol residues in Japanese ginger, scallions, onions, carrots, radishes, wormwood, cucumbers and other vegetables are "uniformly regulated" (i.e. 0.01 mg kg-1). The United States, the European Union, South Korea and other countries also require that the paclobutrazol residues in the above vegetables be "not detectable".

[0004] At present, the method for detecting paclobutrazol mainly includes liquid chromatography, liquid chromatography tandem mass spectrometry, enzyme-linked immunosorbent assay, immunoaffinity chromatography column and electrochemical sensor, but these methods all have the disadvantages of complicated operation, time-consuming, expensive cost, etc., and cannot realize the rapid detection of a large number of samples. Therefore, it is of great significance to establish a quick and simple detection method. Therefore, the present invention provides a structurally stable antigen, and the colloidal gold test strip prepared by the antigen can realize the accurate and rapid detection of paclobutrazol. Summary of the invention

[0005] The purpose of the present invention is to provide a paclobutrazol hapten and a synthesis method and application thereof, so as to achieve accurate detection of paclobutrazol and have no cross reaction with other drugs.

[0006] To achieve the above object, the present invention is implemented through the following technical solutions: One of the purposes of the present invention is to provide a paclobutrazol hapten, the structure of which is shown in Formula I: Formula I.

[0007] The second object of the present invention is to provide a method for preparing the above-mentioned paclobutrazol hapten, the steps are as follows: S1. Accurately weigh the paclobutrazol raw material, dissolve it in anhydrous dichloromethane, protect with nitrogen, cool to 0°C, add succinyl chloride, slowly add aluminum chloride, maintain the temperature at 45°C; stir and react for 2 hours; S2. After 2 hours, the mixed solution obtained in step S1 was monitored by TLC to confirm that the reaction was complete, cooled to 0°C, and purified water was added dropwise for quenching, separated, the organic phase was retained, and 10% sodium carbonate was added for extraction twice, the aqueous phase was retained, the acid was adjusted to free, and ethyl acetate was extracted twice, the organic phases were combined, and dried over anhydrous sodium sulfate to obtain a crude paclobutrazol hapten; S3, purification: the crude product obtained in step S2 is purified on a reverse phase FLASH-C18 column to obtain paclobutrazol hapten.

[0008] Preferably, the detailed steps are as follows: S1. Accurately weigh 1.0 g of paclobutrazol raw material, dissolve it in 10 mL of anhydrous dichloromethane, protect with nitrogen, cool to 0°C, add 555 mg of succinyl chloride, slowly add 1.36 g of anhydrous aluminum chloride, maintain the temperature at 45°C; stir and react for 2 hours; S2. After 2 h, the mixed solution obtained in step S1 was monitored by TLC to confirm that the reaction was complete, cooled to 0°C, and 10 mL of purified water was added dropwise for quenching. The organic phase was retained, and 10 mL of 10% sodium carbonate was added for extraction twice. The aqueous phase was retained, and the acid was adjusted to PH = 3, and 10 mL of ethyl acetate was extracted twice. The organic phases were combined and dried over anhydrous sodium sulfate to obtain 1.36 g of crude paclobutrazol hapten. S3, purification: 1 g of the crude paclobutrazol hapten obtained in S2 was purified on a reverse phase FLASH-C18 column to obtain 680 mg of a paclobutrazol carboxyl derivative with a high purity.

[0009] The third object of the present invention is to provide a complete paclobutrazol antigen, which is prepared by coupling the above paclobutrazol hapten with a carrier protein through a coupling technology. The coupling technology is EDC / NHS coupling technology or mixed anhydride method, and the carrier protein is any one of bovine serum albumin (BSA) or chicken ovalbumin (OVA).

[0010] The fourth object of the present invention is to provide two methods for preparing the above-mentioned complete paclobutrazol antigen.

[0011] Specifically, the method is EDC / NHS coupling technology, and when the carrier protein is BSA or OVA, the preparation method of paclobutrazol complete antigen is as follows: Dissolve the above-mentioned paclobutrazol hapten in DMF, slowly add NHS and EDC, stir at room temperature for 6 hours to obtain paclobutrazol hapten activation solution; weigh BSA or OVA and dissolve it in PBS buffer (0.01 mol / L, pH 7.4), stir evenly, slowly add it dropwise to the activated hapten solution, and stir at room temperature for 16 hours. The obtained reaction solution is dialyzed with 0.01 mol / L PBS buffer with a pH of 7.2, and the buffer is changed every 12 hours. The dialyzation is performed for 72 hours to obtain the paclobutrazol complete antigen with BSA or OVA as the carrier protein.

[0012] Specifically, the method is a mixed anhydride method, and when the carrier protein is BSA or OVA, the preparation method of paclobutrazol complete antigen is as follows: Dissolve the above paclobutrazol hapten in 2 mL of anhydrous methanol, add 300 μL of tri-n-butylamine, stir at 4°C for 30 min; add 60 μL of isobutyl chloroformate, stir for 1 h; weigh BSA or OVA, slowly add the above solution, stir for 4 h. Dialyze with 0.01 mol / L PBS with a pH of 7.2, change the water every 2 h, and dialyze for 48 h to obtain the paclobutrazol complete antigen with BSA or OVA as the carrier protein.

[0013] The fifth object of the present invention is to provide a paclobutrazol colloidal gold detection product, characterized in that the paclobutrazol complete antigen is coated on a nitrocellulose membrane.

[0014] The present invention has the following beneficial effects: 1. The present invention provides a connecting arm with a specific length and containing an active group -COOH derived from the structure of paclobutrazol. After the hapten is connected to the carrier protein, the resulting complete antigen can fully expose the antigenic determinant of paclobutrazol and maintain the spatial conformation of paclobutrazol, and has good immunogenicity.

[0015] 2. The paclobutrazol colloidal gold detection product provided by the present invention has the characteristics of high sensitivity, good specificity and strong stability, can realize rapid and accurate detection of paclobutrazol, and has no cross reaction with other drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0017] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0018] Figure 1 This is the MS spectrum of paclobutrazol hapten; Figure 2 This is the synthetic structure diagram of paclobutrazol hapten; Figure 3 The results of sensitivity test of paclobutrazol in fruit and vegetable extracts are shown; Figure 4 This is the specific test result of paclobutrazol colloidal gold test strip. DETAILED DESCRIPTION

[0019] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are 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.

[0020] Example 1. Preparation of Paclobutrazol Hapten The structure of paclobutrazol hapten is shown in formula I: Formula I.

[0021] The preparation method of paclobutrazol hapten described in this embodiment, its specific operation process is as follows: S1. Accurately weigh 1.0 g of paclobutrazol raw material, dissolve it in 10 mL of anhydrous dichloromethane, protect with nitrogen, cool to 0°C, add 555 mg of succinyl chloride, slowly add 1.36 g of anhydrous aluminum chloride, maintain the temperature at 45°C; stir and react for 2 hours; S2. After 2 h, the mixed solution obtained in step S1 was monitored by TLC to confirm that the reaction was complete, cooled to 0°C, and 10 mL of purified water was added dropwise for quenching. The organic phase was retained, and 10 mL of 10% sodium carbonate was added for extraction twice. The aqueous phase was retained, and the acid was adjusted to PH = 3, and 10 mL of ethyl acetate was extracted twice. The organic phases were combined and dried over anhydrous sodium sulfate to obtain 1.36 g of crude paclobutrazol hapten. S3, purification: 1 g of the paclobutrazol hapten intermediate obtained above was purified on a reverse phase FLASH-C18 column to obtain 680 mg of a paclobutrazol carboxyl derivative with high purity. The paclobutrazol carboxyl derivative was detected by LC-MS. Figure 1 It can be seen that the hapten signal of paclobutrazol is 379.20 and the molecular weight of paclobutrazol hapten is 378, thus determining the hapten structure. Figure 2 shown.

[0022] Preparation of complete paclobutrazol antigen 1. Preparation of paclobutrazol hapten-bovine serum albumin or chicken ovalbumin conjugates (paclobutrazol-BSA or paclobutrazol-OVA) using EDC / NHS conjugation technology S1. Weigh 100 mg of the pure hapten in Example 1, dissolve it in 2 mL of DMF, slowly add 36 mg of NHS and 65 mg of EDC, and stir at room temperature for 6 h to obtain a paclobutrazol hapten activation solution; S2. Weigh 120 mg BSA or OVA, dissolve in 20 mL PBS buffer (0.01 mol / L, pH 7.4), stir evenly, slowly add to the activated hapten solution, and stir at room temperature for 16 hours. Dialyze with 0.01 mol / L PBS buffer with a pH of 7.2, changing the buffer every 12 hours, and dialyze for 72 hours to obtain the complete paclobutrazol antigen with bovine serum albumin or chicken ovalbumin as the carrier protein.

[0023] The reaction route is as follows: or

[0024] 2. Preparation of paclobutrazol hapten-bovine serum albumin or chicken ovalbumin conjugate (paclobutrazol-BSA or paclobutrazol-OVA) using mixed anhydride method S1. Weigh 100 mg of pure hapten, dissolve it in 2 mL of anhydrous methanol, add 300 μL of tri-n-butylamine, and stir at 4°C for 30 min. S2, add 60 μL isobutyl chloroformate and stir for 1 h; S3, weigh 120 mg BSA or OVA, slowly add it to the solution obtained in step S2, and stir for 4 h; S4. Dialyze with 0.01 mol / L PBS with a pH of 7.2, changing the water every 2 hours for 48 hours to obtain the complete paclobutrazol antigen with bovine serum albumin or chicken ovalbumin as the carrier protein.

[0025] Example 3: Colloidal gold immunochromatography for rapid detection of paclobutrazol residues in fruit and vegetable extracts 1. Preparation of paclobutrazol colloidal gold detection reagent (1) Preparation of colloidal gold Take 1 mL of 1% (mass percentage) chloroauric acid solution, add 99 mL of ultrapure water to make a final concentration of 0.01% (mass percentage) chloroauric acid solution, heat to boiling, then take 1.6 mL of 1% (mass percentage) trisodium citrate and quickly add it to the boiling chloroauric acid solution at one time, continue heating until the solution changes from light yellow to blue-black and finally to bright red, continue heating for 5 minutes after the color stabilizes, cool to room temperature, and add pure water to the original volume.

[0026] (2) Preparation of colloidal gold-labeled paclobutrazol monoclonal antibody Adjust the pH value of the colloidal gold solution to 8.0, stir evenly with a constant speed stirrer, and add paclobutrazol monoclonal antibody dropwise at the same time, the labeled antibody amount is 1μg / ml colloidal gold, add PEG equivalent to the antibody amount after 1 hour, fully react for 30 minutes, add BSA equivalent to the antibody amount, and continue stirring for 30 minutes after addition. Centrifuge at 9000rpm for 30 minutes to obtain a uniform gold-labeled antibody precipitate, then resuspend in PBS for use, and obtain colloidal gold-labeled paclobutrazol monoclonal antibody.

[0027] (3) Preparation of paclobutrazol colloidal gold test strips 0.2 mg / ml paclobutrazol-coated antigen solution was sprayed on the nitrocellulose membrane to form a detection line T line, and 0.2 mg / ml goat anti-mouse IgG was sprayed to form a control line C line. The detection line and the control line were parallel to each other, and the distance between the two was 0.65 cm. Then, on the bottom plate, the sample pad, the detection line T line sprayed with paclobutrazol-coated antigen, and the control line C line sprayed with goat anti-mouse IgG were overlapped and adhered in sequence on the bottom plate in the same direction.

[0028] The detection line is close to the sample pad, the control line is close to the absorbent paper, the glued detection test paper is cut into test strips of equal width and assembled into a test paper card. Colloidal gold-labeled paclobutrazol monoclonal antibody is added into the microwell and freeze-dried; the microwell and the test strip are sealed and dried for storage.

[0029] 2. Sample Testing Competitive immunochromatography technology is used to qualitatively detect paclobutrazol residues in samples. The test line (T line) on the nitrocellulose membrane is coated with paclobutrazol antigen, and the corresponding paclobutrazol monoclonal antibody is labeled on colloidal gold. During the test, when there is no paclobutrazol in the sample to be tested, the colloidal gold particles will bind to the paclobutrazol antigen coated on the membrane when chromatographed to the test line position, and the T line will develop color. When paclobutrazol is present in the sample to be tested, it will bind to the gold-labeled specific monoclonal antibody to form an immune complex. When the complex is chromatographed to the test line position, it will not bind to the paclobutrazol coated antigen coated on the membrane, and the T line will not develop color or the color will become lighter.

[0030] (1) Testing steps: The method is as follows: take 200 μL of fruit and vegetable extract in the microwell, slowly aspirate and mix with the colloidal gold-labeled paclobutrazol monoclonal antibody in the microwell, incubate at 20-25℃ for 3 minutes, then take 100 μL of the mixture and drop it into the sample well of the test paper card, and read the result after chromatography at 20-25℃ for 5 minutes. If the T line and the C line show purple-red bands at the same time and the T line is stronger than the C line, the result is negative; if the T line is lighter than the C line or the C line is colored but the T line is not colored, the result is positive; if both the C line and the T line are not colored, the detection reagent has expired.

[0031] (2) Sensitivity test of paclobutrazol colloidal gold test strips: The fruit and vegetable extracts were spiked to a final concentration of 1, 3, 5, and 10 μg / kg. Figure 3 As shown, the sensitivity of the paclobutrazol colloidal gold detection device of the present invention can reach 3-4 μg / kg, and the CV value is less than 15%.

[0032] (3) Specificity detection of paclobutrazol colloidal gold test strips: In the negative fruit and vegetable extracts, 500 μg / kg of triadimenol, triazophos, triadimefon and other drugs were added to verify the specificity of the test strips. The specificity test results are shown in Figure 4 The test results were all negative, indicating that paclobutrazol has excellent specificity and no cross-reaction with the above added drugs.

[0033] (4) Product stability test The paclobutrazol colloidal gold product of the present invention was subjected to accelerated tests at 4°C and 37°C, and the paclobutrazol standard product 1 μg / kg was detected at 0d, 7d, 14d, and 30d, respectively, to evaluate the stability performance of the product. The results are shown in Table 1. When aged to 30 days, the paclobutrazol colloidal gold detection product at 4°C and 37°C still had good sensitivity in detecting the 1 μg / kg standard product, and there was no significant difference from the test results on the first day, and the CV was <10%, indicating that the test strip provided by the present invention has good stability.

[0034] Table 1 Stability of paclobutrazol colloidal gold test strips

[0035] In summary, the colloidal gold test strip of the present invention has the characteristics of high sensitivity, strong specificity and good stability, and can be applied to the colloidal gold immunochromatography method to quickly detect paclobutrazol residues in fruit and vegetable extracts.

Claims

1. A paclobutrazol hapten, characterized in that: The structure of the paclobutrazol hapten is shown in Formula I, Formula I.

2. A method for preparing the paclobutrazol hapten according to claim 1, characterized in that: S1. Accurately weigh the paclobutrazol raw material, dissolve it in anhydrous dichloromethane, protect with nitrogen, then cool to 0°C, add succinyl chloride, slowly add aluminum chloride, maintain the temperature at 45°C, and stir to react for 2 hours; S2. After 2 hours, the mixed solution obtained in step S1 was monitored by TLC to confirm that the reaction was complete, cooled to 0°C, and purified water was added dropwise for quenching, separated, the organic phase was retained, and 10% sodium carbonate was added for extraction twice, the aqueous phase was retained, the acid was adjusted to free, and ethyl acetate was extracted twice, the organic phases were combined, and dried over anhydrous sodium sulfate to obtain a crude paclobutrazol hapten; S3, purification: the crude product obtained in step S2 is purified on a reverse phase FLASH-C18 column to obtain paclobutrazol hapten.

3. A complete paclobutrazol antigen, characterized in that: The paclobutrazol hapten according to claim 1 is coupled with a carrier protein through a coupling technology, wherein the coupling technology is EDC / NHS coupling technology or a mixed anhydride method, and the carrier protein is any one of bovine serum albumin and chicken ovalbumin.

4. A method for preparing the complete paclobutrazol antigen according to claim 3, characterized in that: The method is an EDC / NHS coupling technology, and when the carrier protein is bovine serum albumin (BSA) or chicken ovalbumin (OVA), the method comprises: The paclobutrazol hapten described in claim 1 is dissolved in DMF, NHS and EDC are slowly added, and the mixture is stirred at room temperature for 6 hours to obtain a paclobutrazol hapten activation solution; BSA or OVA is weighed and dissolved in a PBS buffer (0.01 mol / L, pH 7.4), stirred evenly, and slowly added dropwise to the activated hapten solution, and stirred at room temperature for 16 hours; the obtained reaction solution is dialyzed with a 0.01 mol / L PBS buffer having a pH of 7.2, during which the buffer is changed every 12 hours, and the dialyzation is performed for 72 hours to obtain a paclobutrazol complete antigen with a carrier protein of BSA or OVA.

5. A method for preparing the complete paclobutrazol antigen according to claim 3, characterized in that: The method is a mixed anhydride method. When the carrier protein is BSA or OVA, the method comprises: The paclobutrazol hapten described in claim 1 is dissolved in 2 mL of anhydrous methanol, 300 μL of tri-n-butylamine is added dropwise, and the mixture is stirred at 4°C for 30 min; 60 μL of isobutyl chloroformate is added, and the mixture is stirred for 1 h; BSA or OVA is weighed, and the mixture is slowly added into the above solution, and the mixture is stirred for 4 h; the mixture is dialyzed with 0.01 mol / L PBS having a pH of 7.2, during which the water is changed every 2 h, and the dialyzation is performed for 48 h to obtain the paclobutrazol complete antigen with BSA or OVA as the carrier protein.

6. Provide a paclobutrazol colloidal gold detection product, characterized in that The nitrocellulose membrane is coated with the complete paclobutrazol antigen according to claim 4 or claim 5.

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