CN104833781B - Magnetic molecular imprinting bionic ELISA (enzyme-linked immuno sorbent assay) detecting method of malachite green - Google Patents

Abstract

The invention discloses a magnetic molecular imprinted bionic ELISA detection method of aquatic product malachite green, which includes the preparation of magnetic molecular imprinted polymer and the establishment of direct competition ELISA method and other experimental steps. Malachite Green Magnetic Molecularly Imprinted Polymers (MG‑MMIPs) with uniform diameter were investigated, and their adsorption properties were investigated. Using this MG‑MMIPs as a biomimetic antibody, it was used to competitively adsorb MG antigens and enzyme-labeled MG antigens to establish direct competition ELISA detection method. The sensitivity of the standard curve established by this method under the optimal reaction conditions is 20.14μg L ^‑1 , and the minimum detection limit is 0.12μg L ^‑1 , which is 2 ppb(ng/g) lower than the detection limit of the malachite green rapid test card At the same time, the method has high selectivity for MG, and the cross-reactivity rates for two structural analogs of MG (methyl violet and brilliant green) are 7.4% and 3.9%, respectively.

Description

A magnetic molecular imprinting bionic ELISA detection method for aquatic product malachite green

technical field

The invention relates to a relatively large range, in particular to a magnetic molecular imprinting bionic ELISA detection method of malachite green, an aquatic product.

Background technique

Malachite Green (Malachite Green, MG) is a triphenylmethane substance, also known as base block green, basic malachite green, malachite green, basic green, Chinese green or aniline green. Green crystalline solid. Malachite green has been used in many industries, such as leather industry, food dyeing industry, pottery industry, printing and dyeing industry, etc. Since MG can play a very good role in the prevention and treatment of aquatic animal diseases, it has been widely used in aquaculture since 1936. Malachite green can also be used as a fungicide, which has a good effect on preventing the growth of mold in fish eggs and the secondary pollution of fungi. However, malachite green has the disadvantages of high toxicity, high residue, high carcinogenicity and mutagenesis. In May 2002, our country designated it as an illegal drug for aquatic organisms, but because of its low price and remarkable bactericidal effect, there are still illegal merchants still using it. Because it can achieve a good bactericidal effect with a small amount of addition, it brings certain difficulties to the detection of MG in aquatic products. Therefore, it is urgent and of great practical significance to establish a detection method for MG residues.

At present, the detection methods of MG mainly include high performance liquid chromatography, liquid chromatography-mass spectrometry, and gas chromatography-mass spectrometry. These methods have the advantages of reliable results, high sensitivity, high selectivity and good repeatability in the determination and analysis of malachite green residues in aquatic products. However, these methods are cumbersome and time-consuming in the sample pretreatment process, and the equipment is expensive and the detection cost is high; not only need to consume a lot of solvent and a lot of time, but also easily cause secondary pollution, affecting the accuracy of the test results . Therefore, it is of great significance to develop a fast and efficient detection method for MG to control the quality and safety of aquatic products.

Molecularly imprinted polymers have the remarkable characteristics of predetermined structure and activity, specific recognition and wide applicability, while enzyme-linked immunoassay is fast and sensitive. Combining the two, using synthetic molecularly imprinted polymers as biomimetic antibodies not only has selective specificity, but also can be reused.

Contents of the invention

The purpose of the present invention is to provide a bionic ELISA detection method of magnetic molecular imprinting of aquatic product malachite green, so as to solve the problems raised in the above-mentioned background technology.

To achieve the above object, the present invention provides the following technical solutions:

A magnetic molecular imprinting bionic ELISA detection method for aquatic product malachite green, the specific steps are as follows:

(1) Synthesis of Fe ₃ O ₄ : Weigh 0.30-0.36g polyethylene glycol, add 7-9mL ultrapure water, sonicate until polyethylene glycol is completely dissolved, weigh 2.00-2.06g FeCl ₃ and add to polyethylene glycol Ultrasonic dissolve in diol solution, then weigh 1.4~1.6g FeCl ₂ and add to the above solution until completely dissolved, then add 0.4~0.6mL0.1mol L ^-1 dilute hydrochloric acid immediately, pass nitrogen to deoxygenate for 12~18min, under 60℃ water bath Quickly add 55-65mL of ammonia water while stirring, adjust the speed to 1500r min ^-1 , react for 1h, adsorb with a magnet, elute with ultrapure water for 4-6 times, freeze-dry in vacuum and store for later use;

(2) Preparation of pre-polymerization solution: Add 0.08-0.12g Fe ₃ O ₄ into 0.8-1.2mL oleic acid and stir evenly with a glass rod, add 8-12mL 0.05mol L ^-1 MG chloroform solution and 43-170μL functional monolayer body, stirred at a speed of 400r min ^-1 for 25-35min, and stood at 4°C for 1.5-2.5h to form A liquid;

(3) Preparation of pre-emulsified solution: add 0.4-0.6 mL of Span-80 and tween-80 to 22-28 mL of ultrapure water, then add 475-950 μL of cross-linking agent, and stir at 400 r min ^-1 for 25-35 min Form B liquid;

(4) Preparation of MG magnetic molecularly imprinted polymer: slowly add liquid A to liquid B under stirring at 400r min ^-1 , deoxygenate with nitrogen for 18-22min, raise the temperature to 60°C, add 0.08-1.2g Azobisisobutyronitrile initiates polymerization for 16-20 hours. After the reaction is completed, use 22-28 mL of methanol to break the emulsification. After suction filtration, a coarse malachite green magnetic molecularly imprinted polymer is obtained. The product uses a volume ratio of 8-10:1 methanol- Soxhlet extraction with acetic acid mixture until no malachite green template is washed out, then washed with methanol until neutral, finally washed with ultrapure water to remove methanol, vacuum freeze-dried for later use;

(5) Synthesis of hapten: Dissolve 850-950mg of 4-formylbenzoic acid, 2.2-2.6mL of N,N-dimethylaniline and 2.2-2.6g of anhydrous ZnCl ₂ in 60mL of absolute ethanol. Heating under reflux for 22-26 hours, after cooling at room temperature, add 28-32mL of methanol, dropwise add ammonia water until precipitation occurs, filter and rinse with water, dry with KOH in vacuum to obtain carboxylated recessive malachite green; chloroform dissolves carboxyl groups The recessive malachite green, add tetrachloro-p-benzoquinone and glacial acetic acid, stir and react at 25℃ for 1-2h, the reaction product is washed 2-3 times with an equal volume of chloroform-tetrachloromethane, and vacuum-dried to obtain half Antigen carboxylated malachite green powder;

(6) Synthesis of enzyme-labeled antigen: Dissolve 1.8-2.2 mg of hapten carboxylated malachite green CMG powder in 0.8-1.2 mL of 20% N,N-dimethylformamide solution, and add 1.8-2.2 mg mL ^-1 EDC 32-24 μL, adjust pH to 4.5-5.0, react for 18-22 minutes, quickly add 7.0-8.0 mg horseradish peroxidase HRP, adjust pH to 7.8-7.2, react at room temperature for 2.5-3.5 hours to obtain CMG -HRP conjugates, stored at 20°C;

(7) Detection: Take 18-22 mg of magnetic molecularly imprinted polymers and place them in a 10-mL centrifuge tube, add 2.8-3.2 mL of MG-PBS buffer solution with a concentration of 0.1-10000 μgL ^-1 , and then immediately add a dilution of 1:1400-1600 2.8-3.2 mL of the enzyme-labeled antigen solution, and the centrifuge tube without MG solution was used as the control group, which was shaken and adsorbed in an ice bath for 1.5-2.5 h; After 35 min, 45-55 μL of stop solution was added to each tube, read on a microplate reader immediately, and calculated the inhibition rate of MG on the antigen-antibody binding reaction at each concentration.

As a further solution of the present invention: the functional monomer in the step (2) is α-methacrylic acid, acrylamide or acrylic acid.

As a further solution of the present invention: the crosslinking agent in the step (3) is ethylene glycol dimethacrylate, divinylbenzene or trimethylolpropane trimethacrylate.

As a further solution of the present invention: the substrate color developing solution in the step (7) is o-phenylenediamine.

As a further solution of the present invention: the stop liquid in the step (7) is 2mol L ⁻¹ sulfuric acid.

Compared with the prior art, the beneficial effects of the present invention are: the magnetic molecularly imprinted polymer of the present invention has the remarkable characteristics of specific recognition and fast separation of solid and liquid, and the enzyme-linked immunoassay has the characteristics of rapidity and sensitivity, and the two Combining them, synthetic molecularly imprinted polymers are used as biomimetic antibodies, which not only have selective specificity, but also can be reused.

Description of drawings

Fig. 1 is the scanning electron microscope picture of magnetic molecularly imprinted polymer in the present invention;

Fig. 2 is the Fourier transform infrared spectrogram of the magnetic molecularly imprinted polymer in the present invention;

Fig. 3 is the hysteresis loop of the magnetic molecularly imprinted polymer in the present invention;

Figure 4 and Figure 5 are thermogravimetric diagrams of magnetic molecularly imprinted polymers in the present invention;

Fig. 6 is the ultraviolet spectrogram of enzyme-labeled antigen in the present invention;

Fig. 7 is the hapten CMG infrared spectrogram in the present invention;

Fig. 8 is MG direct competition ELISA standard curve in the present invention;

Figure 9 shows the consistency of the results of the determination of MG in samples by ELISA and HPLC.

detailed description

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Embodiment of the invention

A magnetic molecular imprinting bionic ELISA detection method for aquatic product malachite green, the specific steps are as follows:

(1) Synthesis of Fe ₃ O ₄ : Weigh 0.33g of polyethylene glycol, add 8mL of ultrapure water, ultrasonicate until the polyethylene glycol is completely dissolved, weigh 2.03g of FeCl ₃ and add it to the polyethylene glycol solution for ultrasonic dissolution , then weighed 1.5g FeCl ₂ and added it to the above solution until it was completely dissolved, then immediately added 0.5mL 0.1mol L ^-1 dilute hydrochloric acid, passed nitrogen to remove oxygen for 15min, and quickly added 60mL ammonia water while stirring in a water bath at 60°C, and adjusted the speed to 1500r min ^-1 , reacted for 1 hour, adsorbed with a magnet, eluted with ultrapure water for 5 times, and stored in vacuum after freeze-drying;

(2) Preparation of pre-polymerization solution: add 0.1g Fe ₃ O ₄ into 1mL oleic acid and stir evenly with a glass rod, add 10mL 0.05mol L ^-1 MG chloroform solution and 110μL functional monomer, and then add 0.05mol L ^-1 MG chloroform solution and 110μL functional monomer, and then add 0.1g Fe 3 O 4 into 1mL oleic acid and stir evenly with a glass rod Stir at low temperature for 30 minutes, and stand at 4°C for 2 hours to form A liquid;

(3) Preparation of pre-emulsified solution: Add 0.5 mL of Span-80 and tween-80 to 25 mL of ultrapure water, then add 700 μL of cross-linking agent, and stir at 400 r min ^-1 for 30 min to form B liquid;

(4) Preparation of MG magnetic molecularly imprinted polymer: slowly add liquid A to liquid B under stirring at 400r min ^-1 , deoxygenate with nitrogen for 20min, raise the temperature to 60°C, add 0.1g of azobisiso Butyronitrile initiated the polymerization for 18 hours. After the reaction was completed, 25 mL of methanol was used to break the emulsification. After suction filtration, the crude malachite green magnetic molecularly imprinted polymer was obtained. The product was Soxhlet-extracted with a methanol-acetic acid mixture with a volume ratio of 9:1 until no malachite was present. The green template was washed out, and then washed with methanol until neutral, and finally the methanol was washed away with ultrapure water, and then vacuum freeze-dried for later use;

(5) Synthesis of hapten: Dissolve 900mg of 4-formylbenzoic acid, 2.4mL of N,N-dimethylaniline and 2.4g of anhydrous ZnCl ₂ in 60mL of absolute ethanol, heat and reflux for 24h under nitrogen protection, room temperature After cooling, add 30mL of methanol, add ammonia water dropwise until precipitation occurs, filter, rinse with water, and dry with KOH in vacuum to obtain carboxylated recessive malachite green; chloroform dissolves carboxylated recessive malachite green, add four Chloro-p-benzoquinone and glacial acetic acid were stirred and reacted at 25°C for 1.5 hours, the reaction product was washed twice with an equal volume of chloroform-tetrachloromethane, and vacuum-dried to obtain hapten carboxylated malachite green powder;

(6) Synthesis of enzyme-labeled antigen: Dissolve 2 mg of hapten carboxylated malachite green CMG powder in 1 mL of 20% N,N-dimethylformamide solution, add 2 mg mL ^-1 EDC 33 μL while magnetically stirring, and adjust the pH to 4 .5～5.0, after reacting for 20 minutes, quickly add 7.5 mg of horseradish peroxidase HRP, adjust the pH to 7.0, react at room temperature for 3 hours to obtain the CMG-HRP conjugate, and store in a freezer at 20°C;

(7) Detection: Take 20 mg of magnetic molecularly imprinted polymer and place it in a 10 mL centrifuge tube, add 3 mL of MG-PBS buffer solution with a concentration of 0.1-10000 μgL ^-1 , and then immediately add 3 mL of enzyme-labeled antigen solution with a dilution ratio of 1:1500, The centrifuge tube without MG solution was used as the control group, and was shaken and adsorbed in an ice bath for 2 h; the upper layer solution was discarded, and 150 μL of the substrate color development solution o-phenylenediamine was added to each tube, and 50 μL of stop solution 2 mol L ^-1 was added to each tube after color development for 30 min. Sulfuric acid, read on the microplate reader immediately, calculate the inhibition rate of MG to the antigen-antibody binding reaction at each concentration,

Inhibition rate (B/B ₀ ) = sample absorbance (B)/negative absorbance (B ₀ );

Draw the MG direct competition ELISA standard curve as shown in Figure 5.

Preferably, the functional monomer in the step (2) is α-methacrylic acid, acrylamide or acrylic acid.

Preferably, the crosslinking agent in the step (3) is ethylene glycol dimethacrylate, divinylbenzene or trimethylolpropane trimethacrylate.

As shown in Figure 1, the scanning electron microscope images of the prepared magnetic molecularly imprinted polymer (left) and non-imprinted polymer (right) show that the morphology of the polymer is regular and the particle size is uniform, about 600nm.

It can be seen from Figure 2 that the infrared spectra of MIP and NIP are basically consistent, which indicates that the template molecules are almost completely eluted. The main functional groups in the polymer can be reflected by the corresponding vibration peaks, among which the stretching vibration peak of the methyl group (-CH ₃ ) is at 2959 cm ^-1 , and the stretching vibration peak of the carbonyl group (C=O) of the polymer is at 1737 cm ^-1 , 1641cm ^-1 is the stretching vibration peak of the double bond (C=C) in the polymer, and 584cm ^-1 is the stretching vibration peak of the Fe-O bond.

It can be seen from Figure 3 that the magnetic molecularly imprinted polymer has very good superparamagnetism, and the saturation magnetic intensity is 54.1emu/g. It can be seen that the prepared MMIPs can be rapidly separated from the sample under the action of an external magnetic field.

It can be seen from Figure 4 and Figure 5 that when the temperature is lower than 265°C, NIP is in a relatively stable state with almost no mass loss; when the temperature rises to 265°C, the mass of NIP begins to decrease a lot, when the temperature reaches At 405°C, the mass reduction of NIP reaches 80%; MIP is very stable when the temperature is lower than 268°C, and there is almost no mass loss; when the temperature is higher than 268°C, mass loss begins, and when the temperature reaches 412°C, the mass The reduction reached 70%, indicating that the magnetic molecularly imprinted polymer has good thermal stability.

As can be seen from Figure 6, CMG has characteristic absorption peaks at 629, 429, 320, and 251nm, HRP has characteristic absorption peaks at 403 and 268nm, and enzyme-labeled antigen has characteristic absorption peaks at 628, 291, and 226nm, while CMG and Some characteristic absorption peaks of HRP disappeared, indicating that the enzyme-labeled antigen was successfully coupled.

As shown in Figure 7, the main functional groups in carboxylated malachite green can be reflected by the corresponding vibration peaks, in which 3462cm ^-1 is the stretching vibration peak of OH, and 1559cm ^-1 and 1449cm ^-1 are the C= on the benzene ring. The stretching vibration peaks of C, 955cm ^-1 and 691cm ^-1 are the stretching vibrations of the benzene ring.

The specificity verification of the established ELISA method: select malachite green and its structural analogs methyl violet and brilliant green as analytes, establish a dcELISA standard curve, obtain the corresponding IC50 values, and calculate the cross-reactivity rate (CR), as The results in Table 1 show that the prepared magnetic molecularly imprinted polymer has a high selectivity to malachite green.

Table 1 _IC50 values and cross-reactivity ratios of MG and its analogues

Addition recovery experiment of actual samples: 5 μg L ^-1 , 10 μg L ^-1 , 50 μg L ^-1 malachite green standard solution was added to sea bass and prawn samples, and then the established dcELISA was used to determine the recovery rate of the samples, as shown in Table 2 The experimental results shown show that the established method can be applied to the actual sample detection, and the detection effect is better.

Table 2 Addition and recovery experiment of MG in samples by ELISA method

Validation of the accuracy of the ELISA method: 5 μg L ^-1 , 10 μg L ^-1 , 50 μg L ^-1 malachite green standard solutions were added to sea bass and prawn samples, and then directly injected by high performance liquid chromatography for determination, as The measurement results shown in Table 3 and Figure 8 show that the established ELISA method and HPLC method have relatively good consistency in the determination results of the perch and prawn samples added with different concentrations of MG, and the established biomimetic ELISA detection The method has high accuracy and practicability.

Table 3 ELISA method and HPLC method to the addition recovery determination result of MG in the sample

Claims (5)

1. a kind of magnetic molecularly imprinted bionical ELISA detection method of aquatic products malachite green oxalate, it is characterised in that concrete steps are such as Under：

(1)Fe₃O₄Synthesis：0.30～0.36g Polyethylene Glycol is weighed, 7～9mL ultra-pure waters are added, ultrasound is until Polyethylene Glycol is complete CL, weighs 2.00～2.06g FeCl₃Ultrasonic dissolution in polyglycol solution is added to, then weighs 1.4～1.6g FeCl₂ It is added in above-mentioned solution to being completely dissolved, adds 0.4～0.6mL 0.1mol L immediately^-1Dilute hydrochloric acid, letting nitrogen in and deoxidizing 12～ 18min, is rapidly added 55～65mL ammonia under 60 DEG C of water-baths while stirring, turns speed to 1500r min^-1, 1h is reacted, Magnet is used Adsorbed, and with ultrapure water elution 4～6 times, saved backup after vacuum lyophilization；

(2) preparation of pre-polymer solution：By 0.08～0.12g Fe₃O₄Stir in adding 0.8～1.2mL Oleic acid and with Glass rod Uniformly, 8～12mL 0.05mol L are added^-1MG chloroformic solutions and 43～170 μ L function monomers, in 400r min^-1Rotating speed under 25～35min of stirring, stands 1.5～2.5h and forms A liquid at 4 DEG C；

(3) preparation of pre-emulsification solution：Span-80 and tween-80 each 0.4～0.6mL is added in 22～28mL ultra-pure waters, 475～950 μ L cross-linking agent are added, in 400r min^-125～35min of lower stirring forms B liquid；

(4) preparation of MG magnetic molecularly imprinted polymers：In 400r min^-1Under rotating speed stirring, A liquid is slowly added in B liquid, Temperature is raised to 60 DEG C by nitrogen 18～22min of deoxygenation, adds 0.08～1.2g azodiisobutyronitriles to cause 16～20h of polymerization, instead With 22～28mL methanol breakdowns of emulsion after should terminating, thick malachite green oxalate magnetic molecularly imprinted polymer, product volume after sucking filtration, is obtained Than for 8～10:To washing out without malachite green oxalate template, again with methanol is washed till neutrality to 1 methanol-acetic acid mixed liquor surname extraction, most Methanol is washed away with ultra-pure water afterwards, it is standby after vacuum lyophilization；

(5) haptenic synthesis：By 850～950mg 4- formylbenzoates, 2.2～2.6mL N, accelerine and The anhydrous ZnCl of 2.2～2.6g₂It is dissolved in 60mL dehydrated alcohol, under nitrogen protection, is heated to reflux 22～26h, after room temperature cooling, 28～32mL methanol, Deca ammonia is added to be rinsed with water after filtration, be dried with KOH in vacuum and obtain carboxylic to generation is precipitated The concealed malachite green of base；Chloroform dissolves carboxylated concealed malachite green, adds chloranil, 25 DEG C of glacial acetic acid 1～2h of lower stirring reaction, the isopyknic chloroform of product Jing-tetrachloromethane are washed 2～3 times, and vacuum drying can be obtained The carboxylated malachite green oxalate powder of hapten；

(6) synthesis of enzyme-labelled antigen：1.8～2.2mg hapten carboxylated malachite green oxalate CMG powder is dissolved in into 0.8～1.2mL 20%N, dinethylformamide solution add 1.8～2.2mg mL in magnetic agitation^-132～24 μ L of EDC, adjust pH 4.5 ～5.0, after 18～22min of reaction, 7.0～8.0mg horseradish peroxidase HRP are rapidly added, adjust pH to 7.8～7.2, room temperature 2.5～3.5h of reaction obtains CMG-HRP conjugates, 20 DEG C of freezen protectives；

(7) detect：Take 18～22mg magnetic molecularly imprinted polymers to be placed in 10mL centrifuge tubes, add concentration to be 0.1～10000 μgL^-1MG-PBS 2.8～3.2mL of buffer solution, immediately after add dilution factor be 1：1400～1600 enzyme-labelled antigen solution 2.8～3.2mL, be not added with MG solution centrifuge tube be matched group, ice bath vibration absorption 1.5～2.5h；Upper solution is abandoned, often pipe adds Enter 140～160 μ L substrate nitrite ions, often pipe adds 45～55 μ L terminate liquids after 25～35min of colour developing, reads immediately in microplate reader Number, calculates suppression ratio of the MG to antigen-antibody binding reaction under each concentration.

2. the magnetic molecularly imprinted bionical ELISA detection method of aquatic products malachite green oxalate according to claim 1, its feature It is that the function monomer in step (2) is α-methacrylic acid, acrylamide or acrylic acid.

3. the magnetic molecularly imprinted bionical ELISA detection method of aquatic products malachite green oxalate according to claim 1, its feature It is that the cross-linking agent in step (3) is ethylene glycol dimethacrylate, divinylbenzene or trimethylol propane trimethyl Acrylate.

4. the magnetic molecularly imprinted bionical ELISA detection method of aquatic products malachite green oxalate according to claim 1, its feature It is that the substrate nitrite ion in step (7) is o-phenylenediamine.

5. the magnetic molecularly imprinted bionical ELISA detection method of aquatic products malachite green oxalate according to claim 1, its feature It is that the terminate liquid in step (7) is 2mol L^-1Sulphuric acid.