CN112067799B - Immunomagnetic adsorbent based on phenylboronic acid-directed conjugated antibody and preparation method thereof - Google Patents

Abstract

The present invention relates to an immunomagnetic adsorbent based on phenylboronic acid directional conjugated antibody and a preparation method thereof. The immunomagnetic adsorbent utilizes the specific affinity between the cis-diol structure in the Fc fragment glycoprotein molecule of the antibody and the phenylboronic acid group. To achieve directional coupling of antibodies on the surface of the magnetic carrier. Compared with the traditional method, this method maximizes the utilization rate of the Fab end of the antibody, thereby improving the affinity and adsorption capacity of the immunomagnetic adsorbent for the target, and can effectively control the batch-to-batch difference, which is worthy of further promotion. The prepared immunomagnetic adsorbent can be used for the detection and pretreatment of various food samples, and can be used for the enrichment and purification of hazardous substances such as mycotoxins, pesticide and veterinary drug residues, heavy metals and pathogenic microorganisms in food samples according to the difference of the conjugated antibodies. purification. The purified hazardous substances can be quantitatively or qualitatively detected by methods such as high performance liquid chromatography, immunoassay and enzyme-linked immunosorbent technology.

Description

Immunomagnetic adsorbent based on phenylboronic acid-directed conjugated antibody and preparation method thereof

technical field

The invention belongs to the technical field of food safety detection and pretreatment, and in particular relates to an immunomagnetic adsorbent based on phenylboronic acid directional conjugated antibody and its preparation.

Background technique

In the detection of food hazard factors, pretreatment steps such as sample separation and purification are indispensable and are the key link affecting the reliability of the analysis results. Traditional sample pretreatment methods, such as liquid-phase extraction and solid-phase extraction, are cumbersome to operate and have poor specificity, which affects the detection accuracy. Immunoaffinity chromatography is based on the specific recognition between the ligand and the object to be tested, and its accuracy is good, but the cost is high and time-consuming. The method based on immunomagnetic adsorbent has developed rapidly in the field of sample pretreatment for food safety testing due to its advantages of good specificity, rapidity and high efficiency.

The immunomagnetic adsorbent is composed of a superparamagnetic magnetic carrier and an antibody modified on the magnetic carrier. The target object to be detected is captured on the surface of the magnetic carrier through the specific recognition of the antibody, and then under the action of an external magnetic field, the object to be detected is realized. enrichment and separation.

The basic principle of the current immunomagnetic adsorbent preparation method is usually to use 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) or glutaraldehyde as a coupling reagent to combine the antibody Modified on the surface of carboxylated or aminated magnetic carriers. However, this method has an obvious defect, that is, due to the large number of amino groups at the Fab end (antigen recognition region) and Fc end of the antibody, the orientation of the antibody on the surface of the magnetic carrier is random and uncontrollable. This random orientation reduces the exposure of the Fab end. The number of antibodies reduces the effective utilization of antibodies, resulting in large amounts of antibodies, high cost, low magnetic separation efficiency, and low magnetic separation repeatability and stability.

Therefore, a method of directional coupling of the Fc end of the antibody to the magnetic carrier is developed, so that the Fab end of the antigen recognition end is exposed, reducing the steric hindrance of the binding of the antigen and the antibody, thereby improving the antigen affinity of the immunomagnetic adsorbent and improving the antigen of the test object. High enrichment efficiency, effective control of batch-to-batch difference, and simultaneous reduction of costs are technical problems to be solved urgently in this field.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an immunomagnetic adsorbent based on phenylboronic acid directional conjugated antibody and a preparation method thereof, aiming at the defects of the prior art.

For achieving the above object, the technical scheme adopted in the present invention is:

The present invention provides an immunomagnetic adsorbent based on phenylboronic acid-directed conjugated antibodies, comprising a magnetic carrier and an antibody coupled to the magnetic carrier. The structure-specific affinity of the cis-diol in the directed coupling on the surface of the magnetic carrier.

Further, the magnetic carrier is any superparamagnetic micro-nano particle, preferably the magnetic carrier is a polymer microsphere or a silica microsphere wrapped with Fe ₃ O ₄ nanoparticles.

Further, all or part of the surface of the magnetic carrier is phenylboronic acid groups.

The present invention also provides a method for preparing an immunomagnetic adsorbent based on phenylboronic acid directional coupling antibody. First, a magnetic carrier with surface-modified phenylboronic acid group is prepared, and then the phenylboronic acid group is combined with the glycoprotein molecule on the antibody Fc fragment. The specific affinity of the cis-diol structure in the target immobilizes the antibody on the magnetic carrier. The specific process steps are as follows:

The first step, the preparation of phenylboronated magnetic carrier, the scheme can be one of the following two.

One of the preparation schemes of the magnetic carrier is to synthesize the magnetic carrier modified with the surface carboxyl group or amino group, and then carry out the phenylboronation modification on its surface. For example, by using EDC as a coupling agent to modify aminophenylboronic acid on the surface of carboxylated magnetic beads to prepare phenylboronated magnetic carriers; or by using EDC as a coupling agent to modify 4-carboxyphenylboronic acid on the surface of aminated magnetic beads to prepare phenylboronic acid Magnetic carrier.

The second preparation scheme of magnetic carrier, 1) see the existing literature to synthesize oil-soluble magnetic nanoparticles; 2) then pass common polymers such as polystyrene maleic anhydride (PSMA), polymaleic anhydride-octadecene copolymer ( PMAO) etc. to modify phenylboronic acid groups to prepare polymers containing phenylboronic acid groups, for example, acylation of 3-aminophenylboronic acid and polymaleic anhydride octadecyl ester under the catalysis of 4-dimethylaminopyridine Reaction to synthesize phenylboronic acid-containing polymer; 3) Then encapsulate magnetic nanoparticles in a phenylboronic acid group-containing polymer by emulsification-emulsion solvent evaporation method to prepare a phenylboronic acid magnetic microsphere carrier, specifically, an oil-soluble magnetic microsphere carrier is prepared Magnetic nanoparticles, polymers, certain volatile non-polar solvents such as toluene, chloroform, etc. are used as the oil phase, and an aqueous solution containing surfactants such as sodium lauryl sulfate is used as the water phase, and water is prepared by emulsification by ultrasound, stirring, etc. oil-in-oil emulsion, and then the non-polar solvent in the emulsion droplets is removed by volatilization or rotary evaporation to obtain the phenylboronated magnetic carrier.

The second step is the directional coupling of the antibody to the magnetic carrier. The phenylboronated magnetic carrier synthesized in the first step and the antibody were mixed in a buffer (0.02M phosphate buffer, pH 5.5-8.0, and the optimal pH of different antibodies was different) at room temperature and 25 ℃ for 10-20min to achieve the antibody lysis. Directional coupling, and then adding glucose or OVA solution to continue incubation at room temperature for 10 to 20 minutes to block the excess phenylboronic acid groups on the magnetic carrier.

The immunomagnetic adsorbent provided by the invention can be used for pre-detection treatment of various food samples, and can be used for the enrichment of hazardous substances such as mycotoxins, pesticide and veterinary drug residues, heavy metals and pathogenic microorganisms in food samples according to the difference of the conjugated antibodies. and purification. The purified hazardous substances can be quantitatively or qualitatively detected by high performance liquid chromatography, immunoassay and other methods.

Compared with the prior art, the beneficial effects of the present invention are:

Compared with the non-directionally conjugated immunomagnetic adsorbent of traditional antibodies, the immunomagnetic adsorbent based on phenylboronic acid directional conjugated antibody and its preparation provided by the present invention, the Fc end of the antibody is directionally coupled to the magnetic carrier, the Fab end is exposed, Reduce the steric hindrance of antigen and antibody binding, thereby improving the antigen affinity of the immunomagnetic adsorbent, improving the enrichment efficiency of the antigen to be tested, effectively controlling the difference between batches, and reducing the cost due to the reduction of antibody dosage.

Description of drawings

Fig. 1 is the synthesis schematic diagram of traditional immunomagnetic adsorbent;

Figure 2 is a schematic diagram of the synthesis of immunomagnetic adsorbents based on phenylboronic acid-directed conjugated antibodies.

Fig. 3 is a scheme of modification of phenylboronic acid by polystearyl maleic anhydride.

Detailed ways

The present invention will be further described in detail below with reference to specific embodiments, which are only used to illustrate the specific content of the present invention and are not intended to limit the scope of the present invention.

Example 1

The preparation of the immunomagnetic adsorbent based on phenylboronic acid-directed conjugated antibody Salmonella in this embodiment includes the preparation of phenylboronated magnetic carrier, the coupling of antibody and magnetic carrier, and the specific process steps are as follows:

1) preparation of phenylboronated magnetic carrier,

a. Synthesis of Fe ₃ O ₄ magnetic beads, add 300 mL of ultrapure water to a 500 mL three-necked flask, pass N ₂ to remove oxygen in the water, and preheat at 50 °C for 15 min at the same time. Add 3.2g FeCl ₂ ·H ₂ O, 5.2g FeCl ₃ magnetic stirring and mixing, then add 25mL ammonia water, the yellow solution quickly turns black, and the reaction is kept at 50℃ for 30min. The synthesized magnetic beads were separated by magnetic suction, washed with ultrapure water for 3-5 times until the pH of the solution reached neutral after magnetic suction, and then redissolved in 300 mL of ultrapure water.

b. Oleic acid modification of Fe ₃ O ₄ magnetic beads, add the magnetic beads synthesized in the previous step into a three-necked flask, pass N ₂ , and add 2.4 mL of oleic acid dropwise while stirring. Since the oleic acidified magnetic beads are hydrophobic, they are adsorbed on the stirrer or the wall of the flask. After 3 hours of reaction at 70 °C, the black solution gradually becomes clear. The reaction is stopped, the solution in the beaker is poured out, and 300 mL of ethanol is added to elute the adsorbed oleic acidified magnetic beads. After 2 min, the ethanol solvent was discarded, and this step was repeated 3-5 times until there was no oleic acid layer floating on the surface of the ethanol.

c. Synthesis of magnetic microspheres, synthesizing magnetic microspheres by emulsification-emulsion solvent evaporation method, dispersing the oleic acid magnetic beads synthesized in the previous step in 30 mL of cyclohexane to form an oil phase, 1.5 g of sodium dodecyl sulfate ( SDS) was dissolved in 500 mL of ultrapure water to form an aqueous phase, and the above mixture was sonicated for 10 min to form an oil-in-water microemulsion. Then placed at 60°C overnight to volatilize cyclohexane to obtain SDS-stabilized magnetic microspheres. It was re-dispersed in 60 mL of Tween-80 solution, and then Tween-80-modified magnetic microspheres were obtained by magnetic suction.

d. Synthesis of Fe ₃ O ₄ @SiO ₂ composite nano-microspheres, 1000 mL of ethanol, 30 mL of ammonium hydroxide, 20 mL of ultrapure water and 6 mL of LTEOS were pre-reacted at 30 °C for 20 min to pre-hydrolyze TEOS. Then, 60 mL of magnetic microspheres synthesized in the previous step were added to the mixing system, and by controlling the reaction time, magnetic microspheres coated with an appropriate thickness of _SiO2 layer were obtained.

e. The surface of Fe ₃ O ₄ @SiO ₂ composite nano-microspheres was modified by carboxylation. 10 mg of Fe ₃ O ₄ @SiO ₂ composite nano-microspheres synthesized in the previous step were added to 10 mL of ethanol, sonicated for 1 h to make the dispersion uniform, and 800 μL was added. 3-(triethoxysilyl)propyl succinic anhydride (TEPSA) reagent was modified on the surface of microspheres by ultrasonic for 1 h. After magnetic separation, wash with ultrapure water 3-5 times to remove ethanol and unreacted TEPSA reagent, then add alkaline ultrapure water with pH 9.0 and shake overnight to hydrolyze acid anhydride to generate carboxyl group.

f. Fe ₃ O ₄ @SiO ₂ @-COOH composite nanospheres were surface-modified with phenylboronic acid, and the carboxylated magnetic microspheres synthesized in the previous step were dispersed in EDC-containing buffer (0.02M phosphate buffer, pH 5.5 ), the activation reaction was carried out at room temperature of 25 °C for 30 min. Subsequently, the pH of the system was adjusted to 8.0, 3-aminophenylboronic acid/4-aminophenylboronic acid was added, and the coupling reaction was carried out at room temperature of 25 °C for 30 min. Then, glucosamine or OVA solution was added and incubated at room temperature for 10-20 min to block the remaining carboxyl groups on the magnetic carrier. After the reaction was completed, magnetic microspheres modified with phenylboronic acid were obtained by magnetic separation.

2) Conjugation of anti-Salmonella antibody and magnetic carrier:

2.1 Conjugation of anti-Salmonella antibody and phenylboronic acid magnetic carrier:

Take 1.5 mg of the phenylboronated magnetic carrier (15 mg/mL) synthesized in step 1) with a particle size of 180 nm and disperse it in a buffer (0.02 M phosphate buffer, pH 6) containing 100 μg of anti-Salmonella antibody (2 mg/mL), After 10 min incubation at room temperature 25°C. Add 10uL of glucose and continue to incubate at room temperature for 10min to block the remaining boronic acid groups on the magnetic carrier. After the reaction is completed, remove the supernatant by magnetic separation, and redissolve the magnetic beads in 0.1% BSA, 0.05% Tween-20, 0.01% NaN ₃ in PB7.4 buffer (concentration of 1 mg/mL), stored at 4°C for later use.

2.2 Conjugation of anti-Salmonella antibody and carboxylated magnetic carrier (this is the control of traditional method):

Take 1.5 mg of traditional carboxylated magnetic adsorbent (15 mg/mL) with a particle size of 180 nm and disperse it in a buffer (0.02 M phosphate buffer, pH 6) containing 220 μg anti-Salmonella antibody (2 mg/mL), and add 50 μg EDC to After 90 min incubation at room temperature 25°C. Add 10 mg BSA and continue to incubate at room temperature for 10 min to block the remaining carboxyl groups on the magnetic carrier. After the reaction is over, remove the supernatant by magnetic separation, and redissolve the magnetic beads in 0.1% BSA, 0.05% Tween-20, 0.01% NaN ₃ PB7.4 buffer (concentration of 1 mg/mL), stored at 4 °C for later use.

3) Enrichment of Salmonella by the above two immunomagnetic adsorbents

Add 20 mg of pork minced meat to 200 mL of medium and mix well. Inoculate a certain concentration of Salmonella, the temperature is 37 ℃, the time is 12h shaking culture. The bacterial concentration was adjusted to 10 ⁴ CFU/mL, 10 ⁵ CFU/mL, 10 ⁶ CFU/mL, and 10 ⁷ CFU/mL. Take 1 mL of bacterial liquid of each concentration and 1 mL of minced meat sample solution to be tested, add 10 μL of the two types of immunomagnetic adsorbents after blocking, and mix and incubate for 60 min at 37 °C and 15 rpm. After incubation, magnetic separation was performed for 35 min, the supernatant was discarded, washed with PBS, and reconstituted in PBS.

4) Visually inspect the samples and use the instrument to measure the results using the double-antibody sandwich method

Dilute the two kinds of immunomagnetic adsorbents that captured bacteria to a concentration of 50 μg/mL, and add 70 μL dropwise to the sample wells of commercial Salmonella immunochromatographic test strips, read and record with an immunochromatographic analyzer for 10 minutes. The values of T-line absorbance, C-line absorbance and T/C value, take the concentration of different bacteria as the abscissa, and draw the standard curve with the T-line absorbance and T/C value respectively. At the same time, qualitative analysis was carried out by observing the results with the naked eye. If the T line was colored, it indicated that there was bacteria in the sample, and the detection limit was about 10 ⁴ CFU/mL. 10 ⁴ CFU/mL.

Refer to the standard curve plot made to determine the amount of Salmonella in the sample. The results showed that the enrichment efficiencies of the EDC covalently modified immunomagnetic adsorbents reached 10 ⁴ CFU/mL, 10 ⁵ CFU/mL, 10 ⁶ CFU/mL and 10 ⁷ CFU/mL, respectively. The enrichment rate of phenylboronic acid-directed conjugated immunomagnetic adsorbents reached 58-63%, indicating that the enrichment effect of phenylboronic acid-directed conjugated immunomagnetic adsorbents was obvious under the condition of less antibody consumption. better.

Example 2

The preparation of the OTA immunomagnetic adsorbent based on the phenylboronic acid-directed conjugated antibody in this example includes the preparation of the phenylboronated magnetic carrier, the coupling of the antibody and the magnetic carrier, the enrichment and detection of OTA, and the specific application of the immunomagnetic adsorbent. The process steps are as follows:

1) Preparation of phenylboronated magnetic carrier

Steps a-d are the same as in Example 1.

e. The surface of Fe ₃ O ₄ @SiO ₂ composite nano-microspheres was modified by amination. 10 mg of Fe ₃ O ₄ @SiO ₂ composite nano-microspheres synthesized in the previous step were added to 10 mL of ethanol, ultrasonicated for 1 h to make the dispersion uniform, and 800 μL of 3 -Aminopropyl triethoxysilane (APTES) reagent, modified on the surface of the microspheres by ultrasonic for 1h, and washed with ultrapure water for 3-5 times after magnetic separation to remove ethanol and unreacted APTES reagent.

f. Fe ₃ O ₄ @SiO ₂ @-NH2 composite nanospheres were surface-modified with phenylboronic acid, first dissolve 2-carboxyphenylboronic acid/4-carboxyphenylboronic acid in a buffer containing EDC (0.02M phosphate buffer, pH is 5.5),

The reaction was activated at room temperature of 25°C for 30 min. Subsequently, the pH of the system was adjusted to 8.0, the aminated magnetic microspheres synthesized in the previous step were added, and the coupling reaction was carried out at room temperature of 25 °C for 30 min. After the reaction is completed, magnetic microspheres modified with phenylboronic acid are obtained by magnetic separation.

2) Coupling of anti-OTA antibody and magnetic carrier:

2.1 Coupling of anti-OTA antibody and phenylboronic acid magnetic carrier:

Take 1.5 mg of the phenylboronated magnetic adsorbent (15 mg/mL) synthesized in step 1) with a particle size of 180 nm and disperse it in a buffer (0.02 M phosphate buffer, pH 6) containing 50 μg of anti-OTA antibody (2 mg/mL). , after incubating for 10 min at room temperature 25 °C. Add 10uL of glucose and continue to incubate at room temperature for 10min to block the remaining boronic acid groups on the magnetic carrier. After the reaction is completed, remove the supernatant by magnetic separation, and redissolve the magnetic beads in 0.1% BSA, 0.05% Tween-20, 0.01% NaN ₃ in PB7.4 buffer (concentration of 1 mg/mL), stored at 4°C for later use.

2.2 Conjugation of anti-OTA antibody and carboxylated magnetic carrier:

Take 1.5 mg of carboxylated magnetic adsorbent (15 mg/mL) with a particle size of 180 nm and disperse it in a buffer (0.02 M phosphate buffer, pH 6) containing 300 μg anti-OTA antibody (2 mg/mL), and add 50 μg EDC at room temperature. After 90 min incubation at 25°C. Add 10 mg BSA and continue to incubate at room temperature for 10 min to block the remaining carboxyl groups on the magnetic carrier. After the reaction is over, remove the supernatant by magnetic separation, and redissolve the magnetic beads in 0.1% BSA, 0.05% Tween-20, 0.01% NaN ₃ PB7.4 buffer (concentration of 1 mg/mL), stored at 4 °C for later use.

3) Enrichment and detection of OTA by two immunomagnetic adsorbents:

Take 1mL (concentration of 1mg/mL) of the two prepared immunomagnetic adsorbents in a 5mL centrifuge tube, then add 1mL of the sample to be tested, and prepare the OTA standard with PBS buffer to the concentration of (10ng/mL, 25ng/mL, respectively). , 50ng/mL, 75ng/mL OTA solution as the sample to be tested, and PBS buffer as the blank sample to be tested), mix well, capture for 10min, remove the supernatant after magnetic separation. Finally, 1 mL of eluent was added to elute, the eluent was collected, and the content of OTA in the sample to be tested was detected by HPLC according to "GB5009.96-2016".

The results showed that when the amount of the standard substance was between 10ng, 25ng, 50ng and 75ng, the OTA enriched by EDC covalently modified immunomagnetic adsorbent eluted with the eluent was 8.60ng, 21.4ng, 44.0, and 65.7ng, respectively. The recovery rates of the carboxylated immunomagnetic adsorbents all reached 83-88%; the OTA enriched by the immunomagnetic adsorbents eluting the phenylboronic acid-directed conjugated antibodies with the eluent were 8.81ng, 22.2ng, 45.7, and 66.7ng, respectively; The recovery rates of immunomagnetic adsorbents of boronic acid-directed conjugated antibodies were all 88-92%. The above results show that the OTA enrichment effect of the phenylboronic acid-directed conjugated immunomagnetic adsorbent is better when the amount of antibody is less.

Add 5.0 μg/kg OTA to the blank PBS buffer, add 5 in parallel, and use the immunomagnetic adsorbent of phenylboronic acid-directed conjugated antibody. After the above steps, the addition recovery rate is 88.1%, 89.7%, 89.5% , 90.9%, 89.8%, indicating that the immunomagnetic adsorbent has good regeneration.

4) How to use the phenylboronic acid directional conjugated antibody immunomagnetic adsorbent:

4.1 Sample pretreatment

Weigh a sample of 10.0 g of corn (accurate to 0.01 g), add 50 mL of chloroform and 5 mL of 0.1 mol/L phosphoric acid aqueous solution, and vortex. Shake and extract on a shaker for 3 min to 5 min, filter the extract with qualitative filter paper, take 10 mL of the lower filtrate into a 100 mL flat-bottomed flask, rotate it in a 40°C water bath with a rotary evaporator until it is nearly dry, dissolve the residue with 20 mL of petroleum ether, and add 10 mL of it. The solution was extracted with a vortex shaker for 3 min to 5 min. After standing for stratification, the lower layer solution was taken out and filtered with filter paper.

4.2 Immunomagnetic sorbent capture of phenylboronic acid-directed conjugated antibodies

Take 2 mL of the immunomagnetic adsorbent in a 10 mL centrifuge tube, rinse with 5 mL of deionized water, and separate the washes with a magnetic separation rack each time; add 5 mL of the treated filtrate to the rinsed immunomagnetic adsorbent, mix well, Capture at room temperature for 10 min, separate the immunomagnetic adsorbent with a magnetic separation rack, and rinse the immunomagnetic adsorbent twice with 2 mL of deionized water

4.3 Immunomagnetic sorbent elution of phenylboronic acid-directed conjugated antibodies

Wash the immunomagnetic adsorbent of the phenylboronic acid-directed conjugated antibody with 1 mL of eluate, mix well, separate the immunomagnetic adsorbent with a magnetic separation rack, and recover the eluent, which is the OTA sample after enrichment and purification.

5) Determination

The HPLC reference conditions are listed below:

a) Chromatographic column: C1: column, column length 150mm, inner diameter 4.6mm, particle size 5μm, or equivalent column:

b) column temperature: 30C;

c) Injection volume: 10pL;

d) flow rate: 1mL./min;

e) Detection wavelength: excitation wavelength: 333nm, emission wavelength: 460nm;

f) Mobile phase and elution conditions:

Mobile phase: A: glacial acetic acid-water (2+100), B: acetonitrile;

Isocratic elution conditions: A-B (50+50)

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (2)

1. An immunomagnetic adsorbent based on phenylboronic acid-directed conjugated antibodies, comprising a magnetic carrier and an antibody coupled to the magnetic carrier, wherein the antibody passes through the phenylboronic acid group on the surface of the magnetic carrier and the antibody Fc fragment glycoprotein molecule The specific affinity of the cis-diol structure in the directional coupling on the surface of the magnetic carrier; The magnetic carrier is a polymer microsphere or a silica microsphere wrapped with Fe ₃ O ₄ nanoparticles; The nanoparticles are Fe ₃ O ₄ nanoparticles; All or part of the surface of the magnetic carrier is a phenylboronic acid group; The preparation method of the immunomagnetic adsorbent based on the phenylboronic acid directional conjugated antibody comprises the following steps: 1) Preparation of phenylboronated magnetic carriers: firstly synthesize oil-soluble magnetic nanoparticles, then modify the polymer with phenylboronic acid to prepare a polymer containing phenylboronic acid groups. Particles, polymers, and volatile non-polar solvents are used as the oil phase, and the aqueous solution containing surfactants is used as the water phase. The oil-in-water emulsion is prepared by emulsification by ultrasonication and stirring, and then the non-polar solvents in the emulsion droplets are evaporated or spun The method of steaming is removed to obtain a phenylboronated magnetic microsphere carrier; The phenylboronic acid group-containing polymer is modified by using polystyrene maleic anhydride PSMA or polymaleic anhydride-octadecene copolymer PMAO to carry out phenylboronic acid group modification; The phenylboronic acid modification is to modify aminophenylboronic acid on the surface of the carboxylated magnetic carrier through an amide bond; 2) Directional coupling of the antibody: Add the antibody to the phenylboronated magnetic carrier buffer and incubate at room temperature to make it directionally coupled to the magnetic carrier; then add excess substance containing cis-diol structure to block the excess on the magnetic carrier. The phenylboronic acid site; magnetic separation and washing to remove excess antibody and blocking agent to obtain immunomagnetic adsorbent; The cis-diol structure-containing substance is ovalbumin OVA. 2 . The application of the phenylboronic acid-directed conjugated antibody-based immunomagnetic adsorbent according to claim 1 , wherein it is used for enrichment and purification of target hazardous substances in food samples. 3 .

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