CN112067799B - Immune magnetic adsorbent based on phenylboronic acid directional coupling antibody and preparation method thereof - Google Patents
Immune magnetic adsorbent based on phenylboronic acid directional coupling antibody and preparation method thereof Download PDFInfo
- Publication number
- CN112067799B CN112067799B CN202010913502.9A CN202010913502A CN112067799B CN 112067799 B CN112067799 B CN 112067799B CN 202010913502 A CN202010913502 A CN 202010913502A CN 112067799 B CN112067799 B CN 112067799B
- Authority
- CN
- China
- Prior art keywords
- magnetic
- phenylboronic acid
- antibody
- magnetic carrier
- carrier
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
- G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Hematology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Nanotechnology (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention relates to an immune magnetic adsorbent based on a phenylboronic acid directional coupling antibody and a preparation method thereof. Compared with the traditional method, the method improves the utilization rate of the Fab end of the antibody to the maximum extent, further improves the affinity and the adsorption capacity of the immune magnetic adsorbent to the target object, can effectively control the batch difference, and is worthy of further popularization and application. The prepared immune magnetic adsorbent can be used for the detection pretreatment of various food samples, and can be used for enriching and purifying hazardous substances in the food samples, such as mycotoxin, pesticide and veterinary drug residues, heavy metals, pathogenic microorganisms and the like according to the difference of the coupled antibodies. The purified hazardous substances can be quantitatively or qualitatively detected by methods such as high performance liquid chromatography, immunoassay, enzyme-linked immunosorbent assay and the like.
Description
Technical Field
The invention belongs to the technical field of food safety detection and pretreatment, and particularly relates to an immune magnetic adsorbent based on a phenylboronic acid directional coupling antibody and a preparation method thereof.
Background
In the detection of food hazard factors, pretreatment steps such as separation and purification of samples are indispensable and are key links influencing the reliability of analysis results. The traditional sample pretreatment methods such as liquid phase extraction, solid phase extraction and the like have the disadvantages of complex operation and poor specificity, and influence on the detection accuracy. The immunoaffinity chromatography is based on the specificity recognition between the ligand and the object to be detected, and has good accuracy, high cost and long time consumption. The method based on the immune magnetic adsorbent is rapidly developed in the field of pretreatment of food safety detection samples due to the advantages of good specificity, rapidness and high efficiency.
The immune magnetic adsorbent is composed of a magnetic carrier with superparamagnetism and an antibody modified on the magnetic carrier, a target object to be detected is captured on the surface of the magnetic carrier through the specific recognition function of the antibody, and then the enrichment and separation of the object to be detected are realized under the action of an external magnetic field.
The basic principle of the preparation method of the current immune magnetic adsorbent is that 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) or glutaraldehyde and the like are used as coupling reagents to modify antibodies on the surface of a carboxylated or aminated magnetic carrier. However, the method has an obvious defect that the Fab end (antigen recognition region) and the Fc end of the antibody both have a large amount of amino groups, so that the orientation of the antibody on the surface of the magnetic carrier is random and uncontrollable, the random orientation reduces the exposed quantity of the Fab end, reduces the effective utilization rate of the antibody, and has the consequences of large dosage of the antibody, overhigh cost, low magnetic separation efficiency, low magnetic separation repeatability and stability and the like.
Therefore, the development of a method for directionally coupling the Fc end of the antibody to the magnetic carrier allows the Fab end of the antigen recognition end to be exposed, and reduces the steric hindrance of the combination of the antigen and the antibody, so that the antigen affinity of the immunomagnetic adsorbent is improved, the enrichment efficiency of the antigen of the object to be detected is improved, the lot difference is effectively controlled, and the cost is reduced.
Disclosure of Invention
The invention aims to provide an immune magnetic adsorbent based on a phenylboronic acid directional coupling antibody and a preparation method thereof, aiming at the defects of the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides an immune magnetic adsorbent based on a phenylboronic acid directional coupling antibody, which comprises a magnetic carrier and an antibody coupled on the magnetic carrier, wherein the antibody is directionally coupled on the surface of the magnetic carrier through the affinity effect of the phenylboronic acid group on the surface of the magnetic carrier and the specificity of a cis-diol structure in an antibody Fc fragment glycoprotein molecule.
Further, the magnetic carrier is any micro-nano particle with superparamagnetism, and preferably the magnetic carrier is coated with Fe3O4Polymeric microspheres or silica microspheres of nanoparticles.
Further, the surface of the magnetic carrier is completely or partially provided with phenylboronic acid groups.
The invention also provides a preparation method of the immune magnetic adsorbent based on the phenylboronic acid directional coupling antibody, which comprises the steps of firstly preparing a magnetic carrier with the surface modified with the phenylboronic acid group, and then directionally fixing the antibody on the magnetic carrier through the affinity effect of the phenylboronic acid group and the cis-diol structure specificity in the glycoprotein molecules on the Fc fragment of the antibody. The specific process steps are as follows:
in the first step, the benzene borated magnetic carrier is prepared by one of the following two schemes.
According to one preparation scheme of the magnetic carrier, the magnetic carrier with surface carboxyl or amino modification is synthesized, and then phenylboronic acid modification is carried out on the surface of the magnetic carrier. For example, EDC is used as a coupling agent to modify aminophenylboronic acid on the surface of carboxylated magnetic beads to prepare a phenylboronic acid magnetic carrier; or modifying 4-carboxyl phenylboronic acid on the surface of the aminated magnetic bead by using EDC as a coupling agent to prepare the phenylboronic acid magnetic carrier.
The second preparation scheme of the magnetic carrier, 1) the synthesis of oil-soluble magnetic nanoparticles is disclosed in the prior literature; 2) then, modifying phenylboronic acid groups of common polymers such as polystyrene maleic anhydride (PSMA), polymaleic anhydride-octadecene copolymer (PMAO) and the like to prepare the phenylboronic acid group-containing polymer, for example, synthesizing the phenylboronic acid-containing polymer by acylation reaction of 3-aminophenylboronic acid and polymaleic anhydride octadecanol ester under the catalysis of 4-dimethylaminopyridine; 3) then encapsulating the magnetic nanoparticles in a polymer containing phenylboronic acid groups by an emulsification-emulsion solvent volatilization method to prepare the phenylboronic acid magnetic microsphere carrier, specifically, emulsifying an aqueous solution containing oil-soluble magnetic nanoparticles, a polymer and a certain volatile nonpolar solvent such as toluene, chloroform and the like as an oil phase and a surfactant such as sodium dodecyl sulfate as an aqueous phase by ultrasonic, stirring and other modes to prepare an oil-in-water emulsion, and removing the nonpolar solvent in emulsion droplets by volatilization, rotary evaporation and other modes to obtain the phenylboronic acid magnetic carrier.
And secondly, directionally coupling the antibody with the magnetic carrier. Mixing the phenylboronic acid magnetic carrier synthesized in the first step with the antibody in a buffer solution (0.02M phosphate buffer solution, pH is 5.5-8.0, the optimal pH values of different antibodies are different), incubating at room temperature of 25 ℃ for 10-20 min to realize directional coupling of the antibody, adding glucose or OVA solution, continuing to incubate at room temperature for 10-20 min to seal redundant phenylboronic acid groups on the magnetic carrier, and obtaining the immune magnetic adsorbent for directional coupling of the antibody through magnetic separation after the reaction is finished.
The immune magnetic adsorbent provided by the invention can be used for carrying out detection pretreatment on various food samples, and can be used for enriching and purifying hazardous substances in the food samples, such as mycotoxin, pesticide and veterinary drug residues, heavy metals, pathogenic microorganisms and the like according to different coupled antibodies. The purified hazardous substance can be quantitatively or qualitatively detected by methods such as high performance liquid chromatography, immunoassay and the like.
Compared with the prior art, the invention has the beneficial effects that:
compared with the traditional immune magnetic adsorbent for non-directional coupling of antibodies, the immune magnetic adsorbent based on the phenylboronic acid directional coupling antibody and the preparation method thereof provided by the invention have the advantages that the Fc end of the antibody is directionally coupled to the magnetic carrier, the Fab end is exposed, and the steric hindrance caused by combination of the antigen and the antibody is reduced, so that the antigen affinity of the immune magnetic adsorbent is improved, the enrichment efficiency of the antigen of an object to be detected is improved, the batch difference is effectively controlled, and meanwhile, the cost is reduced due to the reduction of the using amount of the antibody.
Drawings
FIG. 1 is a schematic diagram of the synthesis of a conventional immunomagnetic adsorbent;
FIG. 2 is a schematic diagram of the synthesis of an immunomagnetic adsorbent based on a phenylboronic acid directional coupling antibody.
FIG. 3 shows the scheme of modifying phenylboronic acid with poly (octadecyl maleate) ester.
Detailed Description
The present invention is described in further detail below with reference to specific examples, which are provided only for illustrating the specific contents of the present invention and are not intended to limit the scope of the present invention.
Example 1
The preparation of the immune magnetic adsorbent based on the phenylboronic acid directional coupling antibody salmonella in the embodiment comprises the preparation of a phenylboronic acid magnetized carrier, and the coupling of the antibody and the magnetized carrier, and the specific process steps are as follows:
1) the preparation of the phenylboronic acid magnetized carrier,
a.Fe3O4synthesis of magnetic beads, 300mL of ultrapure water was placed in a 500mL three-necked flask, and N was introduced thereinto2To remove oxygen from the water while preheating at 50 ℃ for 15 min. Adding 3.2g FeCl2·H2O，5.2gFeCl3After the mixture is stirred evenly by magnetic force, 25mL ammonia water is added, the yellow solution quickly turns into black, and the reaction is carried out for 30min at the constant temperature of 50 ℃. And (3) separating the synthesized magnetic beads through magnetic attraction, washing the magnetic beads for 3-5 times by using ultrapure water until the pH value of the solution is neutral after the magnetic attraction, and redissolving the solution in 300mL of ultrapure water.
b.Fe3O4Oleic acid modification of magnetic beads, adding the magnetic beads obtained in the previous step into a three-neck flask, and introducing N22.4mL of oleic acid was added dropwise with stirring. The oil acidified magnetic beads are hydrophobic and adsorbed on the wall of a stirrer or a flask, the black solution is gradually clear after reacting for 3 hours at 70 ℃, the reaction is stopped, the solution in the beaker is poured out, 300mL of ethanol is added to elute the adsorbed oil acidified magnetic beads, the ethanol solvent is removed after the magnetic attraction is carried out for 2min, and the step is repeated for 3-5 times until the liquid level of the ethanol does not float the oleic acid layer.
c. Magnetic microsphere synthesis, namely synthesizing magnetic microspheres by an emulsion-emulsion solvent volatilization method, dispersing the synthesized oleic acid magnetic beads in 30mL of cyclohexane to form an oil phase, dissolving 1.5g of Sodium Dodecyl Sulfate (SDS) in 500mL of ultrapure water to form an aqueous phase, and carrying out ultrasonic treatment on the mixture for 10min to form an oil-in-water microemulsion. Then placing at 60 ℃ to volatilize cyclohexane overnight to obtain the magnetic microsphere with stable SDS. And re-dispersing the magnetic microspheres in 60mL of Tween-80 solution, and then obtaining the Tween-80 modified magnetic microspheres by magnetic attraction.
d.Fe3O4@SiO2Synthesis of composite nanospheres, 1000mL of ethanol, 30mL of ammonium hydroxide, 20mL of ultrapure water and 6mL of TEOS were pre-reacted at 30 ℃ for 20min to pre-hydrolyze TEOS. Then adding the 60mL magnetic microspheres synthesized in the previous step into a mixed system, and controlling the reaction time to obtain SiO with a proper coating thickness2Magnetic microspheres of the layer.
e.Fe3O4@SiO2Performing carboxylation modification on the surface of the composite nano microsphere, and taking 10mg of Fe synthesized in the previous step3O4@SiO2Adding the composite nano-microspheres into 10mL of ethanol, performing ultrasonic treatment for 1h to uniformly disperse the composite nano-microspheres, adding 800 mu L of 3- (triethoxysilyl) propyl succinic anhydride (TEPSA) reagent, and performing ultrasonic treatment for 1h to modify the composite nano-microspheres on the surfaces of the microspheres. After magnetic separation, washing with ultrapure water 3-5 times to remove ethanol and unreacted TESPSA reagent, followed by addition of basic ultrapure water at pH 9.0 and shaking overnight to hydrolyze acid anhydride to generate carboxyl groups.
f.Fe3O4@SiO2Modifying phenylboronic acid on the surface of the @ COOH composite nano microsphere, dispersing the carboxylated magnetic microsphere synthesized in the previous step in a buffer solution (0.02M phosphate buffer solution, pH 5.5) containing EDC, and carrying out activation reaction at room temperature of 25 ℃ for 30 min. Then, the pH value of the system is adjusted to 8.0, 3-aminophenylboronic acid/4-aminophenylboronic acid is added, and the coupling reaction is carried out for 30min at the room temperature of 25 ℃. And then adding glucosamine or OVA solution, continuing incubation for 10-20 min at room temperature to seal the residual carboxyl groups on the magnetic carrier, and obtaining the magnetic microsphere modified with phenylboronic acid by magnetic separation after the reaction is finished.
2) Coupling of anti-salmonella antibodies to magnetic carriers:
2.1 coupling of anti-salmonella antibody with phenylboronic acid magnetic carrier:
1.5mg of the phenylboronated magnetic carrier (15mg/mL) with the particle size of 180nm synthesized in step 1) was dispersed in a buffer (0.02M phosphate buffer, pH 6) containing 100. mu.g of an anti-Salmonella antibody (2mg/mL), and incubated at 25 ℃ for 10 min. Adding 1Incubating the 0uL glucose for 10min at room temperature to seal the residual boric acid groups on the magnetic carrier, removing the supernatant by magnetic separation after the reaction is finished, and re-dissolving the magnetic beads in 0.1% BSA, 0.05% Tween-20 and 0.01% NaN3The buffer solution (1 mg/mL) of PB7.4 was stored at 4 ℃ until use.
2.2 coupling of anti-Salmonella antibodies to carboxylated magnetic carriers (this is a traditional method control):
1.5mg of a conventional carboxylated magnetic adsorbent (15mg/mL) having a particle size of 180nm was dispersed in a buffer (0.02M phosphate buffer, pH 6) containing 220. mu.g of an anti-Salmonella antibody (2mg/mL), and 50. mu.g of EDC was added thereto and incubated at 25 ℃ for 90 minutes. Adding 10mgBSA, incubating for 10min at room temperature to block the residual carboxyl groups on the magnetic carrier, removing the supernatant by magnetic separation after the reaction is finished, and re-dissolving the magnetic beads in 0.1% BSA, 0.05% Tween-20 and 0.01% NaN3The buffer solution (1 mg/mL) of PB7.4 was stored at 4 ℃ until use.
3) The two immunomagnetic adsorbents are used for enriching salmonella
Adding 20mg of pork paste into 200mL of culture medium, and uniformly mixing. Inoculating salmonella with certain concentration, and performing shake culture at 37 ℃ for 12 h. Adjusting the concentration of the bacterial liquid to 104CFU/mL、105CFU/mL、106CFU/mL.107CFU/mL. And respectively adding 10 mu L of the two sealed immune magnetic adsorbents into 1mL of bacterial liquid with various concentrations and 1mL of meat paste sample solution to be detected, mixing and incubating for 60min at the temperature of 37 ℃ and the rotating speed of 15 rpm. After incubation and magnetic separation for 35min, the supernatant was discarded, washed with PBS and redissolved in PBS.
4) Visual inspection of samples by double antibody sandwich method and measurement of results by using instrument
Diluting the two immunomagnetic adsorbents of the captured bacteria to the concentration of 50 mug/mL, dripping 70 mug L of the two immunomagnetic adsorbents into a sample adding hole of a commercial salmonella immunochromatographic test strip, reading the two immunomagnetic adsorbents by using an immunochromatographic analyzer for 10min, recording the absorbance of a T line, the absorbance of a C line and the value of T/C, and drawing a standard curve by taking the concentrations of different bacteria as abscissa and the absorbance of the T line and the value of T/C as ordinate. Simultaneously, qualitative analysis is carried out by observing results with naked eyes, and if the T line has colorThen the bacteria exist in the sample, and the detection limit is about 104CFU/mL, no color development of the T line indicates no Salmonella in the sample or less than 10 Salmonella in the sample4CFU/mL。
The number of Salmonella in the sample was determined by reference to the standard curve chart. The results show that: when the standard substance amount is 104CFU/mL、105CFU/mL、106CFU/mL、107Between CFU/mL, the enrichment efficiency of EDC covalent modification immune magnetic adsorbent respectively reaches 44-50%, and the enrichment rate of phenylboronic acid directional coupling immune magnetic adsorbent reaches 58-63%, which shows that the enrichment effect of phenylboronic acid directional coupling immune magnetic adsorbent is obviously better under the condition of less antibody consumption.
Example 2
The preparation of the OTA immune magnetic adsorbent based on the phenylboronic acid directional coupling antibody in this embodiment includes the preparation of the phenylboronic acid magnetized carrier, the coupling of the antibody and the magnetized carrier, the enrichment and the detection of OTA, and the specific process steps of the application of the immune magnetic adsorbent are as follows:
1) preparation of phenylboronic acid magnetic carrier
The steps a to d are the same as in example 1.
e.Fe3O4@SiO2Performing amination modification on the surface of the composite nano microsphere, and taking 10mg of Fe synthesized in the previous step3O4@SiO2Adding the composite nano-microspheres into 10mL of ethanol, performing ultrasonic treatment for 1h to uniformly disperse the composite nano-microspheres, adding 800 μ L of 3-Aminopropyltriethoxysilane (APTES) reagent, performing ultrasonic treatment for 1h to modify the composite nano-microspheres on the surfaces of the microspheres, performing magnetic separation, and washing with ultrapure water for 3-5 times to remove ethanol and unreacted APTES reagent.
f.Fe3O4@SiO2The surface modification phenylboronic acid of the @ NH2 composite nano microsphere is prepared by dissolving 2-carboxyphenylboronic acid/4-carboxyphenylboronic acid in a buffer solution (0.02M phosphate buffer solution, pH 5.5) containing EDC,
activating reaction at 25 deg.c for 30 min. And then, adjusting the pH value of the system to 8.0, adding the aminated magnetic microsphere synthesized in the previous step, and performing coupling reaction at room temperature of 25 ℃ for 30 min. And after the reaction is finished, obtaining the magnetic microspheres for modifying the phenylboronic acid through magnetic separation.
2) Coupling of anti-OTA antibody to magnetic carrier:
2.1 coupling of anti-OTA antibody with phenylboronic acid magnetic carrier:
1.5mg of the phenylboronated magnetic adsorbent (15mg/mL) with a particle size of 180nm synthesized in step 1) was dispersed in a buffer (0.02M phosphate buffer, pH 6) containing 50. mu.g of anti-OTA antibody (2mg/mL) and incubated at 25 ℃ for 10 min. Adding 10uL glucose, incubating at room temperature for 10min to block the residual boric acid groups on the magnetic carrier, removing the supernatant by magnetic separation after the reaction is finished, and re-dissolving the magnetic beads in 0.1% BSA, 0.05% Tween-20 and 0.01% NaN3The buffer solution (1 mg/mL) of PB7.4 was stored at 4 ℃ until use.
2.2 coupling of anti-OTA antibody to carboxylated magnetic carriers:
1.5mg of carboxylated magnetic adsorbent (15mg/mL) having a particle size of 180nm was dispersed in a buffer (0.02M phosphate buffer, pH 6) containing 300. mu.g of anti-OTA antibody (2mg/mL), and 50. mu.g of EDC was added thereto and incubated at 25 ℃ for 90 minutes. Adding 10mgBSA, incubating at room temperature for 10min to block the residual carboxyl groups on the magnetic carrier, removing the supernatant by magnetic separation after the reaction is finished, and re-dissolving the magnetic beads in 0.1% BSA, 0.05% Tween-20 and 0.01% NaN3The buffer solution (1 mg/mL) of PB7.4 was stored at 4 ℃ until use.
3) Enrichment and detection of OTA by two immunomagnetic adsorbents:
taking 1mL (concentration is 1mg/mL) of the prepared two immune magnetic adsorbents into a 5mL centrifuge tube, then adding 1mL of samples to be detected, respectively preparing OTA standard products into OTA solutions with concentrations of (10ng/mL, 25ng/mL, 50ng/mL and 75ng/mL as samples to be detected and PBS buffer as blank samples to be detected), mixing uniformly, capturing for 10min, and removing supernatant after magnetic separation. And finally, adding 1mL of eluent for elution, collecting the eluent, and detecting the content of the OTA in the sample to be detected by an HPLC method according to GB 5009.96-2016.
The results show that: when the standard substance amount is between 10ng, 25ng, 50ng and 75ng, the amounts of OTA which is enriched by the EDC covalent modification immune magnetic adsorbent and is eluted by eluent are respectively 8.60ng, 21.4ng, 44.0 and 65.7ng, and the recovery rates of the carboxylation immune magnetic adsorbent reach 83-88%; the OTA enriched by the immune magnetic adsorbent for eluting the phenylboronic acid directional coupling antibody by using the eluent is 8.81ng, 22.2ng, 45.7 ng and 66.7ng respectively; the recovery rate of the immune magnetic adsorbent of the phenylboronic acid directional coupling antibody reaches 88-92%. The results show that the OTA enrichment effect of the phenylboronic acid directional coupling immunomagnetic adsorbent is better under the condition of less antibody dosage.
After 5 pieces of the immunomagnetic adsorbing agent of the phenylboronic acid directional coupling antibody are added into a blank PBS buffer solution by 5.0 mu g/kg of OTA in parallel, and the above steps are carried out, the addition recovery rates are 88.1%, 89.7%, 89.5%, 90.9% and 89.8%, which shows that the immunomagnetic adsorbing agent has good reproducibility.
4) The application method of the phenyl boronic acid directional coupling antibody immunomagnetic adsorbent comprises the following steps:
4.1 sample pretreatment
A sample of 10.0g of corn (accurate to 0.01g) was weighed into 50mL of chloroform and 5mL of 0.1mol/L aqueous phosphoric acid and vortexed. Shaking and extracting for 3-5 min on a shaker, filtering the extracting solution by using qualitative filter paper, taking 10mL of lower-layer filtrate to a 100mL flat-bottomed flask, rotating swallow hair to be nearly dry by using a rotary evaporator in 40C water bath, dissolving residues by using 20mL of petroleum ether, adding 10mL of the extracting solution, shaking and extracting for 3-5 min by using a vortex shaker, standing for layering, taking the lower-layer solution, and filtering by using filter paper.
4.2 Immunomagnetic adsorbent Capture of Phenylboronic acid Directional coupling antibodies
2mL of the immune magnetic adsorbent is taken to be put into a 10mL centrifuge tube, 5mL of deionized water is used for rinsing, and a magnetic separation frame is used for separating the washing liquid each time; adding 5mL of the treated filtrate into the rinsed immunomagnetic adsorbent, mixing, capturing at room temperature for 10min, separating the immunomagnetic adsorbent with a magnetic separation rack, rinsing the immunomagnetic adsorbent with 2mL of deionized water for 2 times
4.3 Immunomagnetic adsorbent elution of Phenylboronic acid directed conjugate antibody
And washing the immune magnetic adsorbent of the phenylboronic acid directional coupling antibody by using 1mL of eluent, uniformly mixing, separating the immune magnetic adsorbent by using a magnetic separation frame, and recovering eluent, namely the enriched and purified OTA sample.
5) Measurement of
The reference conditions for high performance liquid chromatography are listed below:
a) chromatographic column C1 column, column length 150mm, inner diameter 4.6mm, particle size 5 μm, or equivalent column:
b) the column temperature is 30C;
c) the sample injection amount is 10 pL;
d) the flow rate is 1 mL./min;
e) the detection wavelength is 333nm of excitation wavelength and 460nm of emission wavelength;
f) mobile phase and elution conditions:
the mobile phase A is glacial acetic acid-water (2+100), and B is acetonitrile;
isocratic elution conditions A-B (50+50)
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Claims (2)
1. The immune magnetic adsorbent based on the phenylboronic acid directional coupling antibody comprises a magnetic carrier and an antibody coupled on the magnetic carrier, and is characterized in that the antibody is directionally coupled on the surface of the magnetic carrier through the affinity effect of the phenylboronic acid group on the surface of the magnetic carrier and the specificity of a cis-diol structure in an antibody Fc fragment glycoprotein molecule;
the magnetic carrier is wrapped Fe3O4Polymeric or silica microspheres of nanoparticles;
the nano particles are Fe3O4A nanoparticle;
the whole or part of the surface of the magnetic carrier is a phenylboronic acid group;
the preparation method of the immune magnetic adsorbent based on the phenylboronic acid directional coupling antibody comprises the following steps:
1) preparation of phenylboronic acid magnetic carrier: firstly synthesizing oil-soluble magnetic nanoparticles, then carrying out phenylboronic acid modification on the polymer to prepare a polymer containing phenylboronic acid groups, finally preparing an oil-in-water emulsion by using an emulsification-emulsion solvent volatilization method and taking the magnetic nanoparticles, the polymer and a volatile nonpolar solvent as an oil phase and an aqueous solution containing a surfactant as an aqueous phase through ultrasonic and stirring emulsification, and then removing the nonpolar solvent in emulsion droplets in a volatilization or rotary evaporation mode to obtain a phenylboronic acid magnetic microsphere carrier;
the polymer containing the phenylboronic acid group is modified by using polystyrene maleic anhydride PSMA or polymaleic anhydride-octadecene copolymer PMAO;
the phenylboronic acid modification is to modify aminophenylboronic acid on the surface of the carboxylated magnetic carrier through an amido bond;
2) directional coupling of antibodies: adding the antibody into a buffer solution containing a phenylboronic acid magnetic carrier for incubation at room temperature, and directionally coupling the antibody to the magnetic carrier; then adding excessive substances containing cis-diol structures to seal redundant phenylboronic acid sites on the magnetic carrier; magnetic separation washing is carried out to remove redundant antibodies and blocking agents, and an immune magnetic adsorbent is obtained;
the substance containing the cis-diol structure is ovalbumin OVA.
2. The application of the immunomagnetic adsorbing agent based on the phenylboronic acid directional conjugated antibody according to claim 1, wherein the immunomagnetic adsorbing agent is used for enriching and purifying target hazardous substances in food samples.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010913502.9A CN112067799B (en) | 2020-09-03 | 2020-09-03 | Immune magnetic adsorbent based on phenylboronic acid directional coupling antibody and preparation method thereof |
| PCT/CN2021/108825 WO2022048350A1 (en) | 2020-09-03 | 2021-07-28 | Immunomagnetic adsorbent based on phenylboronic acid directional coupling antibody and preparation method therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010913502.9A CN112067799B (en) | 2020-09-03 | 2020-09-03 | Immune magnetic adsorbent based on phenylboronic acid directional coupling antibody and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112067799A CN112067799A (en) | 2020-12-11 |
| CN112067799B true CN112067799B (en) | 2022-06-14 |
Family
ID=73666661
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010913502.9A Active CN112067799B (en) | 2020-09-03 | 2020-09-03 | Immune magnetic adsorbent based on phenylboronic acid directional coupling antibody and preparation method thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN112067799B (en) |
| WO (1) | WO2022048350A1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112067799B (en) * | 2020-09-03 | 2022-06-14 | 南昌大学 | Immune magnetic adsorbent based on phenylboronic acid directional coupling antibody and preparation method thereof |
| CN113024669B (en) * | 2021-03-12 | 2022-06-14 | 北京华科泰生物技术股份有限公司 | RBP antibody marked by amino-group or carboxyl-group-containing substance, human RBP immunochromatography detection kit and preparation method thereof |
| CN113184861B (en) * | 2021-04-29 | 2022-11-01 | 胡晓青 | Mesoporous silica, carboxylated mesoporous silica, drug-loading system, and preparation method and application thereof |
| CN113687063B (en) * | 2021-07-29 | 2023-03-24 | 南昌大学 | Glycoprotein dynamic light scattering immunization method based on phenylboronic acid crosslinking agent |
| CN113667721B (en) * | 2021-07-29 | 2024-02-09 | 南昌大学 | Analysis method for high-sensitivity instant detection of miRNA |
| CN114460310A (en) * | 2022-04-12 | 2022-05-10 | 天津康博尔生物基因技术有限公司 | Colored latex microsphere and preparation method and application thereof |
| CN115364830B (en) * | 2022-07-11 | 2023-09-12 | 苏州赛分科技股份有限公司 | Synthesis method of boric acid affinity separation material and separation material |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684005A (en) * | 2008-09-28 | 2010-03-31 | 复旦大学 | Nano magnetic material of surface modified boric acid base group, preparation method and application thereof |
| CN103043647A (en) * | 2012-12-26 | 2013-04-17 | 复旦大学 | Aminophenylboronic acid surface-modified nano-carbon material, as well as preparation method and application thereof |
| CN106475068A (en) * | 2015-09-01 | 2017-03-08 | 中国科学院大连化学物理研究所 | The graphene oxide composite nano materials of phenylboric acid functionalization and its preparation and application |
| CN108355625A (en) * | 2018-02-09 | 2018-08-03 | 江苏大学 | A kind of glycoprotein surface imprinted polymer and its preparation method and application affine based on team's boron |
Family Cites Families (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6218160B1 (en) * | 1997-10-31 | 2001-04-17 | Roche Diagnostics Corporation | Site-specific conjugation of glycoproteins |
| US6623982B1 (en) * | 1999-07-12 | 2003-09-23 | Immunivest Corporation | Increased separation efficiency via controlled aggregation of magnetic nanoparticles |
| EP1494028A1 (en) * | 2003-07-03 | 2005-01-05 | Labsoft Diagnostics AG | Immunomagnetic separation of specific target cells |
| US20080135490A1 (en) * | 2005-01-07 | 2008-06-12 | Board Of Trustees Of The University Of Arkansas | Quantum dot biolabeling and immunomagnetic separation for detection of contaminants |
| CN100425627C (en) * | 2007-01-04 | 2008-10-15 | 吉林大学 | Preparation process of magnetic partical/polymer/silicon dioxide structure magnetic microball |
| TW201028692A (en) * | 2009-01-23 | 2010-08-01 | Nat Univ Tsing Hua | Method for immobilizing protein |
| KR101256628B1 (en) * | 2009-11-24 | 2013-04-19 | 한국생명공학연구원 | Method of Rapid Detection of Microorganisms Using Nanoparticles |
| CN102500296B (en) * | 2011-11-04 | 2013-10-30 | 同济大学 | Preparation method for mesoporous silicon oxide hollow microspheres with magnetic nanoparticles embedded in shell layers |
| CN103353495B (en) * | 2011-11-15 | 2014-11-26 | 南昌大学 | Method for enriching leucomalachite green and leucogentian violet in aquatic products |
| CN102558463B (en) * | 2012-01-04 | 2013-05-29 | 福州大学 | One-pot synthesis of phenylboronic acid polymer magnetic nano composite material and preparation method and application thereof |
| TWI458732B (en) * | 2012-06-27 | 2014-11-01 | Univ Nat Chiao Tung | Borate moiety-contained linker and bio-sensing element containing the same |
| JP6532202B2 (en) * | 2014-09-08 | 2019-06-19 | Jsr株式会社 | Magnetic particle, ligand binding particle, method for detecting or separating target substance, and method for producing the magnetic particle |
| CN104459126A (en) * | 2014-12-16 | 2015-03-25 | 四川大学 | Method of gathering salmonella based on immunomagnetic beads |
| US10605806B2 (en) * | 2015-05-19 | 2020-03-31 | Universiti Brunei Darussalam | Electrochemical immunosensor and method of use for analyte detection |
| CN107435039A (en) * | 2017-06-13 | 2017-12-05 | 安徽安龙基因医学检验所有限公司 | A kind of preparation method and storage method of the immunomagnetic beads for sorting leucocyte |
| CN107490681A (en) * | 2017-09-29 | 2017-12-19 | 安徽安龙基因科技有限公司 | A kind of preparation method of EpCAM antibody immune magnetic beads |
| US11585777B2 (en) * | 2018-04-27 | 2023-02-21 | Innotech Alberta Inc. | Method and device for detecting a component in a sample |
| CN112067799B (en) * | 2020-09-03 | 2022-06-14 | 南昌大学 | Immune magnetic adsorbent based on phenylboronic acid directional coupling antibody and preparation method thereof |
-
2020
- 2020-09-03 CN CN202010913502.9A patent/CN112067799B/en active Active
-
2021
- 2021-07-28 WO PCT/CN2021/108825 patent/WO2022048350A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684005A (en) * | 2008-09-28 | 2010-03-31 | 复旦大学 | Nano magnetic material of surface modified boric acid base group, preparation method and application thereof |
| CN103043647A (en) * | 2012-12-26 | 2013-04-17 | 复旦大学 | Aminophenylboronic acid surface-modified nano-carbon material, as well as preparation method and application thereof |
| CN106475068A (en) * | 2015-09-01 | 2017-03-08 | 中国科学院大连化学物理研究所 | The graphene oxide composite nano materials of phenylboric acid functionalization and its preparation and application |
| CN108355625A (en) * | 2018-02-09 | 2018-08-03 | 江苏大学 | A kind of glycoprotein surface imprinted polymer and its preparation method and application affine based on team's boron |
Non-Patent Citations (3)
| Title |
|---|
| Fabrication of Oriented Antibody-Conjugated Magnetic Nanoprobes and Their Immunoaffinity Application;Po-Chiao Lin等;《Anal. Chem.》;20091002;第81卷;第8774-8782页 * |
| Po-Chiao Lin等.Fabrication of Oriented Antibody-Conjugated Magnetic Nanoprobes and Their Immunoaffinity Application.《Anal. Chem.》.2009,第81卷8774-8782页. * |
| 磁性荧光微球制备及其在荧光免疫层析检测中的应用;郭亮;《中国博士学位论文全文数据库工程科技Ⅰ辑》;20200115;第39-62页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112067799A (en) | 2020-12-11 |
| WO2022048350A1 (en) | 2022-03-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112067799B (en) | Immune magnetic adsorbent based on phenylboronic acid directional coupling antibody and preparation method thereof | |
| Wang et al. | Immunomagnetic separation: An effective pretreatment technology for isolation and enrichment in food microorganisms detection | |
| US5302532A (en) | Chromatographic supports having an immobilized flocculating agent and their use in immunoassays | |
| Aguilar-Arteaga et al. | Magnetic solids in analytical chemistry: a review | |
| Li et al. | Efficient preparation of surface imprinted magnetic nanoparticles using poly (2-anilinoethanol) as imprinting coating for the selective recognition of glycoprotein | |
| Khodadadi et al. | Aptamer functionalized magnetic nanoparticles for effective extraction of ultratrace amounts of aflatoxin M1 prior its determination by HPLC | |
| Zhou et al. | An enhanced ELISA based on modified colloidal gold nanoparticles for the detection of Pb (II) | |
| Smith et al. | Optimization of antibody-conjugated magnetic nanoparticles for target preconcentration and immunoassays | |
| CN110204735B (en) | Preparation method and application of magnetic core-hollow porous molecularly imprinted polymer satellite assembly of macrolide antibiotics | |
| Liu et al. | Affinity capture of aflatoxin B 1 and B 2 by aptamer-functionalized magnetic agarose microspheres prior to their determination by HPLC | |
| CN110240704B (en) | Preparation method and application of targeted enzyme immobilization carrier based on magnetic molecular imprinting technology | |
| CN111650320B (en) | Detection method and kit for quinolone veterinary drugs based on magnetic solid-phase extraction | |
| Wei et al. | Combining magnetic MOFs as a highly adsorbent with homogeneous chemiluminescent immunosensor for rapid and ultrasensitive determination of Ochratoxin A | |
| JP2001513203A (en) | Particles with polyaldehyde dextran coating with reduced matrix effect | |
| Vergara-Barberan et al. | Recent advances in aptamer-based miniaturized extraction approaches in food analysis | |
| CN109781871B (en) | Aflatoxin B1And B2Magnetic solid phase extraction material, preparation method and application | |
| Chen et al. | Immunomagnetic separation and MS/SPR end-detection combined procedure for rapid detection of Staphylococcus aureus and protein A | |
| Du et al. | L-histidine functionalized multi-walled carbon nanotubes for on-line affinity separation and purification of immunoglobulin G in serum | |
| WO2017206713A1 (en) | Method for coupling magnetic particles with antibody molecules | |
| Pichon | Aptamer-based and immunosorbents | |
| Xu et al. | Aptamer-enhanced fluorescence determination of bisphenol A after magnetic solid-phase extraction using Fe 3 O 4@ SiO 2@ aptamer | |
| Šafarík et al. | Overview of magnetic separations used in biochemical and biotechnological applications | |
| CN114720515B (en) | Construction method and application of linear range adjustable polydopamine mediated modification-free portable conductivity immunosensor | |
| CN112067800B (en) | Immune magnetic adsorbent based on hydrazide directional coupling antibody and preparation method thereof | |
| CN114832793A (en) | Magnetic nano material for detecting tetrahydrocannabinic acid and synthetic cannabinoids drugs and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |