CN110538640A - Preparation method and application of mycotoxin chitosan immunoaffinity adsorbent - Google Patents
Preparation method and application of mycotoxin chitosan immunoaffinity adsorbent Download PDFInfo
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- CN110538640A CN110538640A CN201910921806.7A CN201910921806A CN110538640A CN 110538640 A CN110538640 A CN 110538640A CN 201910921806 A CN201910921806 A CN 201910921806A CN 110538640 A CN110538640 A CN 110538640A
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- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
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Abstract
the invention discloses a preparation method and application of a mycotoxin chitosan immunoaffinity adsorbent, and belongs to the technical field of natural polysaccharide development and application and functional material preparation. Preparing a chitosan solution and an oil phase consisting of liquid paraffin, emulsifier span 80 and petroleum ether, pouring the chitosan solution into a feeding pool of a membrane emulsification instrument, adjusting nitrogen pressure and the magnetic stirring rotating speed of the oil phase to enable the chitosan solution to slowly pass through SPG membrane pores under the nitrogen pressure and be pressed into the oil phase, adding a cross-linking agent for stirring and cross-linking after emulsification is finished, and obtaining chitosan microspheres after petroleum ether washing, ethanol washing, water washing, reduction and water washing; coupling the chitosan microspheres with a mycotoxin antibody, washing with distilled water and PBS (phosphate buffer solution), and finally obtaining the chitosan microsphere immunoaffinity adsorbent. The prepared chitosan microsphere immunoaffinity adsorbent has uniform microsphere particle size and wider pH stability, and can be used for purifying, separating and determining mycotoxin in food samples.
Description
Technical Field
The invention relates to a preparation method and application of a mycotoxin chitosan immunoaffinity adsorbent, belonging to the technical field of natural polysaccharide development and application and functional material preparation.
background
Mycotoxin widely pollutes plant-derived products such as grains, oil plants, food and the like, has great threat to human health, and has carcinogenic, teratogenic and genotropic effects on animals and human beings. The most representative mycotoxins with serious harm are aflatoxin, zearalenone toxin, vomitoxin, fumonisin and the like. The World Health Organization (WHO) also listed mycotoxin contamination of agricultural products as an important source of food-borne diseases in 2002. At present, the mycotoxin residue in agricultural products is mainly detected by HPLC, HPLC-MS and other instrument analysis methods. Because of the complex matrix and low mycotoxin content of the agricultural product sample, the sample is usually required to be purified before the instrumental analysis and detection. Immunoaffinity chromatography (IAC) utilizes antigen-antibody specific reversible binding to realize concentration and separation of target substances, and has the advantages of high selectivity, simple and rapid operation and the like, so the IAC is known as an optimal sample pretreatment method.
Currently, the most commonly used commercial chromatographic filler carrier is agarose gel, has stable performance and good biocompatibility, but needs virulent cyanogen bromide in the activation and regeneration processes, and is too expensive. Therefore, the search for a chromatographic packing carrier with excellent performance and economical utility is the direction of much research in laboratories.
Chitosan, chitin deacetylation products, the second most natural polysaccharide which is second to cellulose in nature, has rich sources, and has the advantages of safety, no toxicity, easy degradation, good biocompatibility and the like. In addition, chitosan surface contains a large number of modifiable functional groups (hydroxyl and amino groups), and various derivatives can be prepared by reactions such as acylation, hydroxylation, etherification, Schiff's reaction, esterification, hydrolysis and the like. Therefore, the chitosan is not only a natural resource, but also a new material, and has good application prospect in the fields of medicine, chemical industry, agriculture and the like. The chromatographic packing using the chitosan microsphere as the carrier prepared at present has some defects in the aspects of microsphere size uniformity, stability under acidic conditions and the like, and the application of the chromatographic packing is limited. In order to solve the problem, the invention optimizes and improves the preparation method of the chitosan microspheres, combines the chitosan microspheres with mycotoxin antibodies to obtain the immunoaffinity adsorbent taking the chitosan microspheres with stable pH and uniform particle size as carriers, and carries out purification separation and analysis determination on mycotoxin residues in agricultural products by utilizing the obtained immunoaffinity adsorbent.
Disclosure of Invention
The invention aims to provide a preparation method of a mycotoxin chitosan immunoaffinity adsorbent, which is used for concentrating and purifying mycotoxin residues in agricultural products.
the preparation of the chitosan microsphere adopts a membrane emulsification method, takes chitosan as a raw material, takes a glutaraldehyde saturated toluene solution as a cross-linking agent, and takes span 80 as an emulsifier. Physical and chemical characterization analysis shows that the chitosan microspheres have uniform particle size and are stable within the pH range of 4-10. The obtained chitosan microsphere is coupled with a mycotoxin antibody to obtain an immunoaffinity adsorbent, and the immunoaffinity adsorbent is used for purifying, separating and analyzing and measuring mycotoxin residues in an actual sample.
The invention is realized by the following technical scheme:
A preparation method of a chitosan microsphere immunoaffinity adsorbent with stable pH and uniform particle size comprises the following steps:
(1) preparing a chitosan solution and an oil phase consisting of liquid paraffin and petroleum ether, pouring the chitosan solution into a feeding pool of a membrane emulsification instrument, adjusting nitrogen pressure and continuous phase magnetic stirring rotation speed to enable the chitosan solution to slowly pass through SPG membrane holes under the nitrogen pressure and be pressed into the oil phase, adding a cross-linking agent after emulsification for stirring and cross-linking, and washing with petroleum ether, ethanol, water, reduction and water again to obtain chitosan microspheres;
(2) Stirring and coupling the chitosan microspheres prepared in the step (1) with a mycotoxin antibody, and washing with distilled water and PBS to obtain the immunoaffinity adsorbent.
In the step (1) of the present invention, the molecular weight of the chitosan is 100kD, and the chitosan concentration is 1% by mass.
In the step (1) of the invention, the volume ratio of the liquid paraffin to the petroleum ether in the oil phase consisting of the liquid paraffin and the petroleum ether is 7:5, and the oil phase contains span 80 with the volume mass percent of 4%.
in the step (1), the volume ratio of the chitosan (water phase) to the oil phase consisting of the liquid paraffin and the petroleum ether is 1: 6.
In step (1) of the present invention, the crosslinking agent is a glutaraldehyde-saturated toluene solution, and the ratio of chitosan (amino group): the molar ratio of glutaraldehyde (aldehyde groups) was 1: 0.5.
In step (1) of the present invention, the stirring speed is 500 rpm.
in the step (1) of the invention, the nitrogen pressure is 0.02 MPa.
In the step (2), the coupling agent is epichlorohydrin, and the volume concentration is 20%.
The prepared mycotoxin chitosan microsphere immunoaffinity adsorbent is used for purifying, separating and analyzing and determining residual mycotoxin in food samples.
The invention has the beneficial effects that: (1) the invention develops a natural polysaccharide chromatographic packing carrier which is rich in source, safe and nontoxic; (2) the chitosan microspheres prepared by the method have uniform particle size and wider pH stability; (3) the chitosan microsphere immunoaffinity adsorbent prepared by the invention can be used for purifying, separating and analyzing and determining the mycotoxin residue in an actual sample.
Drawings
FIG. 1 is a scanning electron microscope image of chitosan microspheres of the present invention;
FIG. 2 is a particle size distribution diagram of the chitosan microspheres of the present invention;
FIG. 3 is a graph of pH stability of chitosan microspheres of the present invention;
FIG. 4 is an appearance diagram of the chitosan microsphere immunoaffinity column of the present invention;
FIG. 5 is a graph showing the evaluation criteria of the ICELISA method of the present invention;
FIG. 6 is a standard curve diagram of the HPLC evaluation method of the present invention.
Detailed Description
The following examples are provided as further illustration of the invention and are not to be construed as limitations or limitations of the invention. The invention is further illustrated by the following examples.
EXAMPLE 1 preparation of mycotoxin chitosan microsphere immunoaffinity adsorbent
(1) Preparing chitosan microspheres with uniform particle size and stable pH:
a. Dissolving 0.5g of chitosan in 50mL of 2% acetic acid solution to prepare 1% chitosan solution;
b. Preparing 300mL of oil phase (containing 4% span 80) formed by mixing liquid paraffin and petroleum ether (v: v,7: 5);
c. Pouring the chitosan solution into a feeding pool of a membrane emulsification instrument, adjusting the nitrogen pressure to be 0.02MPa, and adjusting the magnetic stirring speed of the oil phase to be 500rpm, so that the chitosan solution is pressed into the oil phase through an SPG membrane under the nitrogen pressure;
d. Adding 7.5mL of cross-linking agent for cross-linking reaction for 1h, adding 5mL of 1M sodium hydroxide solution, and continuing to cross-link for 2 h;
e. after the crosslinking, washing with petroleum ether, ethanol and water to neutrality, reducing overnight with 5M NaBH4, washing with distilled water to neutrality, and freeze-drying.
(2) Preparation of immunoaffinity adsorbents, this example is zearalenone:
a. Weighing 0.5g of chitosan microspheres, washing the chitosan microspheres for 2 times by using a coupling buffer solution of NaHCO3 (containing 0.5mol/L NaCl) with pH of 8.3, and then resuspending the precipitates in 31mL of the coupling buffer solution, adding 1mL of 11mg/mL antibody solution, and stirring and adsorbing for 3 hours;
b. Adding coupling agent epichlorohydrin to make the final volume concentration of the epichlorohydrin 20%, and carrying out oscillation crosslinking for 5h at 37 ℃;
c. After the crosslinking, centrifuging at 8000r/min for 3min, taking supernatant, and measuring absorbance at 280nm and 260nm according to the formula: calculating the content of unconjugated antibody by using Cprotein (mg/mL) ═ 1.45A280nm-0.74A260nm, and further calculating the conjugation rate;
d. Wash with copious amounts of water to neutral and then wash 2 times with PBS.
experiment I, optimization and performance characterization of preparation conditions of chitosan microspheres with uniform particle size and stable pH
1. Determination of the conditions of the chitosan microspheres prepared in example 1, which have uniform particle size and stable pH: (1) concentration of chitosan solution: in the experiment, 0.05g, 0.1g and 0.15g of chitosan (molecular weight is 100kDa) are respectively weighed and dissolved in 10ml of 2% acetic acid solution to prepare 0.5%, 1% and 1.5% chitosan solution, the nitrogen pressure is 0.02MPa, the water-phase oil-phase ratio is 1:6, and the stirring speed is 500 rpm; (2) nitrogen pressure: in the experiment, the concentration of the chitosan (molecular weight is 100kDa) solution is 1 percent, the nitrogen pressure is selected from 0.01MPa, 0.02MPa and 0.03MPa, the water-phase oil-phase ratio is 1:6, and the stirring speed is 500 rpm; (3) water phase and oil phase: in the experiment, the concentration of the chitosan (molecular weight is 100kDa) solution is 1 percent, the nitrogen pressure is 0.02MPa, the water phase-oil phase ratio is selected to be 1:6, 1:8 and 1:10, and the stirring speed is 500 rpm; (4) stirring speed: in the experiment, the concentration of the chitosan (molecular weight is 100kDa) solution is 1 percent, the nitrogen pressure is 0.02MPa, the water-phase oil-phase ratio is 1:6, and the stirring speed is selected from 400rpm, 500rpm and 600 rpm. The chitosan microspheres are prepared according to an experimental method, a scanning electron microscope shows that the prepared chitosan microspheres are in a regular spherical shape (figure 1), a laser particle size analyzer is used for measuring the particle size distribution and the SPAN value of the microspheres, and the results are shown in Table 1.
obtaining an optimized condition through a single-factor experiment: the chitosan (molecular weight 100kDa) solution concentration is 1%, the nitrogen pressure is 0.02MPa, the water phase-oil phase ratio is 1:6, the stirring speed is 500rpm, and the prepared chitosan microspheres have the average particle size of 109 micrometers and the particle size distribution SPAN 1.55 (figure 2). The SPAN value was calculated as follows:
SPAN＝(D-D)/D
TABLE 1 influence of different preparation parameters on the particle size distribution of chitosan microspheres
2. Determination of chemical stability of chitosan microspheres with uniform particle size and stable pH prepared in example 1: 0.1g of chitosan microspheres were suspended in 50mM HAc-NaAc (pH 4.0), 50mM PBS (pH 6.0), 50mM Tris-HCl (pH 8.0) and 50mM NaHCO3-Na2CO3(pH 10.0), respectively, and stored at 4 ℃ for 30h, and no significant change in the structure of the chitosan microspheres was observed as a result of scanning electron microscopy (FIG. 3).
experiment II, preparation and application of chitosan microsphere zearalenone immunoaffinity adsorbent
1. Determination of binding capacity of chitosan microsphere zearalenone immunoaffinity adsorbent prepared in example 1:0.5 mL of the prepared immunoaffinity adsorbent is filled in an SPE column to obtain an immunoaffinity column (figure 4), 7mL of 0.5 mu g/mL zearalenone standard solution is filled in the column, one tube is collected from each 1.0mL of sample loading liquid, and the content of zearalenone in each component is determined by indirect competition ELISA (iclELISA). Calculated according to the following formula: column capacity (i.e., total amount of adsorbed zearalenone) — total amount of zearalenone loaded — total amount of zearalenone washed off. The results showed that the 1-6 mL of the sample solution contained no zearalenone, and that 0.428. mu.g of zearalenone was washed off at the 7-mL sample. Thus, the maximum column capacity of an IAC column is about: [6 × 0.5+0.072] × 2 ═ 6.1 μ g/mL gel.
2. The chitosan microsphere zearalenone immunoaffinity adsorbent prepared in example 1 is used for labeling and recovering zearalenone in a corn flour sample: the empty corn flour was normalized to a final zearalenone concentration of 50 and 100 μ g/kg. A sample of corn meal (4g) was weighed into a homogenizer and extracted at high speed for 2 minutes by adding 1g NaCl and 10mL acetonitrile water (80:20, v/v). The extract was filtered and diluted with distilled water at a ratio of 1: 5. A total of 10mL of the diluted extract was passed through a zearalenone immunoaffinity column. Zearalenone was then eluted with 1ml methanol and collected for HPLC detection and the recovery calculated.
TABLE 2 Chitosan microsphere zearalenone immunoaffinity adsorbent for the spiking recovery of zearalenone from corn flour samples
Third, the performance evaluation method of the chitosan microsphere zearalenone immunoaffinity adsorbent
1. Establishment of an iclelisa evaluation method: after blocking, 50. mu.L of standard solutions with a series of concentrations (50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.2, 0.1, 0.05, 0ng/mL) were added, 3 replicates for each concentration, 50. mu.L of primary antibody with the best dilution factor were added, secondary enzyme-labeled antibody was added, absorbance Ai at 450nm and absorbance A0 for the control (i.e., at 0ng/mL) were measured with an enzyme-labeled detector after chromogenic blocking, and plotted with Ai/A0 as ordinate and the logarithm of the standard solution concentration as abscissa. And fitting by Origin software to obtain a standard curve with the linear range of 0.05-50 ng/mL. The standard curve chart of the method is shown in the attached figure 5 in the specification.
2. Establishment of HPLC evaluation method: chromatographic conditions for HPLC: a Shim-pack VP-ODS column (250X 4.6 mm); mobile phase: v (acetonitrile): v (water): v (methanol) 46:46: 8; detecting by a fluorescence detector, wherein the excitation wavelength is 274nm, and the emission wavelength is 440 nm; column temperature: room temperature; sample introduction amount: 50 mu L of the solution; flow rate: 1.0 mL/min.
Standard working solutions (5mg/mL) were prepared in series of concentrations (15.625, 31.25, 62.5, 125, 250, 500ng/mL) and were subjected to HPLC measurements and a linear fit was performed for each concentration and its corresponding peak area. The standard curve has good linear relation, and the linear regression equation is as follows: y is 3085.3x-61878(R2 is 0.9995), and the linear range is 15.625-500 ng/mL. The standard curve chart of the method is shown in the attached figure 6 of the specification.
the above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. That is, all equivalent changes and modifications made according to the content of the claims of the present invention should be within the technical scope of the present invention.
Claims (9)
1. a preparation method of chitosan microsphere immunoaffinity adsorbent with stable pH and uniform particle size is characterized by comprising the following steps:
(1) Preparing a chitosan solution and an oil phase consisting of liquid paraffin and petroleum ether, pouring the chitosan solution into a feeding pool of a membrane emulsification instrument, adjusting nitrogen pressure and continuous phase magnetic stirring rotation speed to enable the chitosan solution to slowly pass through SPG membrane holes under the nitrogen pressure and be pressed into the oil phase, adding a cross-linking agent after emulsification for stirring and cross-linking, and washing with petroleum ether, ethanol, water, reduction and water again to obtain chitosan microspheres;
(2) stirring and coupling the chitosan microspheres prepared in the step (1) with a mycotoxin antibody, and washing with distilled water and PBS to obtain the immunoaffinity adsorbent.
2. The method for preparing a pH stable homogeneous chitosan microsphere immunoaffinity adsorbent according to claim 1, wherein in step (1), the molecular weight of chitosan is 100kD, and the chitosan concentration is 1% by mass.
3. the method for preparing the chitosan microsphere immunoaffinity adsorbent with stable pH and uniform particle size according to claim 1, wherein in step (1), the volume ratio of the liquid paraffin to the petroleum ether in the oil phase consisting of the liquid paraffin and the petroleum ether is 7:5, and the oil phase contains span 80 with the volume mass percent of 4%.
4. The method for preparing the chitosan microsphere immunoaffinity adsorbent with stable pH and uniform particle size according to claim 1, wherein in the step (1), the volume ratio of the chitosan (water phase) to the oil phase consisting of the liquid paraffin and the petroleum ether is 1: 6.
5. the method for preparing the immunoaffinity adsorbent of chitosan microspheres with stable pH and uniform particle size according to claim 1, wherein in step (1), the cross-linking agent is glutaraldehyde-saturated toluene solution, and the ratio of chitosan (amino): the molar ratio of glutaraldehyde (aldehyde groups) was 1: 0.5.
6. the method for preparing the immunoaffinity adsorbent of chitosan microspheres with stable pH and uniform particle size according to claim 1, wherein in step (1), the stirring speed is 500 rpm.
7. The method for preparing the chitosan microsphere immunoaffinity adsorbent with stable pH and uniform particle size according to claim 1, wherein in step (1), the nitrogen pressure is 0.02 MPa.
8. The method for preparing the immunoaffinity adsorbent of chitosan microspheres with stable pH and uniform particle size according to claim 1, wherein in step (2), the coupling agent is epichlorohydrin and the volume concentration is 20%.
9. A pH stable, uniform size chitosan microsphere immunoaffinity adsorbent prepared according to the method of claim 1, wherein the prepared mycotoxin chitosan microsphere immunoaffinity adsorbent is used for the decontamination separation and analytical determination of mycotoxin residues in food samples.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111296724A (en) * | 2020-03-03 | 2020-06-19 | 武汉轻工大学 | Application of cellulose carbonized microspheres and feed |
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Application publication date: 20191206 |