CN102520163B - Determination method for carbohydrate allergen - Google Patents

Abstract

The invention discloses a determination method for a carbohydrate allergen. The method comprises the following steps of: marking the carbohydrate allergen by using zinc oxide quantum dots; immobilizing an agglutinin layer on a chip in an electrolytic cell; specifically binding the carbohydrate allergen marked by the zinc oxide quantum dots with agglutinin on the chip in a series of carbohydrate allergen solutions to be determined of known concentrations; connecting the zinc oxide quantum dots marked on the carbohydrate allergen to the chip; dissolving the zinc oxide quantum dots into zinc ions by using acidic solution; determining concentration of the dissolved zinc ions by using dissolving voltammetry; and establishing relation between the concentration of the zinc ions and the concentration of the carbohydrate allergen so as to determine the concentration of the carbohydrate allergen to be determined.

Description

A method for the determination of carbohydrate allergens

technical field

The invention belongs to the technical field of medicine and biotechnology. Specifically, the invention describes a method for detecting carbohydrate allergens.

Background technique

Food allergy is an IgE-mediated type I allergic reaction to food. The clinical manifestations are various symptoms such as urticaria, asthma, abdominal pain and diarrhea, and severe cases can lead to shock. Generally speaking, food allergens are proteins or glycoproteins with a molecular weight between 10 and 70 kD. With the development of society and changes in people's diet structure, the incidence of allergies is increasing day by day, posing a huge threat to people's health. Among allergic reactions, more than 90% are caused by eight types of foods such as eggs, peanuts, and milk. At present, the detection methods for allergens in some foods are still very limited, mainly based on the immune response of some allergens. However, this method is costly, and for some allergens, the corresponding antibodies or antigens are difficult to obtain, thus hindering the application of immunoassay technology in allergen detection.

Contents of the invention

Technical problem: Aiming at the above technical problems, the present invention provides a stripping voltammetry detection chip for zinc oxide quantum dot-labeled carbohydrate allergens. This method utilizes zinc oxide-labeled carbohydrate allergens to detect low-concentration carbohydrate allergens. Carbohydrate allergens have low detection limit, good selectivity, high sensitivity and good stability.

Technical solution: In the method for measuring carbohydrate allergens of the present invention, zinc oxide quantum dots are used to label carbohydrate allergens; at the same time, a layer of lectin is immobilized on the chip in the electrolytic cell, and a series of known concentrations of the to-be-tested In the sugar allergen solution, the sugar allergen marked with zinc oxide quantum dots is specifically combined with the lectin on the chip, and the zinc oxide quantum dots marked on the sugar allergen are connected to the chip; The acidic solution dissolves the zinc oxide quantum dots into zinc ions, and the concentration of the dissolved zinc ions is measured by stripping voltammetry, and the relationship between the concentration of zinc ions and the concentration of sugar allergens is established, so as to determine the concentration of sugar allergens to be tested.

The specific measurement method is:

a. Disperse 0.5-5 mg of zinc oxide quantum dots into 50-200 microliters of 1 ng/ml sugar allergen solution, shake at room temperature for 10 minutes, centrifuge, and buffer with phosphate with a pH value of 6.8-7.5 The solution was washed three times. Add 500 μL 1~2% w/v bovine serum albumin, shake at 15~35 ^o C for 10~30 minutes, centrifuge, wash with phosphate buffer solution with pH value 6.8~7.5 three times; finally disperse in 50~150 μL 0.05~0.1% v/v Triton X-100 Phosphate Buffer Solution, store in the insulation layer of the refrigerator when not in use;

b. Sonicate the silicon dioxide or glass slices in acetone, ethanol, and water for 5 minutes respectively, and then in NH ₄ OH/H ₂ O ₂ /H ₂ O 1:1:5~7,v at 60~80 °C /v and HCl/H ₂ O ₂ /H ₂ O 1:1:4~6, v/v soaked for 5~10min respectively; take it out, wash it with clean water, and dry it; then put in 5% v/v Soak in the ethanol solution of 3-aminopropylethoxysilane for 2~5 hours, wash and dry, and then add 10~15mL of phosphoric acid containing 1~2% v/v glutaraldehyde with a pH value of 6.8~7.5 Salt buffer solution, let it stand at 4 ^o C for 8-12 h, then wash it with deionized water, dry it, and set it aside;

c. Install the modified chip in an electrolytic cell with a pore size smaller than the side length of the chip, and drop 50 μL of phosphate buffer solution containing 1-2 mg/mL lectin with a pH value of 6.8-7.5 on the above-mentioned processed chip. , and stood at 4 ^o C for 8-12 h, then washed with deionized water; then soaked the chip in a solution containing 0.5-1% w/v bovine serum albumin and 0.05-0.1% v/v Tween-20 0.5~1 h, then wash with deionized water and set aside;

d. Drop the processed sample to be tested on the prepared chip, place it for 15-50min, and clean the chip surface 3 times with deionized water;

e. Inject a hydrochloric acid solution with a pH value of 1~3 into the electrolytic cell to dissolve the zinc oxide quantum dots on the chip, and wash it three times with a hydrochloric acid solution with a pH value of 1~3, combine the dissolving solution and washing solution, and add a pH value of 6.8~ 7.5 phosphate buffer solution to 1~3 ml;

f. Use a platinum electrode as the counter electrode, a silver/silver chloride electrode as the reference electrode, and a mercury film electrode as the working electrode, enrich at -0.8~-1.5 volts for 90~150 seconds, and measure the dissolution of zinc by square wave voltammetry Peak, the potential is about -1.1 volts;

g. Change the sugar allergen concentration in the first step, repeat steps e, f, and g, measure the peak current of the stripping voltammetry peak of zinc corresponding to a series of sugar allergen concentrations, and establish the peak current and sugar allergen Correspondence between concentrations.

The chip is a silicon dioxide chip or a glass chip.

The correspondence between the stripping voltammetric peak current of zinc measured in the above process and the concentration of carbohydrate allergens is the important basic data of the inventive method, and provides a theoretical basis for concentration judgment for carrying out actual clinical detection with this inventive method, Using the method of the invention to perform rapid and accurate clinical detection has extremely important significance.

 The above-mentioned lectins and carbohydrate allergens are concanavalin and its egg mucin.

The above-mentioned chip is silicon dioxide or glass.

Beneficial effect: compared with the prior art, the present invention has the following advantages:

1. The labeling process and dissolution process of zinc oxide quantum dots on carbohydrate allergens of the present invention are simple in operation and mild in conditions, and the combination of quantum dots and carbohydrate allergens is stable and firm. It is very beneficial to maintain the biological activity of carbohydrate allergens.

2. The present invention uses stripping voltammetry to measure the concentration of zinc ions, which is easy for the enrichment of zinc on the mercury electrode, the stripping peak of zinc is easy to measure and the peak current is strong, and it is easy to establish an accurate relationship between the peak current and the concentration of carbohydrate allergens. curve relationship. It is beneficial to reduce the detection limit of sugar allergen detection and improve detection sensitivity. It makes it possible to quickly and accurately detect low-concentration sugar allergens.

Description of drawings

The following drawings are used to illustrate specific embodiments of the present invention, but are not intended to limit the scope of the present invention defined by the claims.

Figure 1 is a schematic diagram of the assembly of the allergen detection chip.

Fig. 2 is the detection result of the allergen chip of the standard sample. 

Detailed ways

(Figure 1)

1. Disperse 0.5 mg of zinc oxide quantum dots into 50 microliters of 1 ng/ml chicken ovomucin solution, shake at room temperature for 10 minutes, centrifuge, and wash three times with a phosphate buffer solution with a pH value of 7.0. Add 500 μL of 1% (w/v) bovine serum albumin, shake at room temperature for 10 minutes, centrifuge, and wash three times with phosphate buffer solution with a pH value of 7.0. Finally, disperse in 50 μL 0.1% (v/v) Triton X-100 phosphate buffer solution with a pH value of 7.0, and store it in the insulation layer of the refrigerator when not in use.

2. Chip modification, silicon wafer (1x1x0.2mm) was sonicated in acetone, ethanol and water for 5min respectively, and then in NH ₄ OH/H ₂ O ₂ /H ₂ O (1:1:7, v/ v) and HCl/H ₂ O ₂ /H ₂ O (1:1:6, v/v) were soaked for 10 minutes respectively. After taking it out, wash it with clean water and let it dry. Then soak in 5% (v/v) 3-aminopropylethoxysilane ethanol solution for 5 h, wash and dry, and then put in 2% (v/v) glutaraldehyde pH value 7.0 phosphate buffer solution, let stand at 4 ^o C for 12 h, then wash with deionized water, dry and set aside.

3. Detection chip preparation, the modified chip was installed in an electrolytic cell with a pore size smaller than the side length of the chip, and 50 μL of phosphate buffer solution containing 1 mg/mL concanavalin with a pH value of 7.0 was dropped on the above-mentioned processed chip. Chips were placed at 4 ^° C for 12 h, and then washed with deionized water. Then the chip was soaked in 1% (w/v) bovine serum albumin and 0.05% (v/v) Tween-20 for 1 h, then washed with deionized water and set aside.

4. Detection method: drop the treated egg mucin sample on the prepared concanavalin chip, leave it for 15 minutes, and wash the surface of the chip with deionized water for 3 times.

5. Inject 1800 μL of hydrochloric acid solution with a pH value of 2 into the electrolytic cell and sonicate for 10 minutes to dissolve the zinc oxide quantum dots on the chip, then transfer the solution to the electrolytic cell and add 2000 μL of phosphate buffer solution with a pH value of 7.01 in sequence , 4uL 10 ppm Mercury(II) Nitrate. 50 μL of sodium hydroxide solution (0.32 M). Then by electrochemical detection,

6. Use a platinum electrode as the counter electrode, a silver/silver chloride electrode as the reference electrode, and a mercury film electrode as the working electrode, and enrich at -1.3 V for 2 min. Use square wave voltammetry to measure the dissolution peak of zinc, and the potential is about at -1.1 volts.

7. Change the concentration of chicken ovomucin in the first step, repeat steps 4, 5, and 6, measure the peak current of the stripping voltammetry peak of zinc corresponding to a series of chicken ovomucin concentrations, and establish the peak current and chicken ovomucin Correspondence between concentrations (Figure 2).

Claims (1)

1. A method for measuring carbohydrate allergens, characterized in that it utilizes zinc oxide quantum dots to label carbohydrate allergens; at the same time, a layer of lectin is immobilized on the chip in the electrolytic cell, and is tested in a series of known concentrations. In the sugar allergen solution, the sugar allergen marked with zinc oxide quantum dots is specifically combined with the lectin on the chip, and the zinc oxide quantum dots marked on the sugar allergen are connected to the chip; The acidic solution dissolves the zinc oxide quantum dots into zinc ions, measures the concentration of the dissolved zinc ions by stripping voltammetry, establishes the relationship between the concentration of zinc ions and the concentration of sugar allergens, thereby determining the concentration of sugar allergens to be measured; The assay method of the carbohydrate allergen specifically comprises the following steps: a. Disperse 0.5-5 mg of zinc oxide quantum dots into 50-200 microliters of 1 ng/ml sugar allergen solution, shake at room temperature for 10 minutes, centrifuge, and buffer with phosphate with a pH value of 6.8-7.5 Wash the solution three times, add 500 μL 1-2% w/v bovine serum albumin, shake at 15-35°C for 10-30 minutes, centrifuge, wash three times with phosphate buffer solution with a pH value of 6.8-7.5; finally disperse in 50-150 μL. .05～0.1%v/v Triton X-100 Phosphate Buffer Solution, stored in the insulation layer of the refrigerator when not in use; b. Sonicate the silicon dioxide or glass slices in acetone, ethanol and water for 5 minutes respectively, and then in NH ₄ OH/H ₂ O ₂ /H ₂ O1:1:5～7, v/v at 60-80°C Soak with HCl/H ₂ O ₂ /H ₂ O1:1:4~6, v/v for 5~10 minutes respectively; take it out, wash it with clean water, and dry it; then put in 5% v/v 3-amine Soak in ethanol solution of propylethoxysilane for 2-5 hours, wash and dry, then put 10-15mL phosphate buffer solution containing 1-2% v/v glutaraldehyde with pH value of 6.8-7.5, Stand at 4°C for 8-12 hours, then wash with deionized water, dry in the air, and set aside; c. Install the modified chip in an electrolytic cell with a pore size smaller than the side length of the chip, drop 50 μL of phosphate buffer solution containing 1-2 mg/mL lectin with a pH value of 6.8-7.5 on the above-mentioned treated chip, And stand at 4°C for 8-12 hours, then wash with deionized water; then soak the chip with 0.5-1% w/v bovine serum albumin and 0.05-0.1% v/v Tween-20 for 0.5-1 hour, Then rinse with deionized water and set aside; d. Drop the processed sample to be tested on the prepared chip, place it for 15-50 minutes, and clean the surface of the chip with deionized water for 3 times; e. Inject a hydrochloric acid solution with a pH value of 1 to 3 into the electrolytic cell to dissolve the zinc oxide quantum dots on the chip, and wash it three times with a hydrochloric acid solution with a pH value of 1 to 3, combine the dissolving solution and the washing solution, and add a pH value of 6.8 to 6.8. 7.5 phosphate buffer solution to 1-3 ml; f. Use a platinum electrode as the counter electrode, a silver/silver chloride electrode as the reference electrode, and a mercury film electrode as the working electrode, enrich at -0.8 to -1.5 volts for 90 to 150 seconds, and measure the dissolution of zinc by square wave voltammetry Peak, the potential is about -1.1 volts; g. Change the sugar allergen concentration in the first step, repeat steps e, f, and g, measure the peak current of the stripping voltammetry peak of zinc corresponding to a series of sugar allergen concentrations, and establish the peak current and sugar allergen Correspondence between concentrations.

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