CN115792206A - Application of parylene in improving color development performance of ELISA solid phase carrier - Google Patents

Abstract

The invention discloses application of parylene in improving color rendering property of an ELISA solid phase carrier, and relates to the field of bioanalysis detection. According to the invention, the parylene is loaded on the surface of the ELISA solid-phase carrier, so that the ELISA solid-phase carrier with good antigen/antibody adsorption performance can be obtained, and the color rendering performance of ELISA detection is improved. The preparation process of the parylene-loaded ELISA solid phase carrier provided by the invention is simple, ELISA can be carried out without purchasing the whole kit, and the parylene C film can be removed and then recycled, so that the cost is reduced. Parylene is evaporated on the surface of a solid phase carrier, and can be used together with equipment such as a mobile phone, a spectrometer, POCT and the like to carry out portable detection on disease markers.

Description

Application of parylene in improving color rendering property of ELISA solid phase carrier

Technical Field

The invention relates to the field of bioanalysis detection, in particular to application of parylene in improving the color rendering property of an ELISA solid phase carrier.

Background

The accurate detection of the disease marker can provide important references for early diagnosis of diseases, judgment of benign and malignant tumors, treatment of diseases and prognosis detection. Enzyme-linked immunosorbent assays (ELISA) are considered to be one of the most reliable disease marker detection methods for the qualitative and quantitative determination of pathogen antigens or antibodies in various biological samples. In ELISA, antigen or antibody is firstly combined on the surface of a solid-phase enzyme label plate, after a sample to be detected or diluent thereof is added, the antibody (or antigen) in the sample is specifically combined with a coating antigen (or antibody), a substance which is not combined is washed and removed, and then a labeled antibody (or antigen) of enzyme is added to form an antigen-antibody-enzyme complex with the labeled antibody (or antigen); adding a substrate chromogenic reagent of the enzyme for reaction and color development, and finally determining whether the corresponding antibody or antigen exists in the sample to be detected by naked eye interpretation or scanning of an enzyme labeling instrument.

The ELISA plate is an important solid phase carrier for ELISA, and the solid phase polystyrene surface as the carrier plays an important role in the adsorption of antigen, antibody or antigen-antibody complex. Antigens, antibodies and other biomolecules are adsorbed to the carrier surface by a variety of mechanisms, including passive adsorption through hydrophobic, water/ionic bonds. The existing enzyme label plate is usually contained in a complete kit for sale, is expensive, is basically a 48-well plate and a 96-well plate, and has certain limitation on use.

Disclosure of Invention

The invention aims to provide application of parylene C (parylene C) in improving the color rendering property of an ELISA solid phase carrier, so as to solve the problems in the prior art, and the parylene C is loaded on the surface of the ELISA solid phase carrier, so that the ELISA solid phase carrier with good antigen/antibody adsorption property can be obtained, and the color rendering property of ELISA detection is improved.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides application of parylene in improving color rendering property of an ELISA solid phase carrier.

The invention also provides a method for improving the color rendering property of the ELISA solid phase carrier, which comprises the step of loading a layer of parylene film on the surface of the ELISA solid phase carrier.

Further, the method for loading is evaporation.

Further, the evaporation comprises a step of carrying out evaporation operation by using a vacuum evaporation instrument.

Further, the ELISA solid phase carrier is prepared from a transparent substrate material.

Further, the transparent substrate material is glass or plastic.

The invention also provides an ELISA solid-phase carrier, and a layer of parylene film is loaded on the surface of the ELISA solid-phase carrier.

The invention also provides the application of the ELISA solid phase carrier in preparing an ELISA kit.

The invention discloses the following technical effects:

(1) According to the invention, parylene C is loaded on the surface of the ELISA solid-phase carrier, so that the ELISA solid-phase carrier with good antigen/antibody adsorption performance can be obtained, and the color rendering performance of ELISA detection is improved.

(2) The preparation process of the parylene C-loaded ELISA solid phase carrier provided by the invention is simple, ELISA can be carried out without purchasing the whole kit, and the parylene C film can be removed and then recycled, so that the cost is reduced.

(3) The ELISA solid phase carrier can be diversified, is not limited to 48 or 96 pore plates, can be designed by self and made of various structures and materials, and can be prepared by using transparent substrate materials, such as glass, plastic and the like. Parylene C is evaporated on the surface of the solid phase carrier, and can be used together with equipment such as a mobile phone, a spectrometer, POCT and the like to carry out portable detection on the disease marker.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a color development of a common 96-well plate after completion of ELISA;

FIG. 2 is a color development of 96-well plate evaporated with parylene C after ELISA;

FIG. 3 is a color development of a well plate in an ELISA kit after completion of ELISA.

FIG. 4 shows the color development results of different experimental groups.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every intervening value, to the extent any stated value or intervening value in a stated range, and any other stated or intervening value in a stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. It is intended that the specification and examples be considered as exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.

The general 96-well plate used in the following examples was purchased from Shanghai Lingyo Biotech Co., ltd, and the ELISA kit was purchased from Solebao Biotech Co., ltd.

Example 1

Parylene c was evaporated onto a common 96-well plate:

(1) Weighing 10g of parylene C material, and filling the material into a material port of a parylene C evaporation plating instrument by using tinfoil paper;

(2) Placing a common 96-hole plate on a sample stage of a parylene C vacuum evaporation instrument, and cleaning dust at each interface;

(3) Opening an instrument and starting evaporation;

(4) And (5) after the evaporation is finished, taking out the sample when the temperature is reduced to the room temperature, and obtaining the 96-hole plate for evaporating parylene C.

In order to realize the reuse of the above 96-well plate, it is necessary to remove the parylene c film evaporated on the substrate after the use. The used 96-pore plate for evaporating parylene C is soaked in tetrahydrofuran for 60min, and then is washed by deionized water, so that the parylene C film with the antigen or the antibody can be completely removed.

Effect verification

1. Method for producing a composite material

Experimental group 1: common 96-well plates.

Experimental group 2: the resulting 96-well plate of evaporated parylene c was prepared in example 1.

Experimental group 3: well plate in ELISA kit.

The ELISA detection experiments are carried out by using the 96-well plate respectively and divided into the three experimental groups, and the experimental method is as follows:

coating, adding protein, adding detection antibody, adding HRP standard antibody, and developing.

(1) Diluting Anti-IgG coated antibody with diluent according to a certain proportion, adding the diluted Anti-IgG coated antibody into reaction holes, incubating at 4 ℃ overnight, wherein each reaction hole is 100 microliters;

(2) Washing the pore plate for 3 times by PBS, adding a sealing solution into the pores, and incubating for 2 hours at room temperature, wherein each pore is 200 microliters;

(3) Washing the pore plate for 3 times by PBS, adding IgG protein standard substance with 100 microliters per pore, and reacting for 1 hour at room temperature;

(4) Washing the pore plate for 3 times by PBS, adding an enzyme-labeled antibody into the reaction holes, reacting for 45 minutes at room temperature of 100 microliters per hole;

(5) Washing the pore plate for 3 times by PBS, adding 100 microliters of developing solution into each pore, and reacting for 10 minutes at room temperature;

(6) The well plate was washed 3 times with PBS, and 50. Mu.l of stop solution was added to each well, and photographed by cell phone immediately.

(7) Pictures were processed with ImageJ software and the colorimetric values Δ E were calculated. Delta E is calculated by the formula (1),

and plotting according to the corresponding relation of the chromatic value and the concentration gradient.

2. Results

The color development results for each group are shown in Table 1 and FIGS. 1-4. The result shows that the common 96-pore plate of the experimental group 1 has unobvious color development, and the detection range and the sensitivity are not high; compared with the common 96-well plate, the color rendering property of the 96-well plate of the experiment group 2 is greatly improved, and is lighter than that of the ELISA kit of the experiment group 3 by one well, and specific numerical values are shown in FIG. 4. The method proves that the adsorption of the antigen/antibody can be effectively improved by evaporating parylene C onto a common 96-pore plate, so that the color development performance is improved, and the method has great application prospect. Besides, parylene C can be removed, the enzyme label plate can be recycled, the manufacturing process is simple, and the cost is low.

TABLE 1

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (8)

1. Application of parylene in improving color development performance of ELISA solid phase carrier.

2. A method for improving color development performance of an ELISA solid carrier is characterized by comprising the step of loading a layer of parylene film on the surface of the ELISA solid carrier.

3. The method of claim 2, wherein the loading is performed by evaporation.

4. The method of claim 3, wherein the evaporation comprises a step of performing an evaporation operation using a vacuum evaporator.

5. The method of claim 2, wherein the ELISA solid support is prepared from a transparent substrate material.

6. The method of claim 5, wherein the transparent substrate material is glass or plastic.

7. An ELISA solid phase carrier is characterized in that a parylene film is loaded on the surface of the ELISA solid phase carrier.

8. Use of the ELISA solid support of claim 7 in the preparation of an ELISA kit.

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