CN111521775A - A method for preparing paper-based microfluidic chip for bisphenol A detection based on wax spray printing technology - Google Patents

Abstract

The invention discloses a method for preparing a paper-based micro-fluidic chip for quantitative detection of bisphenol A based on a wax-spraying printing technology. The method comprises the following steps: placing paper printed by a wax-spraying printer in an oven to melt wax, and preparing a paper-based microfluidic chip with a hydrophilic channel and a hydrophobic channel on a paper substrate; carrying out surface modification on the paper substrate reaction area to ensure that the paper substrate reaction area is covalently coupled with the antibody; the sample and the enzyme-labeled antigen are subjected to antigen-antibody reaction through direct competition with the antibody, and then color development is performed through TMB; after color development, a mobile phone is used for photographing, and data processing and gray level analysis are carried out through software such as Excel and Image J, so that a novel method for rapidly, accurately and highly sensitively quantitatively detecting bisphenol A is established and used for quantitatively detecting bisphenol A. The whole detection process is simple to operate, high in analysis speed and small in required sample volume, can detect a plurality of samples simultaneously, and provides a new detection method for detecting other target objects.

Description

A paper-based microfluidic chip for the detection of bisphenol A based on wax spray printing technology Methods

Technical field:

The invention relates to the technical field of detection, in particular to a paper-based microfluidic chip prepared based on a wax spray technology for the detection of bisphenol A.

Background technique:

Microfluidic analysis technology can integrate multiple analysis platforms, and to a great extent integrate the functions of the entire analysis platform into small analytical instruments, even on micron-scale microfluidic chips. As a liquid transport platform at the microscopic scale, the microfluidic chip can quantitatively and stably control the liquid flow, so as to realize biochemical analysis, DNA measurement, protein screening, microdroplet manipulation, cell separation, material synthesis, etc. a function. Microfluidic chip technology has the advantages of low consumption, short sample processing time, high detection sensitivity and high resolution. It can also integrate sample processing, separation, reaction and other analysis-related processes, which greatly improves the efficiency of analysis. Compared with traditional laboratories, microfluidic technology has the advantages of miniaturization, portability, rapid reaction, small sample requirements, and integration of various functions. Because of these advantages, microfluidic technology is an ideal platform to transform the concept of on-site rapid and real-time detection into reality, and has great research value and application value. Paper-based core products shine in the field of microfluidic chips with their unique advantages.

Paper-based Microfluidic Device was originally proposed by the Whitesites research group in 2007, using filter paper as the base material to build a "Lab-on-paper", using lithography, spraying It is made of wax printing, flexographic printing and other technologies, and has been used in biological analysis, colorimetric analysis, electrochemical analysis and other research fields. Based on filter paper, the hydrophilic/hydrophobic micro-channel network and related analysis devices are formed through various processing techniques, and a "paper-based micro-lab" is constructed. Compared with the traditional microfluidic chip technology, the paper-based microfluidic chip has both The advantages of paper: low cost, simple process, simple and pollution-free post-processing, strong biocompatibility, etc.

Bisphenol A is mainly used in the production of polycarbonate plastics and epoxy resins. It is one of the most common endocrine disruptors, which can be released through a variety of ways, posing a threat to human health. Animal experiments have shown that bisphenol A has the effect of environmental hormones, and even low doses of bisphenol A can have adverse effects on animals. BPA can also enter organisms through the food chain and interact with estrogen receptors, thereby affecting reproductive, immune, nervous and endocrine systems. At the same time, many studies have shown that bisphenol A has certain teratogenicity and embryotoxicity, which can significantly increase the probability of animals suffering from cancer. In addition, bisphenol A can also synergize with ultraviolet light or cadmium, increasing the harm to the human body. As BPA is widely used in the production of plastic packaging materials, it is often released and transferred to the environment and food, so its presence cannot be ignored.

So far, the detection of bisphenol A mainly relies on physical and chemical analysis methods such as high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-multi-stage mass spectrometry, etc. The detection methods used generally have the characteristics of high sensitivity, good selectivity and high precision, but they need to be completed in the laboratory and require trained professionals. Most of the instruments used are expensive, complicated to operate, and time-consuming. , it is difficult to meet the needs of high-throughput and rapid detection of a large number of samples. Therefore, it is of great scientific and practical significance to study a specific, sensitive, convenient and efficient rapid detection method for bisphenol A, and to develop the corresponding detection technology and equipment.

SUMMARY OF THE INVENTION

The invention discloses a method for rapidly detecting bisphenol A by preparing a paper-based microfluidic chip based on a wax spray technology. The use of wax spray printing technology can realize flexible design patterns, combined with paper-based microfluidics technology that is easy to process, easy to operate, and portable, and the stable combination of bisphenol A antibody and sodium carboxymethyl cellulose adsorption, and obtain bisphenol A standard The curve established a fast, accurate and highly sensitive method for the detection of BPA.

The object of the present invention is achieved in this way. A method for preparing a paper-based microfluidic chip for bisphenol A detection based on a wax spray printing technology, comprising the following steps:

(1) Using chromatographic paper as the base, the template drawn by CAD is printed by a wax spray printer. The template includes a hydrophobic channel and a hydrophilic channel, and the hydrophilic channel includes a reaction area for immune reaction. The zone is a closed reaction zone; the chromatographic paper with the printed pattern is placed in an oven to bake to obtain a paper-based microfluidic chip;

(2) Modification treatment in the reaction zone: the paper-based microfluidic chip in the reaction zone is subjected to surface modification treatment;

(3) adding the coating antibody corresponding to the antigen to be tested in the reaction zone;

(4) adding blocking agent in the reaction zone;

(5) The detection of bisphenol A is realized by colorimetric method.

Preferably, the chromatographic paper with the printed pattern is placed in an oven at 110° C. for 5-8 minutes.

Preferably, the paper-based microfluidic chip obtained in step (1) adopts a 12×6 array, the reaction area is a small hole with a diameter of 5mm, and the hole spacing is 8mm. In order to suppress the flow of the sample between the papers, use AutoCAD on a white background The hydrophobic barrier is designed to be black, and the advantages of using a 12×6 array are: the first three rows of 12×3 columns can make a standard curve at one time, reducing the human error caused by repeated operations, and the last three rows of 12×3 columns can be used for multiple samples It can improve the detection accuracy by ensuring that the samples and the standard curve are operated under the same conditions. The invention designs a 12×6 square array of microfluidic chips, which has the advantage of being able to detect multiple samples at one time, saving time and repeating the detection of the samples to improve the detection accuracy;

Preferably, the chromatography paper used in step (1) is whatman chromatography paper. The quality and uniformity of whatman chromatography paper is good.

Preferably, in step (2), the paper-based microfluidic chip is surface-modified with sodium carboxymethyl cellulose, so that the surface of the paper-based microfluidic chip has carboxyl groups. The specific steps are as follows: The sodium carboxymethyl cellulose is dissolved in deionized water, dialyzed to a salt-free state with a 10-12kDa filter membrane, and then freeze-dried for 5-10 minutes. The sodium carboxymethyl cellulose obtained after freeze-drying is similar to marshmallows. Dissolve the purified carboxymethyl cellulose in calcium chloride solution, and drop 10-15 μL of 0.2 g/L sodium carboxymethyl cellulose into the prepared paper-based microfluidic well. By modifying the surface of the paper-based microfluidic chip, the surface of the paper-based microfluidic chip has carboxyl groups, which can improve the coupling rate of antibodies and provide more binding sites for subsequent samples and enzyme-labeled antigens. .

The paper-based modification usually uses chitosan glutaraldehyde, while the present invention uses sodium carboxymethyl cellulose to surface functionalize the paper-based cellulose, which is selected because of its chemical and physical properties. That is, it has carboxyl groups (for EDC/NHS chemistry) and is irreversibly adsorbed on paper-based cellulose fibers under suitable conditions. The irreversible adsorption is not only due to the reduced charge repulsion but also due to the hydrogen bonding between the unsubstituted glucopyranoside of sodium carboxymethylcellulose and the glucopyranoside of cellulose. In addition to the charge, the addition of sodium carboxymethyl cellulose also changed other physical properties of the paper-based cellulose, such as surface swelling and hydration. The enhanced swelling properties and hydrogel-like structure can facilitate immobilization and stability of biomolecules, thereby favoring a hydrophilic environment. Antibodies are then covalently linked to sodium carboxymethylcellulose by irreversible adsorption of sodium carboxymethylcellulose. This will provide a stable cellulose biointerface for subsequent immunoassays. The stable binding of bisphenol A antibody to the adsorption of sodium carboxymethyl cellulose was achieved, and the standard curve of bisphenol A was obtained. At the same time, the surface modification of paper base with sodium carboxymethyl cellulose can also prevent non-specific protein adsorption.

Preferably, in step (3), a coating antibody corresponding to the antigen to be tested is added to the reaction area, and the coating antibody is cross-linked with the carboxyl groups on the surface of the paper-based microfluidic chip. The specific steps are: in the modified paper-based microfluidic chip The surface of the control chip was activated with 3-6 μL of 4 mg/mL EDC1-(3-dimethylaminopropyl)-3-ethylcarbodiimide), 3-6 μL of 6 mg/mL NHS (N-N-hydroxysuccinimide)12 -18min, convert the carboxyl group of sodium carboxymethyl cellulose into amine-reactive ester, and then in PBS buffer solution with pH 7.5, covalently bind bisphenol A monoclonal antibody to sodium carboxymethyl cellulose in a certain amount. On the active site of the activity; use pH 8.5 ethanolamine hydrochloride solution to remove unreacted NHS, and then use PBST to remove unconnected bisphenol A antibody.

Preferably, the blocking process in step (4) is as follows: 8-12 μL of 1 mg/ml milk powder blocking solution is dropped into the paper-based microfluidic well for blocking, and then washed three times with PBST to wash the excess blocking solution.

Preferably, the step (5) detection includes the following steps:

(1) Add 5 μL of different concentrations of bisphenol A standard and enzyme-labeled antigen into the microfluidic reaction well at a volume ratio of 1:1, and then wash with 10 μL PBSTbuffer four times;

(2) Color development: Mix substrate A and substrate B, add 5 μL of the mixture to the microfluidic reaction well for color development, and take pictures with a mobile phone after color development;

(3) Analysis: Data processing and grayscale analysis were performed with Excel and ImageJ software, and a standard curve was drawn.

Preferably, the volume ratio of Substrate A to Substrate B is 32.44:1; Substrate A is 0.43g urea hydrogen peroxide, 2.5g β-dextrin and 8.2g anhydrous sodium acetate to adjust the pH to 5.0, and determine with ultrapure water. Make up to 1000ml, store at 4°C, take out from 4°C and return to room temperature before use; Substrate B is 100mg TMB (3,3',5,5'-tetramethylbenzidine) and 10ml DMSO (dimethyl sulfoxide) Mix and store in a brown bottle in a cool place until later.

The principle of the technical solution is that the antibody bound to the surface of the paper base still maintains its immunological activity, and the enzyme-labeled antigen retains both its immunological activity and the enzymatic activity. During detection, the test substance (antigen) and the enzyme-labeled antigen in the sample competitively bind with the immobilized antibody, and the unbound substances are removed by washing, and the amount of the enzyme that can be immobilized is related to the amount of the test substance in the sample. By adding the substrate reacting with the enzyme, the color is developed, and the content of the substance in the sample can be judged according to the depth of the color, and the qualitative or quantitative analysis can be carried out. Due to the high catalytic efficiency of the enzyme, the results of the immune reaction are indirectly amplified, and the assay method achieves high sensitivity.

The paper-based microfluidic core product designed in the present invention takes chromatographic paper as the base, forms hydrophilic/hydrophobic micro-channel network and related analysis devices through wax spray processing technology, and builds a "miniature laboratory on paper". Fluidic chip technology, paper-based microfluidic chip has the advantages of paper: low cost, simple process, simple and pollution-free post-processing, and strong biocompatibility.

Large-scale instrumentation requires advanced and expensive instrumentation, as well as complex, laborious, and time-consuming preprocessing steps. Traditional enzyme-linked immunosorbent assay (ELISA) is relatively expensive because it requires large amounts of analyte samples and reagents (especially antibodies) and an expensive microplate reader to collect data. However, using this method to detect bisphenol A can not only save the amount of analytical reagents (only a few μL of reagents are needed), save the sample pretreatment time and greatly reduce the detection time, but also reduce the detection cost, and can meet the requirements of rapid, sensitive and accurate detection. On-site testing or routine testing needs.

Description of drawings:

Fig. 1 is a kind of working reagent volume used in the preparation of paper-based microfluidic chip based on wax spray printing technology for detecting bisphenol A of the present invention;

2 is a microfluidic chip template for preparing a paper-based microfluidic chip for detecting bisphenol A based on the wax spray printing technology of the present invention;

3 is a working principle of a paper-based microfluidic chip prepared based on the wax spray printing technology for detecting bisphenol A according to the present invention;

FIG. 4 is a standard curve of a paper-based microfluidic chip prepared based on the wax spray printing technology of the present invention for detecting bisphenol A;

FIG. 5 shows the optimization of the amount of antibody coating in the experimental process of preparing a paper-based microfluidic chip based on the wax spray printing technology for the detection of bisphenol A according to the present invention.

Detailed ways:

The following is a further description in conjunction with the accompanying drawings

The working principle of the invention is as follows: a paper-based chip is prepared on a chromatographic paper by using a wax spray printer, the bisphenol A monoclonal antibody is fixed on the modified paper base, and the color-developing substrate is catalyzed according to the specificity of the antigen, antibody and enzyme. In principle, the bisphenol A standard and enzyme-labeled antigen are added to the chip, and the standard and enzyme-labeled antigen competitively bind to the antibody. The color gradually becomes lighter, and the gray value of the color is analyzed by Image J. As can be seen in Figure 4, as the logarithm of the concentration increases, the relative gray value gradually increases, so BPA can be detected by colorimetry.

Example 1

The steps to study the working reagents required for the microfluidic chip are as follows:

(1) Drawing with CAD: A 12×6 array was used, the diameter of the reaction zone was 5mm, and the hole spacing was 8mm. In order to suppress the flow of the sample between the papers, Auto CAD was used to design the hydrophobic channel to be black on a white background.

(2) Wax printer printing: Microfluidic paper-based patterns were printed on chromatographic paper using a wax printer.

(3) Place the printed chromatographic paper in a 110°C oven for 6 minutes to melt the wax.

To determine the required volume of working reagents (PBST buffer, blocking solution, sample, substrate A + substrate B) on the paper-based chip, different volumes (1-10 μL) of crystal violet dye solution [0.025% (w/v) ] were added to the chip and the results were evaluated visually at room temperature. It can be seen from Figure 1 that the maximum amount of reagent that can be tolerated, and the amount of reagent that is just impermeable is 10 μL.

Finally, the PBST buffer required for the microfluidic chip was selected as 10 μL, the sample and enzyme-labeled antigen were mixed at a volume ratio of 1:1 to 5 μL, the blocking solution was 10 μL, and the volume ratio of substrate (substrate A to substrate B) was selected. 32.44: 1) 5μL, the substrate A is 0.43g urea hydrogen peroxide, 2.5g β-dextrin and 8.2g anhydrous sodium acetate, adjust the pH to 5.0, make up to 1000ml with ultrapure water, store at 4°C for later use Take it out from 4°C and return to room temperature before; Substrate B is a mixture of 100 mg TMB and 10 mL DMSO, and is stored in a brown bottle in a cool place for later use.

Example 2

The steps of preparing a paper-based microfluidic chip for detecting bisphenol A based on the wax spray technology of the present invention are as follows:

(1) Drawing with CAD: A 12×6 array was used, the diameter of the reaction zone was 5 mm, and the hole spacing was 8 mm. In order to inhibit the flow of the sample between the papers, the hydrophobic barrier designed on a white background using Auto CAD was black.

(2) Wax printer printing: Microfluidic patterns were printed on whatman chromatography paper using a wax printer.

(3) Place the printed chromatographic paper in a 110°C oven for 6 minutes to melt the wax.

(4) Surface modification of the paper-based reaction zone: surface modification with sodium carboxymethyl cellulose, the specific steps are as follows: dissolving sodium carboxymethyl cellulose in deionized water, and dialyzing it to salt-free with a 11kDa filter membrane After freeze-drying for 8 min, the purified carboxymethyl cellulose was dissolved in calcium chloride solution, and 13 μL of 0.2 g/L sodium carboxymethyl cellulose was dropped into the prepared paper-based microfluidic well.

(5) The antibody was immobilized in the reaction zone: activated on the surface of the modified paper-based microfluidic chip with 5 μL EDC (4 mg/ml) and 5 μL NHS (6 mg/ml) for 15 min, and sodium carboxymethyl cellulose was The carboxyl group was converted into an amine-reactive ester, and then the bisphenol A monoclonal antibody was covalently bound to the activated site modified by sodium carboxymethyl cellulose according to the amount of 0.1 μg/well in PBS buffer solution with pH 7.5; Unreacted NHS was removed with ethanolamine hydrochloride solution at pH 8.5, and unlinked bisphenol A antibody was removed with PBST.

(6) Blocking: Take 10 μL of 1 mg/mL skimmed milk powder blocking solution, drop it into a paper-based microfluidic well for blocking, and then wash with PBST three times to wash the excess blocking solution.

Example 3

According to the method of Example 2, except that the amount of bisphenol A monoclonal antibody added in step (5) is different, A-F indicates that the amount of conjugated antibody is 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125 μg/well, respectively, and proceed to the next step Reactions and three groups were compared, of which 1.4.7 was listed as the control group (the sample added in the reaction was 2.5μL PBS+2.5μL enzyme-labeled antigen); Phenol A standard substance + 2.5 μl enzyme-labeled antigen); 3.6.9 is listed as blank (5 μL PBS was added in the reaction). It can be seen from Figure 5 that the color of the control group is the darkest, the color of the experimental group is slightly lighter, and the color of the blank group is the lightest. Through experiments, it was found that with the gradual reduction of the antibody coupling amount, the degree of color development gradually decreased, and the color difference was not obvious, which would affect the accuracy of the experiment; when the coupling amount was 0.1 μg/well, the background color interference was the lowest, and the experimental group The color development is moderate, and the color is negatively correlated with the sample concentration, and the linear trend is significant; when the antibody coupling amount exceeds 0.1 μg/well, it will cause unnecessary waste of antibody, so the effect is better when the antibody coupling amount is 0.1 μg/well.

Example 4

The steps of preparing a paper-based microfluidic chip for detecting bisphenol A based on the wax spray technology of the present invention are as follows:

(1) Drawing with CAD: 12 × 6 arrays were used, the diameter of the reaction area was 5 mm, and the hole spacing was 8 mm. In order to suppress the flow of the sample between the papers, AutoCAD was used to design the hydrophobic barrier to be black on a white background.

(2) Wax printer printing: Microfluidic patterns were printed on whatman chromatography paper using a wax printer.

(3) Place the printed chromatographic paper in a 110°C oven for 5 minutes to melt the wax.

(4) Surface modification of paper-based reaction zone: surface modification with sodium carboxymethyl cellulose, the specific steps are as follows: dissolving sodium carboxymethyl cellulose in deionized water, and dialyzing it to salt-free with a 10kDa filter membrane After freeze-drying for 5 min, the purified carboxymethyl cellulose was dissolved in calcium chloride solution, and 10 μL of 0.2 g/L sodium carboxymethyl cellulose was dropped into the prepared paper-based microfluidic well.

(5) The antibody was immobilized in the reaction zone: on the surface of the modified paper-based microfluidic chip, 3 μL EDC (4 mg/ml) and 3 μL NHS (6 mg/ml) were used for activation for 10 min. The carboxyl group was converted into an amine-reactive ester, and then the bisphenol A monoclonal antibody was covalently bound to the activated site modified by sodium carboxymethyl cellulose according to the amount of 0.1 μg/well in PBS buffer solution with pH 7.5; Unreacted NHS was removed with ethanolamine hydrochloride solution at pH 8.5, and unlinked bisphenol A antibody was removed with PBST.

(6) Blocking: Take 8 μL of 1 mg/mL skim milk powder blocking solution, drop it into the paper-based microfluidic well for blocking, and then wash with PBST three times to wash the excess blocking solution.

Example 5

The steps of preparing a paper-based microfluidic chip for detecting bisphenol A based on the wax spray technology of the present invention are as follows:

(1) Drawing with CAD: A 12×6 array was used, the diameter of the reaction zone was 5 mm, and the hole spacing was 8 mm. In order to suppress the flow of the sample between the papers, Auto CAD was used to design the hydrophobic barrier to be black on a white background.

(2) Wax printer printing: Microfluidic patterns were printed on filter paper using a wax printer.

(3) Place the printed chromatographic paper in a 110°C oven for 8 minutes to melt the wax.

(4) Surface modification of paper-based reaction zone: surface modification with sodium carboxymethyl cellulose, the specific steps are as follows: dissolving sodium carboxymethyl cellulose in deionized water, and dialyzing it with a 12kDa filter until it is salt-free After freeze-drying for 10 min, the purified carboxymethyl cellulose was dissolved in calcium chloride solution, and 15 μL of 0.2 g/L sodium carboxymethyl cellulose was dropped into the prepared paper-based microfluidic well.

(5) The antibody was immobilized in the reaction zone: activated on the surface of the modified paper-based microfluidic chip with 6 μL EDC (4 mg/mL) and 6 μL NHS (6 mg/mL) for 18 min, and sodium carboxymethyl cellulose was activated for 18 min. The carboxyl group was converted into an amine-reactive ester, and then the bisphenol A monoclonal antibody was covalently bound to the activated site modified by sodium carboxymethyl cellulose according to the amount of 0.1 μg/well in PBS buffer solution with pH 7.5; Unreacted NHS was removed with ethanolamine hydrochloride solution at pH 8.5, and unlinked bisphenol A antibody was removed with PBST.

Example 6 standard curve drawing

(1) Reaction: Different concentrations of bisphenol A standard substance and enzyme-labeled antigen were added to the microfluidic reaction well obtained in Example 2 at a volume ratio of 1:1 for reaction, and then washed four times with PBST buffer solution. Take 1000μg/L, 100μg/L, 33.33μg/L, 11.11μg/L, 3.703μg/L, 1.234μg/L, 0.411μg/L, 0.137μg/L, 0.045μg/L concentration of bisphenol A standard respectively. The product and the enzyme-labeled antigen were mixed at a volume ratio of 1:1 and added to the chip for reaction.

(2) Color development: Mix substrate A and substrate B, add 5 μL of the mixture to the microfluidic reaction well for color development, and take photos with a mobile phone in time after color development.

(3) Analysis: Data processing and grayscale analysis were performed with Excel and Image J software, and a standard curve was drawn.

Obviously, the corresponding colors of the standard products with different concentrations are also different. It can be seen from Figure 4 that the concentration of the standard products is positively correlated with the gray value. According to this method, a standard library with one-to-one correspondence between relative gray values and bisphenol A concentration can be established, so that the concentration of unknown bisphenol A samples can be detected.

The molecular weight of the sodium carboxymethyl cellulose is 250kDa.

The concentration of the ethanolamine hydrochloride solution is 1M.

The detection of embodiment 7 quality control product

Table 1. Precision test for the detection of bisphenol A content in quality control products

It can be seen from the test data in Table 1 that the lowest and highest values of the intra-assay coefficient of variation (CV) are both controlled within 10%, indicating that the precision of the paper-based chip is good.

Claims (10)

1. a method for preparing paper-based microfluidic chip for bisphenol A detection based on wax spray printing technology, is characterized in that: comprise the steps: (1) Using chromatographic paper as the base, the template drawn by CAD is printed by a wax spray printer. The template includes a hydrophobic channel and a hydrophilic channel. The hydrophilic channel is a reaction zone for immune reaction. The reaction zone It is a closed reaction zone; the chromatographic paper with the printed pattern is placed in an oven to bake to obtain a paper-based microfluidic chip; (2) Modification treatment in the reaction zone: the reaction zone in the paper-based microfluidic chip is subjected to surface modification treatment; (3) adding the coating antibody corresponding to the antigen to be tested in the reaction zone; (4) adding blocking agent in the reaction zone; (5) The detection of bisphenol A is realized by colorimetric method. 2. a kind of method for preparing paper-based microfluidic chip for bisphenol A detection based on wax spray printing technology according to claim 1, is characterized in that: step (1) places the chromatographic paper of printed pattern at 110 ℃ Bake in the oven for 5-8min. 3. a kind of method for preparing paper-based microfluidic chip for bisphenol A detection based on wax spray printing technology according to claim 1, is characterized in that: the paper-based microfluidic chip obtained in step (1) adopts 12 × 6 arrays, the reaction area is a small hole with a diameter of 5mm, the hole spacing is 8mm, and the hydrophobic channel is designed to be black on a white background using Auto CAD. 4 . A method for preparing a paper-based microfluidic chip for bisphenol A detection based on wax spray printing technology according to claim 1 , wherein the chromatography paper used in step (1) is whatman chromatography paper. 5 . 5. a kind of method for preparing paper-based microfluidic chip for bisphenol A detection based on wax spray printing technology according to claim 1, is characterized in that: step (2) paper-based microfluidic chip uses carboxymethyl fiber The specific steps are as follows: dissolving sodium carboxymethyl cellulose in deionized water, dialyzing it to a salt-free state with a 10-12kDa filter membrane, and then freeze-drying it for 5-10min. The sodium carboxymethyl cellulose was dissolved in calcium chloride solution, and 10-15 μL of 0.2 g/L sodium carboxymethyl cellulose was dropped in the prepared paper-based microfluidic well. 6. a kind of method for preparing paper-based microfluidic chip for bisphenol A detection based on wax spray printing technology according to claim 1, is characterized in that: step (3) adds the coating corresponding to the antigen to be tested in the reaction zone Antibodies, coated antibodies and carboxyl groups on the surface of the paper-based microfluidic chip are cross-linked and fixed. The specific steps are: on the surface of the modified paper-based microfluidic chip, use 3-6 μL of 4 mg/mL EDC, 3-6 μL of 6 mg/mL NHS was activated for 12-18min, the carboxyl group of sodium carboxymethylcellulose was converted into amine-reactive ester, and then the bisphenol A monoclonal antibody was covalently bound to carboxymethylcellulose in a certain amount in PBS buffer solution with pH 7.5 On the activated site modified with sodium urea; unreacted NHS was removed with ethanolamine hydrochloride solution at pH 8.5, and unconnected bisphenol A antibody was removed with PBST. 7. a kind of method for preparing paper-based microfluidic chip for bisphenol A detection based on wax spray printing technology according to claim 6, is characterized in that: step (3) adds the coating corresponding to the antigen to be tested in the reaction zone Antibodies, coated antibodies and carboxyl groups on the surface of the paper-based microfluidic chip are cross-linked and immobilized. The specific steps are: activate the surface of the modified paper-based microfluidic chip with 5 μL of 4 mg/mL EDC and 5 μL of 6 mg/mL NHS for 15 min , the carboxyl groups of sodium carboxymethylcellulose were converted into amine-reactive esters, and then the bisphenol A monoclonal antibody was covalently bound to sodium carboxymethylcellulose at a concentration of 0.1 μg/well in PBS buffer solution at pH 7.5. On the modified activated site; use pH 8.5 ethanolamine hydrochloride solution to remove unreacted NHS, and then use PBST to remove unlinked bisphenol A antibody. 8. a kind of method for preparing paper-based microfluidic chip for bisphenol A detection based on wax spray printing technology according to claim 1, is characterized in that: step (4) sealing process is: get the milk powder of 1mg/ml 8-12 μL of blocking solution was dropped into paper-based microfluidic wells for blocking, and then washed three times with PBST to wash excess blocking solution. 9. A method for preparing a paper-based microfluidic chip for bisphenol A detection based on wax spray printing technology according to claim 1, wherein the detection in step (5) comprises the following steps: (1) Add different concentrations of bisphenol A standard substance and enzyme-labeled antigen into the microfluidic reaction well at a volume ratio of 1:1 for the reaction. The amount added is 5 μL. After incubating for 15 min, wash with 10 μL PBST buffer for four times. ; (2) Color development: Mix substrate A and substrate B, add 5 μL of the mixture to the microfluidic reaction well for color development, and take pictures with a mobile phone after color development; (3) Analysis: Data processing and grayscale analysis were performed with Excel and Image J software, and a standard curve was drawn. 10. The method for preparing a paper-based microfluidic chip for bisphenol A detection based on wax spray printing technology according to claim 9, wherein the volume ratio of substrate A to substrate B is 32.44:1; Substrate A is 0.43g urea hydrogen peroxide, 2.5g β-dextrin and 8.2g anhydrous sodium acetate to adjust the pH to 5.0, dilute to 1000ml with ultrapure water, store at 4°C, take out from 4°C and restore to room temperature before use; Substrate B is 100 mg of TMB mixed with 10 ml of DMSO and stored in a brown bottle in a cool place until use.

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