CN111521775A - Method for preparing paper-based micro-fluidic chip for bisphenol A detection based on wax-spraying printing technology - Google Patents
Method for preparing paper-based micro-fluidic chip for bisphenol A detection based on wax-spraying printing technology Download PDFInfo
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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
The technical field is as follows:
the invention relates to the technical field of detection, in particular to a paper-based microfluidic chip prepared based on a wax spraying technology and used for detecting bisphenol A.
Background art:
the microfluidic analysis technology can integrate a plurality of analysis platforms, and greatly integrate the functions of the whole analysis platform into a small analysis instrument, even a micron-sized microfluidic chip. The micro-fluidic chip is used as a liquid transport platform under the micro-scale, and can quantitatively and stably control the liquid flow, thereby realizing multiple functions of biochemical analysis, DNA (deoxyribonucleic acid) continuous measurement, protein screening, micro-droplet control, cell separation, material synthesis and the like. The micro-fluidic chip technology has the advantages of less consumption, short sample processing time, high detection sensitivity, high resolution and the like, and can integrate the processes related to analysis, such as sample processing, separation, reaction and the like, thereby greatly improving the analysis efficiency. Compared with the traditional laboratory, the microfluidic technology has the advantages of small volume, portability, rapid reaction, less and quantitative sample requirement and integration of multiple functions. Because of these advantages, the microfluidic technology is an ideal platform for converting the concept of rapid and real-time on-site detection into reality, and has great research value and application value. The paper-based core product has unique advantages and can greatly brighten in the field of microfluidic chips.
Paper-based Microfluidic devices (Paper-based Microfluidic devices) were originally proposed by the Whitesites research group in 2007, a "Lab-on-Paper-based micro laboratory" (Lab-on-Paper) was constructed by using filter Paper as a base material and manufactured by using technologies such as photolithography, wax-spray printing, flexible printing and the like, and the Paper-based Microfluidic devices were already applied to the research fields such as biological analysis, colorimetric analysis, electrochemical analysis and the like. The paper-based micro-fluidic chip has the advantages of paper compared with the traditional micro-fluidic chip technology by using filter paper as a substrate and forming a hydrophilic/hydrophobic micro-channel network and related analytical devices through various processing technologies to construct a micro-laboratory on paper: low cost, simple process, simple post-treatment, no pollution, strong biocompatibility and the like.
Bisphenol a is mainly used for producing polycarbonate plastics and epoxy resin, is one of the most common endocrine disruptors, can be released through various ways, and poses a threat to the health of human bodies. Animal experiments show that bisphenol A has the effect of environmental hormone, and even low dosage of bisphenol A can produce adverse effect on animals. BPA can also enter organisms through the food chain and interact with estrogen receptors, thereby affecting the reproductive, immune, neurological and endocrine systems. Meanwhile, multiple studies show that bisphenol A has certain teratogenicity and embryotoxicity and can obviously increase the probability of cancer of animals. In addition, bisphenol A can also generate synergistic effect with ultraviolet rays or cadmium, and the harm to human bodies is increased. With the widespread use of BPA in the production of plastic packaging materials, it tends to be released and transferred to the environment and to food, and its presence is therefore not negligible.
At present, the detection of bisphenol A mainly depends on physical and chemical analysis means such as High Performance Liquid Chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-multistage mass spectrometry and the like, and the detection method based on chromatography or chromatography-mass spectrometry generally has the characteristics of high sensitivity, good selectivity and high precision, but needs to be completed in a laboratory, needs trained professionals, and most of used instruments are heavy, complex in operation and long in time consumption, and cannot meet the requirements of high-throughput and rapid detection of a large number of samples. Therefore, the research on the bisphenol A rapid detection method with sensitive specificity, convenience and high efficiency and the development of corresponding detection technology and equipment have important scientific and practical significance.
Disclosure of Invention
The invention discloses a method for preparing a paper-based micro-fluidic chip for rapidly detecting bisphenol A based on a wax spraying technology. The method can realize flexible design of patterns by using a wax-spraying printing technology, combines a paper-based micro-fluidic technology which is easy to process, operate and carry with a stable combination of bisphenol A antibody and sodium carboxymethyl cellulose adsorption, and obtains a bisphenol A standard curve to establish a rapid, accurate and highly sensitive bisphenol A detection method.
The object of the invention is thus achieved. A method for preparing a paper-based microfluidic chip for bisphenol A detection based on a wax-spraying printing technology comprises the following steps:
(1) taking chromatographic paper as a substrate, and printing a template drawn by a CAD (computer aided design) through a wax-spraying printer to obtain the template, wherein the template comprises a hydrophobic channel and a hydrophilic channel, the hydrophilic channel comprises a reaction area for generating immunoreaction, and the reaction area is a closed reaction area; placing the chromatographic paper printed with the patterns in an oven for baking to obtain a paper-based microfluidic chip;
(2) and (3) carrying out modification treatment in the reaction zone: the paper-based microfluidic chip in the reaction area is subjected to surface modification treatment;
(3) adding a coating antibody corresponding to the antigen to be detected in the reaction area;
(4) adding a sealing agent into the reaction zone;
(5) the detection of bisphenol A is realized by a colorimetric method.
Preferably, the chromatographic paper printed with the pattern is placed in an oven at 110 ℃ and baked for 5-8 min.
Preferably, the paper-based microfluidic chip obtained in step (1) adopts a 12 × 6 array, the reaction area is small holes with the diameter of 5mm, the hole spacing is 8mm, in order to inhibit the flow of the sample between papers, the hydrophobic barrier is designed to be black on a white background by using AutoCAD, and the 12 × 6 array has the advantages that: the first three rows of 12X 3 columns can make a standard curve at one time, so that human errors caused by repeated operation are reduced, the last three rows of 12X 3 columns can be used for detecting a plurality of samples, the samples and the standard curve are ensured to be operated under the same condition, and the detection accuracy can be improved. The invention designs the 12 x 6 microfluidic chip square column, and has the advantages that a plurality of samples can be detected at one time, so that the time is saved, and the samples are repeatedly detected to improve the detection accuracy;
preferably, the chromatographic paper used in the step (1) is whatman chromatographic paper. The quality and the uniformity of the whatman chromatographic paper are better.
Preferably, the paper-based microfluidic chip in the step (2) is subjected to surface modification treatment by using sodium carboxymethyl cellulose, so that the surface of the paper-based microfluidic chip is provided with carboxyl, and the specific steps are as follows: dissolving sodium carboxymethylcellulose in deionized water, dialyzing with 10-12kDa filter membrane to salt-free state, freeze drying for 5-10min, and freeze drying to obtain sodium carboxymethylcellulose similar to cotton candy state. Dissolving the purified carboxymethyl cellulose in calcium chloride solution, and dripping 10-15 mu L of 0.2g/L sodium carboxymethyl cellulose into the prepared paper-based microfluidic pore. The surface of the paper-based microfluidic chip is modified to make the surface of the paper-based microfluidic chip have carboxyl, so that the coupling rate of the antibody can be improved, and more binding sites are provided for the subsequent addition of a sample and an enzyme-labeled antigen.
The paper base modification usually uses chitosan glutaraldehyde, while the present invention uses sodium carboxymethyl cellulose for surface functionalization of paper base cellulose, which is selected because of its chemical and physical properties. That is, it has carboxyl groups (suitable for EDC/NHS chemistry) and adsorbs irreversibly on paper-based cellulose fibers under suitable conditions. Irreversible adsorption is not only due to the reduction in charge repulsion but also due to 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 changes other physical properties of the paper-based cellulose, such as surface swelling, and hydration can occur. The enhanced swelling characteristics and hydrogel-like structure facilitate the immobilization and stability of biomolecules, thereby favoring a hydrophilic environment. The antibody is then covalently linked to the sodium carboxymethyl cellulose by irreversible adsorption of the sodium carboxymethyl cellulose. This will provide a stable cellulose biological interface for subsequent immunoassays. The stable combination of the bisphenol A antibody and the absorption of the sodium carboxymethyl cellulose is realized, and a bisphenol A standard curve is obtained. Meanwhile, the surface of the paper base is modified by the sodium carboxymethyl cellulose, and non-specific protein adsorption can be prevented.
Preferably, in the step (3), a coating antibody corresponding to the antigen to be detected is added into the reaction area, and the specific steps of crosslinking and fixing the coating antibody and carboxyl on the surface of the paper-based microfluidic chip are as follows: activating 3-6 mu L of 4mg/mL EDC1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) and 3-6 mu L of 6mg/mL NHS (N-N-hydroxysuccinimide) for 12-18min on the surface of the modified paper-based microfluidic chip to convert carboxyl of sodium carboxymethylcellulose into amine reaction ester, and then covalently bonding a bisphenol A monoclonal antibody on the modified activated site of the sodium carboxymethylcellulose according to a certain amount in a PBS (phosphate buffered saline) buffer solution with the pH value of 7.5; unreacted NHS was removed using ethanolamine hydrochloride solution pH8.5 and unbound bisphenol A antibody was removed using PBST.
Preferably, the step (4) of closing process is as follows: taking 8-12 mu L of milk powder sealing solution of 1mg/ml to drop in the paper-based microfluidic well for sealing, then washing with PBST three times, and washing excess sealing solution.
Preferably, the step (5) of detecting comprises the following steps:
(1) adding bisphenol A standard products with different concentrations and enzyme-labeled antigen into a microfluidic reaction hole in a volume ratio of 1:1 for reaction, and then washing the reaction product for four times by using 10 mu LPBSTbuffer;
(2) color development: mixing the substrate A and the substrate B, adding 5 mu L of the mixed solution into the microfluidic reaction hole for color development, and photographing by using a mobile phone after the color development;
(3) and (3) analysis: and (4) performing data processing and gray level analysis by using Excel and ImageJ software, and drawing a standard curve.
Preferably, the volume ratio of substrate a to substrate B is 32.44: 1; the substrate A is 0.43g of carbamide peroxide, 2.5g of beta dextrin and 8.2g of anhydrous sodium acetate, the pH value is adjusted to 5.0, the volume is adjusted to 1000ml by ultrapure water, the substrate A is preserved at 4 ℃, and the substrate A is taken out from 4 ℃ before use and is recovered to the room temperature; substrate B was 100mg of TMB (3,3',5,5' -tetramethylbenzidine) and 10ml of DMSO (dimethyl sulfoxide) and stored in a brown bottle in a cool place.
The principle of the technical scheme is as follows: the antibody combined on the surface of the paper base still keeps the immunological activity, and the enzyme-labeled antigen keeps the immunological activity and the activity of the enzyme. In the detection, a substance to be detected (antigen) in a sample and an enzyme-labeled antigen are competitively bound to an immobilized antibody, and the unbound substance is removed by washing, whereby the amount of the immobilized enzyme is correlated with the amount of the substance to be detected in the sample. The content of the substance in the sample can be judged according to the shade of the color through color development after adding the substrate which reacts with the enzyme, and qualitative or quantitative analysis is carried out. The catalytic efficiency of the enzyme is high, so that the result of immune reaction is indirectly amplified, and the measuring method achieves high sensitivity.
The paper-based microfluidic core product designed by the invention takes chromatographic paper as a substrate, forms a hydrophilic/hydrophobic micro-channel network and related analytical devices by a wax-spraying processing technology, and constructs a 'micro laboratory on paper', and compared with the traditional microfluidic chip technology, the paper-based microfluidic chip has the advantages of paper: low cost, simple process, simple post-treatment, no pollution, strong biocompatibility and the like.
Large instrumentation requires advanced and expensive instrumentation, as well as complex, laborious, and time-consuming pre-processing steps. Conventional enzyme-linked immunoassays (ELISAs) are relatively expensive because they require large sample volumes of analyte and reagents (particularly antibodies) as well as expensive microplate readers to collect the data. However, the method for detecting bisphenol A can not only save the using amount of an analytical reagent (only a few mu L of reagent is 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 field detection or daily test.
Description of the drawings:
FIG. 1 shows the volume of working reagents used for detecting bisphenol A in a paper-based microfluidic chip prepared based on wax-jet printing technology according to the present invention;
FIG. 2 is a micro-fluidic chip template for detecting bisphenol A of the paper-based micro-fluidic chip prepared based on the wax-spraying printing technology;
FIG. 3 shows the working principle of the paper-based microfluidic chip prepared based on wax-spraying printing technology for detecting bisphenol A according to the present invention;
FIG. 4 is a standard curve for detecting bisphenol A by a paper-based microfluidic chip prepared based on wax-jet printing technology;
FIG. 5 shows the optimization of the amount of the antibody coating in the experimental process of the paper-based microfluidic chip prepared based on the wax-spraying printing technology and used for detecting bisphenol A.
The specific implementation mode is as follows:
the following further description is made with reference to the accompanying drawings
The working principle of the invention is as follows: a paper-based chip is prepared on chromatographic paper by using a wax-spraying printer, a bisphenol A monoclonal antibody is fixed on a modified paper base, a bisphenol A standard substance and an enzyme-labeled antigen are added into the chip according to the principle of antigen antibody and enzyme-specific catalytic substrate color development, the standard substance and the enzyme-labeled antigen are competitively combined with the antibody, the combination of the enzyme-labeled antigen and the antibody on the paper base is gradually reduced along with the increase of the concentration of the standard substance, the color is gradually lightened, the gray value of the color is analyzed through Image J, as can be seen from figure 4, the relative gray value is gradually increased along with the increase of the logarithm of the concentration, so that the bisphenol A can be detected by a colorimetric method.
Example 1
The steps for researching the working reagent required by the microfluidic chip are as follows:
(1) drawing with CAD: using a 12X 6 array, the reaction zone diameter was 5mm, the hole spacing was 8mm, and to inhibit sample flow between papers, the hydrophobic channels were designed to be black on a white background using Auto CAD.
(2) Printing by a wax printer: the microfluidic paper-based pattern was printed on chromatography paper using a wax printer.
(3) And (3) baking the chromatographic paper printed with the patterns in an oven at 110 ℃ for 6min, and melting the wax.
To determine the volume of working reagent (PBST buffer, blocking solution, sample, substrate A + substrate B) required on paper-based chips, different volumes (1-10. mu.L) of crystal violet dye solution [ 0.025% (w/v) ] were added to the chips and the results were visually evaluated at room temperature. From FIG. 1, it can be seen that the maximum amount of reagent that can be tolerated, and the just impermeable amount of reagent, is 10. mu.L.
Finally selecting 10 muL of PBST buffer required by the microfluidic chip, mixing the sample and the enzyme-labeled antigen in a volume ratio of 1:1 to obtain 5 muL, mixing the blocking solution to obtain 10 muL, adjusting the pH of the substrate A to 5.0 by using 0.43g of carbamide peroxide, 2.5g of beta dextrin and 8.2g of anhydrous sodium acetate to obtain 5 muL of a substrate (the volume ratio of the substrate A to the substrate B is 32.44: 1), diluting the volume to 1000ml by using ultrapure water, storing the volume at 4 ℃ for later use, and taking out the sample from 4 ℃ to restore to room temperature before use; substrate B was 100mg TMB mixed with 10mL DMSO and stored in a brown bottle in the shade until use.
Example 2
The invention discloses a paper-based microfluidic chip prepared based on wax spraying technology and used for detecting bisphenol A, which comprises the following steps:
(1) drawing with CAD: using a 12X 6 array, the reaction zone diameter was 5mm, the hole spacing was 8mm, and to inhibit sample flow between papers, the hydrophobic barrier designed on a white background using Auto CAD was black.
(2) Printing by a wax printer: the microfluidic patterns were printed on whatman chromatographic paper using a wax printer.
(3) And (3) baking the chromatographic paper printed with the patterns in an oven at 110 ℃ for 6min, and melting the wax.
(4) Modifying the surface of the paper-based reaction zone: the surface modification is carried out by using sodium carboxymethyl cellulose, and the specific steps are as follows: dissolving sodium carboxymethylcellulose in deionized water, dialyzing with 11kDa filter membrane to salt-free state, freeze-drying for 8min, dissolving purified carboxymethylcellulose in calcium chloride solution, and dripping 13 μ L of sodium carboxymethylcellulose 0.2g/L into the prepared paper-based microfluidic pore.
(5) Antibody immobilization in the reaction zone: activating the surface of the modified paper-based micro-fluidic chip with 5 mu L of EDC (4 mg/ml) and 5 mu L of NHS (6mg/ml) for 15min, converting sodium carboxyl of the carboxymethyl cellulose into amine reaction ester, and then covalently bonding a bisphenol A monoclonal antibody on the modified activated site of the sodium carboxymethyl cellulose according to the amount of 0.1 mu g/well in PBS (phosphate buffer solution) with the pH value of 7.5; unreacted NHS was removed using ethanolamine hydrochloride solution pH8.5 and unbound bisphenol A antibody was removed using PBST.
(6) And (3) sealing: taking 10 mu L of skim milk powder sealing solution of 1mg/mL, dropping the solution in a paper-based microfluidic well for sealing, then washing the solution three times by using PBST, and washing excessive sealing solution.
Example 3
Following the procedure of example 2 except that the amount of bisphenol A monoclonal antibody added in step (5) was varied, and A-F indicated that the amount of the coupled antibody was 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125. mu.g/well, respectively, and the next reaction was performed and three controls were made, in which 1.4.7 was listed as a control (2.5. mu.L PBS + 2.5. mu.L enzyme-labeled antigen was added to the reaction); 2.5.8 is listed as an experimental group (2.5. mu.L of bisphenol A standard substance with 1ppm and 2.5. mu.L of enzyme-labeled antigen are added in the reaction); 3.6.9 is listed as blank (5. mu.L PBS was added to the reaction). It can be seen from FIG. 5 that the control group developed the darkest color, the experimental group developed the lighter color, and the blank group developed the lightest color. Experiments show that along with the gradual reduction of the antibody coupling amount, the color development degree is gradually reduced, the color difference is not obvious, and the accuracy of the experiment is influenced; when the coupling amount is 0.1 mu g/well, the interference of background color is the lowest, the color development of an experimental group is moderate, and the color and the sample concentration form a negative correlation linear trend which is obvious; when the amount of antibody coupled exceeds 0.1. mu.g/well, unnecessary antibody waste is caused, so that the effect is preferable when the amount of antibody coupled exceeds 0.1. mu.g/well.
Example 4
The invention discloses a paper-based microfluidic chip prepared based on wax spraying technology and used for detecting bisphenol A, which comprises the following steps:
(1) drawing with CAD: using a 12X 6 array, the reaction zone diameter was 5mm, the hole spacing was 8mm, and to inhibit sample flow between papers, the hydrophobic barrier was designed to be black on a white background using AutoCAD.
(2) Printing by a wax printer: the microfluidic patterns were printed on whatman chromatographic paper using a wax printer.
(3) And (3) baking the chromatographic paper printed with the patterns in an oven at 110 ℃ for 5min, and melting the wax.
(4) Modifying the surface of the paper-based reaction zone: the surface modification is carried out by using sodium carboxymethyl cellulose, and the specific steps are as follows: dissolving sodium carboxymethylcellulose in deionized water, dialyzing with 10kDa filter membrane to salt-free state, freeze-drying for 5min, dissolving purified carboxymethylcellulose in calcium chloride solution, and dripping 10 μ L of sodium carboxymethylcellulose 0.2g/L into the prepared paper-based microfluidic pore.
(5) Antibody immobilization in the reaction zone: activating the surface of the modified paper-based micro-fluidic chip with 3 mu L of EDC (4 mg/ml) and 3 mu L of NHS (6mg/ml) for 10min, converting sodium carboxyl of the carboxymethyl cellulose into amine reaction ester, and then covalently bonding a bisphenol A monoclonal antibody on an activated site modified by the sodium carboxymethyl cellulose according to the amount of 0.1 mu g/well in a PBS (phosphate buffer solution) with the pH value of 7.5; unreacted NHS was removed using ethanolamine hydrochloride solution pH8.5 and unbound bisphenol A antibody was removed using PBST.
(6) And (3) sealing: and (3) taking 8 mu L of defatted milk powder sealing solution of 1mg/mL, dropping the solution into a paper-based microfluidic well for sealing, then washing the solution three times by using PBST, and washing excessive sealing solution.
Example 5
The invention discloses a paper-based microfluidic chip prepared based on wax spraying technology and used for detecting bisphenol A, which comprises the following steps:
(1) drawing with CAD: using a 12X 6 array, the reaction zone diameter was 5mm, the hole spacing was 8mm, and to inhibit sample flow between papers, the hydrophobic barrier was designed to be black on a white background using Auto CAD.
(2) Printing by a wax printer: the microfluidic pattern was printed on the filter paper using a wax printer.
(3) And (3) baking the chromatographic paper printed with the patterns in an oven at 110 ℃ for 8min, and melting the wax.
(4) Modifying the surface of the paper-based reaction zone: the surface modification is carried out by using sodium carboxymethyl cellulose, and the specific steps are as follows: dissolving sodium carboxymethylcellulose in deionized water, dialyzing with 12kDa filter membrane to salt-free state, freeze-drying for 10min, dissolving purified carboxymethylcellulose in calcium chloride solution, and dripping 15 μ L of sodium carboxymethylcellulose 0.2g/L into the prepared paper-based microfluidic pore.
(5) Antibody immobilization in the reaction zone: activating the surface of the modified paper-based micro-fluidic chip with 6 mu L of EDC (4 mg/mL) and 6 mu L of NHS (6mg/mL) for 18min, converting sodium carboxyl of the carboxymethyl cellulose into amine reaction ester, and then covalently bonding a bisphenol A monoclonal antibody on an activated site modified by the sodium carboxymethyl cellulose according to the amount of 0.1 mu g/well in a PBS (phosphate buffer solution) with the pH value of 7.5; unreacted NHS was removed using ethanolamine hydrochloride solution pH8.5 and unbound bisphenol A antibody was removed using PBST.
Example 6 Standard Curve plotting
(1) Reaction: bisphenol A standard and enzyme-labeled antigen with different concentrations are added into the microfluidic reaction well obtained in example 2 in a volume ratio of 1:1 for reaction, and then washed with PBST buffer solution for four times. The bisphenol A standard substance with the concentration of 1000 mug/L, 100 mug/L, 33.33 mug/L, 11.11 mug/L, 3.703 mug/L, 1.234 mug/L, 0.411 mug/L, 0.137 mug/L and 0.045 mug/L and the enzyme labeled antigen are mixed and added into the chip for reaction according to the volume ratio of 1: 1.
(2) Color development: and mixing the substrate A and the substrate B, adding 5 mu L of the mixed solution into the microfluidic reaction hole for color development, and taking a picture by using a mobile phone in time after color development.
(3) And (3) analysis: and (4) performing data processing and gray level analysis by using Excel and Image J software, and drawing a standard curve.
Obviously, the colors corresponding to the samples with different concentrations are also different, and as can be seen from fig. 4, the concentrations of the samples are positively correlated with the gray values. According to the method, a standard library with the one-to-one correspondence of the relative gray value and the concentration of the bisphenol A can be established, so that the concentration of an unknown bisphenol A sample can be detected.
The molecular weight of the sodium carboxymethyl cellulose is 250 kDa.
The concentration of the ethanolamine hydrochloride solution is 1M.
Example 7 detection of quality control Material
TABLE 1 detection precision of bisphenol A content in quality control product
As can be seen from the detection data in Table 1, the lowest value and the highest value of the Coefficient of Variation (CV) in batches are both controlled within 10%, which indicates that the precision of the paper-based chip is better.
Claims (10)
1. A method for preparing a paper-based microfluidic chip for bisphenol A detection based on a wax-spraying printing technology is characterized by comprising the following steps: the method comprises the following steps:
(1) taking chromatographic paper as a substrate, and printing the chromatographic paper by a wax-spraying printer through a template drawn by a CAD (computer aided design), wherein the template comprises a hydrophobic channel and a hydrophilic channel, the hydrophilic channel is a reaction area for generating immunoreaction, and the reaction area is a closed reaction area; baking the chromatographic paper printed with the patterns in an oven to obtain a paper-based microfluidic chip;
(2) and (3) carrying out modification treatment in the reaction zone: the reaction area in the paper-based microfluidic chip is subjected to surface modification treatment;
(3) adding a coating antibody corresponding to the antigen to be detected in the reaction area;
(4) adding a sealing agent into the reaction zone;
(5) the detection of bisphenol A is realized by a colorimetric method.
2. The method for preparing the paper-based microfluidic chip for detecting the bisphenol A based on the wax-spraying printing technology according to the claim 1, which is characterized in that: and (1) baking the chromatographic paper printed with the pattern in an oven at 110 ℃ for 5-8 min.
3. The method for preparing the paper-based microfluidic chip for detecting the bisphenol A based on the wax-spraying printing technology according to the claim 1, which is characterized in that: the paper-based microfluidic chip obtained in the step (1) adopts a 12 x 6 array, a reaction area is a small hole with the diameter of 5mm, the hole interval is 8mm, and a hydrophobic channel is designed to be black on a white background by using Auto CAD.
4. The method for preparing the paper-based microfluidic chip for detecting the bisphenol A based on the wax-spraying printing technology according to the claim 1, which is characterized in that: the chromatographic paper used in the step (1) is whatman chromatographic paper.
5. The method for preparing the paper-based microfluidic chip for detecting the bisphenol A based on the wax-spraying printing technology according to the claim 1, which is characterized in that: the paper-based micro-fluidic chip in the step (2) is subjected to surface modification treatment by using sodium carboxymethyl cellulose, and the specific steps are as follows: dissolving sodium carboxymethylcellulose in deionized water, dialyzing with 10-12kDa filter membrane to salt-free state, freeze-drying for 5-10min, dissolving purified sodium carboxymethylcellulose in calcium chloride solution, and dripping 10-15 μ L of sodium carboxymethylcellulose 0.2g/L into the prepared paper-based microfluidic pore.
6. The method for preparing the paper-based microfluidic chip for detecting the bisphenol A based on the wax-spraying printing technology according to the claim 1, which is characterized in that: and (3) adding a coating antibody corresponding to the antigen to be detected into the reaction area, wherein the specific steps of crosslinking and fixing the coating antibody and carboxyl on the surface of the paper-based microfluidic chip are as follows: activating the surface of the modified paper-based micro-fluidic chip with 3-6 mu L of 4mg/mL EDC and 3-6 mu L of 6mg/mL NHS for 12-18min to convert carboxyl of sodium carboxymethylcellulose into amine reaction ester, and then covalently bonding a bisphenol A monoclonal antibody on the modified activation site of the sodium carboxymethylcellulose according to a certain amount in a PBS (phosphate buffered saline) buffer solution with the pH value of 7.5; unreacted NHS was removed using ethanolamine hydrochloride solution pH8.5 and unbound bisphenol A antibody was removed using PBST.
7. The method for preparing the paper-based microfluidic chip for detecting the bisphenol A based on the wax-spraying printing technology according to claim 6, which is characterized in that: and (3) adding a coating antibody corresponding to the antigen to be detected into the reaction area, wherein the specific steps of crosslinking and fixing the coating antibody and carboxyl on the surface of the paper-based microfluidic chip are as follows: activating the surface of the modified paper-based microfluidic chip for 15min by using 5 mu L of 4mg/mL EDC and 5 mu L of 6mg/mL NHS, converting sodium carboxymethyl cellulose carboxyl into amine reaction ester, and then covalently bonding a bisphenol A monoclonal antibody on an activated site modified by the sodium carboxymethyl cellulose according to the amount of 0.1 mu g/well in a PBS (phosphate buffer solution) with the pH value of 7.5; unreacted NHS was removed using ethanolamine hydrochloride solution pH8.5 and unbound bisphenol A antibody was removed using PBST.
8. The method for preparing the paper-based microfluidic chip for detecting the bisphenol A based on the wax-spraying printing technology according to the claim 1, which is characterized in that: the sealing process in the step (4) is as follows: taking 8-12 mu L of milk powder sealing solution of 1mg/ml to drop in the paper-based microfluidic well for sealing, then washing with PBST three times, and washing excess sealing solution.
9. The method for preparing the paper-based microfluidic chip for detecting the bisphenol A based on the wax-spraying printing technology according to the claim 1, which is characterized in that: the detection in the step (5) comprises the following steps:
(1) adding bisphenol A standard products and enzyme-labeled antigens with different concentrations into the microfluidic reaction hole according to the volume ratio of 1:1 for reaction, wherein the adding amount is 5 mu L, incubating for 15min, and washing for four times by using 10 mu L PBST buffer;
(2) color development: mixing the substrate A and the substrate B, adding 5 mu L of the mixed solution into the microfluidic reaction hole for color development, and photographing by using a mobile phone after the color development;
(3) and (3) analysis: and (4) performing data processing and gray level analysis by using Excel and Image J software, and drawing a standard curve.
10. The method for preparing the paper-based microfluidic chip for detecting the bisphenol A based on the wax-spraying printing technology according to claim 9, which is characterized in that: the volume ratio of substrate a to substrate B was 32.44: 1; the substrate A is 0.43g of carbamide peroxide, 2.5g of beta dextrin and 8.2g of anhydrous sodium acetate, the pH value is adjusted to 5.0, the volume is adjusted to 1000ml by ultrapure water, the substrate A is preserved at 4 ℃, and the substrate A is taken out from 4 ℃ before use and is recovered to the room temperature; substrate B was 100mg TMB mixed with 10ml DMSO and stored in a brown bottle in the shade until use.
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