A foldable multi-channel paper-based microfluidic chip and a preparation method and application thereof

Abstract

The application relates to a foldable multi-channel paper-based microfluidic chip and a preparation method and application thereof. The paper-based microfluidic chip comprises a plurality of micro-detection channels. The multi-channel detection capacity enables the microfluidic paper-based chip to simultaneously detect a plurality of pollutant items. The application also discloses application of the paper-based microfluidic chip in simultaneous detection of environmental endocrine disruptors, i.e. bisphenol A, 17beta estradiol and malathion, in pretreated landfill leachate by a solid-phase microextraction technology in combination with a nano-mixed material. The paper-based microfluidic chip has been successfully applied to detection and analysis of environmental endocrine disruptors, and can be completed in a short time only by using a small amount of reaction reagents and samples, and has the characteristics of rapidity, convenience, systematic identification, economy and high efficiency. According to the structure of target analytes, corresponding aptamers can be screened and applied, and a new idea is provided for analysis of other environmental endocrine disruptors or environmental pollutants, and the application has important significance for environmental risk assessment, pollutant control and public health.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microfluidic technology, and particularly relates to a foldable multi-channel paper-based microfluidic chip and a preparation method and application thereof. BACKGROUND

[0002] Landfill leachate is a kind of high-concentration wastewater containing pollutants generated during landfill by physical and chemical reactions. The pollutants contained therein are of various types, and environmental endocrine disruptors are an important component thereof. As environmental endocrine disruptors can cause human function damage and endocrine dysfunction, they have attracted widespread attention. Environmental endocrine disruptors have different effects due to different types of substances. Common types include environmental estrogens, pesticide pollutants and chemical pollutants. Bisphenol A and 17β-estradiol are common environmental estrogens in the environment, and malathion is widely used in agricultural production due to its low price. Therefore, rapid and systematic identification of the three new pollutants in landfill leachate has important practical significance for environmental risk assessment, pollutant control and public health.

[0003] At present, there are various types of methods for detecting bisphenol A, 17β-estradiol and malathion, such as electrochemical method, chemiluminescence method and enzyme-linked immunosorbent assay. However, these technologies have disadvantages such as high cost, trained laboratory personnel and overly complex operation procedures, which limit their application in economically underdeveloped areas and laboratories with relatively simple equipment. At the same time, the complexity of landfill leachate and the high content of pollutants greatly increase the difficulty of detection and analysis, and far from achieving the goal of being used for instant and rapid detection. Therefore, it is of great research value and practical significance to develop a simple and rapid method for endocrine disruptors in landfill leachate in order to obtain the content of target pollutants on site in real time, thereby providing reliable basis for waste management and disposal and engineering maintenance.

[0004] The principle of paper-based microfluidic chip is to make hydrophilic and hydrophobic patterns on paper to promote the formation of micron-scale capillary channels on paper. Common techniques include wax printing and inkjet printing. In practical applications, people will consider the availability and convenience of equipment, materials and manufacturing processes when selecting appropriate techniques. Inkjet printing has good comprehensive performance, and patterns with microfluidic functions can be produced within a few minutes by printing with a machine and heating in an oven, and can be used for testing after simple cutting and assembly. Therefore, developing novel and practical paper-based microfluidic chips by using scientific design and appropriate manufacturing techniques is the key to solving the problem of instant and rapid detection. SUMMARY

[0005] The application aims to provide a folded microfluidic paper-based chip for simultaneously detecting three environmental endocrine disruptors, which is applied to the detection of bisphenol A, 17beta estradiol and malathion in pretreated landfill leachate, and has the characteristics of rapidness, high efficiency and sensitivity.

[0006] To achieve the above-mentioned purpose, the application provides the following scheme.

[0007] A folded multi-channel paper-based microfluidic chip, characterized in that the microfluidic chip comprises two acrylic magnetic plates and a filter paper treated by wax infiltration; the filter paper has a pattern of an equilateral triangle, and a fold line is formed by connecting the midpoints of the three sides of the equilateral triangle, thereby forming four equilateral triangle regions, i.e., four functional layers, which are a color development layer, a transport layer, an aptamer drop layer and a sample injection layer; the color development layer comprises three color development zones and one control zone; the transport layer comprises a central transport zone, a circular transport control zone and three transport channels of the same specification and diverging from the central transport zone, each transport channel is composed of a circular transport zone and a rectangle; the aptamer drop layer comprises three aptamer drop zones and one aptamer drop control zone; the sample injection layer comprises a central sample injection zone and a sample injection control zone; wherein:

[0008] The central sample injection zone is located at the center of the sample injection layer, and the sample flows to the central transport zone of the transport layer through the zone, and then flows to the three color development zones of the color development layer after reaching the end point through the three transport channels;

[0009] The sample injection control zone is located in the region from the center to the edge of the sample injection layer, and is used for dropwise adding the sample, i.e., distilled water, of the control zone, and the sample flows to the corresponding circular transport control zone of the transport layer through the zone, and then flows to the control zone of the color development layer;

[0010] The paper-based is a medium-speed filter paper;

[0011] The color development zone, the control zone, the aptamer drop zone, the aptamer drop control zone, the sample injection control zone, the circular transport zone and the circular transport control zone are all circular shapes of the same size, and the central sample injection zone and the central transport zone are circular shapes of the same size;

[0012] The transport channel is composed of the circular transport zone and the rectangle, and the rectangle connects the central transport zone and the circular transport zone.

[0013] The two pieces of acrylic magnetic sticker board are uniform in size and shape, and are equilateral triangles. Five circular holes are provided on the board, and the positions of the circular holes correspond to one sample adding area, three color developing areas and one control area respectively. The diameter of the central circular hole is 1.50 mm larger than that of the central sample adding area, and the diameters of the four surrounding circular holes are 1.50 mm larger than those of the color developing areas.

[0014] Three corners and the middle area of the two pieces of acrylic magnetic sticker board are respectively provided with circular magnets with a diameter of 4.00 mm and a thickness of 2.00 mm.

[0015] The folding type multi-channel paper-based microfluidic chip is obtained by pressing and assembling in the order of the acrylic magnetic sticker board, the color developing layer, the conveying layer, the aptamer drop adding layer and the acrylic magnetic sticker board, and the sample adding layer is not folded and pressed.

[0016] A preparation method of the paper-based microfluidic chip comprises the following steps:

[0017] 1) The AutoCAD software is used to design the configuration of the paper-based microfluidic chip, so that the color developing layer, the conveying layer, the aptamer drop adding layer and the sample adding layer area are hydrophobic areas, and the rest are hydrophilic areas.

[0018] 2) An A4-sized medium filter paper is cut, and the filter paper is printed using an Xerox 8870 wax jet printer.

[0019] 3) The printed filter paper is placed in an oven at 130 degrees Celsius for 30 seconds, so that the hydrophobic paraffin completely penetrates to the reverse side. The treated filter paper is cut into an equilateral triangle according to the pattern. The midpoints of the three sides of the equilateral triangle are connected to form a fold line, forming four equilateral triangle areas, i.e. four functional layers, which are the color developing layer, the conveying layer, the aptamer drop adding layer and the sample adding layer respectively.

[0020] 4) Three color developing areas and one control area are provided in the color developing layer. One central conveying area, three conveying channels with the same specifications and diverging from the central conveying area, and one circular conveying control area are provided in the conveying layer. Three aptamer drop adding areas and one aptamer drop adding control area are provided in the aptamer drop adding layer. One central sample adding area and one sample adding control area are provided in the sample adding layer.

[0021] 5) The acrylic plate is cut into a designed equilateral triangle pattern. Five circular magnets with a diameter of 4.00 mm and a thickness of 2.00 mm are respectively adhered to the three corners and the middle area of the acrylic plate by using 502 glue.

[0022] 6) Fold the color development layer according to the fold line, fold the aptamer drop layer according to the fold line to the opposite side, and the regions of different layers correspond one by one, finally clamp with two pieces of acrylic magnetic plate, the round holes of the acrylic magnetic plate need to correspond the color development area and the control area of the color development layer, the aptamer drop area and the aptamer drop control area of the aptamer drop layer, and the center sample adding area and the sample adding control area of the sample adding layer.

[0023] The application of the paper-based microfluidic chip in the detection and analysis of bisphenol A, 17 beta estradiol and malathion in landfill leachate.

[0024] The landfill leachate is a pretreated landfill leachate sample, and the pretreatment includes the following steps:

[0025] 1) The filtrate to be treated is subjected to suction filtration and centrifugation to remove most of the suspended solids and impurities;

[0026] 2) Adjust the pH to 3-4, add 2 g / L EDTA, and stir uniformly;

[0027] 3) Perform solid phase extraction using a SAX small column to obtain a concentrated filtrate;

[0028] 4) The collected concentrated liquid is subjected to nitrogen blowing treatment, blown to near dryness, added with water, completely dissolved in an ultrasonic machine, and then constant volume, which is the pretreated landfill leachate sample to be detected.

[0029] The application of the paper-based microfluidic chip in the detection and analysis of bisphenol A, 17 beta estradiol and malathion in landfill leachate, specifically includes the following steps:

[0030] 1) Prepare Au NPs solution with a concentration of 5.75 x 10 -9 M and Ag NPs solution with a concentration of 9.00 x 10 -4 M; after centrifugation, discard 9 / 10 of the supernatant, shake the remaining components uniformly, which are concentrated solutions of Au NPs with a concentration of 5.75 x 10 -8 M and Ag NPs with a concentration of 9.00 x 10 -3 M; mix the two concentrated liquids uniformly in a volume ratio of 1:1 to form Au / Ag NPs nanometer mixed material for standby;

[0031] 2) Prepare bisphenol A aptamer solution with a concentration of 60 μM, 17 beta estradiol aptamer solution with a concentration of 60 μM, and malathion aptamer solution with a concentration of 90 μM, and mix the bisphenol A, 17 beta estradiol and malathion aptamer solutions in equal volumes to prepare mixed aptamer solution with concentrations of 20 μM, 20 μM and 30 μM respectively for standby;

[0032] 3) 20 μM bisphenol A, 20 μM 17β-estradiol and 30 μM malathion aptamer solution were prepared for use;

[0033] 4) 5 μL of 20 μM bisphenol A aptamer solution, 5 μL of 20 μM 17β-estradiol aptamer solution and 5 μL of 30 μM malathion aptamer solution were added dropwise to the three aptamer dropwise areas of the paper-based microfluidic chip respectively, and 5 μL of the mixed aptamer solution was added dropwise to the aptamer dropwise control area, and then dried;

[0034] 5) The paper-based microfluidic chip was reversed, 5 μL of Au / Ag NPs nanometer mixed material was added dropwise to the three color developing areas and one control area of the color developing layer of the paper-based microfluidic chip, and then dried;

[0035] 6) The plastic magnetic plate was removed, the sample layer was folded on the aptamer dropwise layer, and the plastic magnetic plate was clamped and fixed; 12 μL of the sample to be tested was added dropwise to the center sample adding area of the sample layer of the paper-based microfluidic chip, 5 μL of distilled water was added dropwise to the sample adding control area, and then dried;

[0036] 7) 12 μL of 1.5 M sodium chloride solution was added dropwise to the center sample adding area of the sample layer of the paper-based microfluidic chip, 5 μL of 1.5 M sodium chloride solution was added dropwise to the sample adding control area, and then dried for 3-5 min, the color developing result of the color developing layer was photographed, and the result was analyzed by Image J.

[0037] The detection limit of the paper-based microfluidic chip for bisphenol A, 17β-estradiol and malathion in landfill leachate is 0.6 μg / mL -1 , 1.6 μg / mL -1 and 0.9 μg / mL -1 , and the linear range is 5-20 μg / mL -1 , 2-20 μg / mL -1 and 1-50 μg / mL -1 .

[0038] The Au / Ag NPs nanometer mixed material contains an electrostatic force unit.

[0039] The Au / Ag NPs nanometer mixed material is easy to combine with the aptamer of bisphenol A, 17β-estradiol and malathion, and the material and the aptamer have an electrostatic force unit.

[0040] The advantages and beneficial effects of the present application include:

[0041] The application provides a foldable multi-channel paper-based microfluidic chip, which has the characteristics of easy manufacturing, batch production and simple operation.

[0042] The Au / Ag NPs nanometer mixed material is prepared by mixing Au NPs and Ag NPs in equal proportions.

[0043] The application fully utilizes the optical properties of noble metal nanoparticles, and first applies the material to the detection and analysis of environmental endocrine disruptors (bisphenol A, 17 beta estradiol and malathion) in landfill leachate.

[0044] The application has reasonable design, and the prepared foldable multi-channel paper-based microfluidic chip is a new type of instant detection instrument, which can realize accurate detection of environmental endocrine disruptors (bisphenol A, 17 beta estradiol and malathion) in landfill leachate by combining a smart phone and colorimetric software. The application can not only reduce the detection time of bisphenol A, 17 beta estradiol and malathion, but also greatly reduce the detection cost and professional requirements of the operator, and has the characteristics of simple and rapid, instant detection and system identification. The tested paper-based microfluidic chip can be directly discarded to the experimental garbage, and the processing is simple. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 The visible spectrum of the Au / Ag NPs nanometer mixed material prepared in example 1 of the application, TEM characterization and TEM characterization of the aggregated Au / Ag NPs nanometer mixed material are shown in the following table:

[0046] Figure 2 The design drawing of the foldable paper-based microfluidic chip described in example 2 of the application is shown in the following table:

[0047] Figure 3 The design drawing of the acrylic magnetic plate described in example 2 of the application is shown in the following table:

[0048] Figure 4 The structural schematic diagram of the foldable paper-based microfluidic chip in example 2 of the application is shown in the following table:

[0049] Figure 5 The result graph of the foldable paper-based microfluidic chip in example 4 of the application for detecting the mixed solution with the same concentration of bisphenol A, 17 beta estradiol and malathion is shown in the following table: DETAILED DESCRIPTION

[0050] The application will be further described in conjunction with the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the application has no special limitation.

[0051] Example 1

[0052] Au / Ag NPs nanohybrid material and a preparation method thereof, the preparation method comprising the following steps:

[0053] (1) Au NPs preparation: prepare a 0.5886 M tetrachloroauric acid solution, and take 170 μL and drop into a three-necked flask containing 100 mL of distilled water, and oil bath at 120°C. After boiling, add 10 mL of 38.8 mM trisodium citrate, and stir rapidly for 30 minutes. After the reaction is completed, the solution is gradually cooled to room temperature, filtered with a 0.22 μm filter head, and stored in a refrigerator (4°C).

[0054] (2) Ag NPs preparation: under stirring conditions, 1 mL of a 0.10 M silver nitrate solution and 1 mL of a 0.10 M trisodium citrate solution are sequentially added to 90 mL of distilled water. Under the condition of vigorous stirring, 6 mL of a 20 mM sodium borohydride solution is quickly added, and stirred at room temperature for 30 minutes. After the reaction is completed, stand for 8 hours and store in a refrigerator (4°C).

[0055] (3) Preparation of Au NPs and Ag NPs concentrated solution: the above prepared Au NPs solution with a concentration of 5.75 x 10 -9 M and the Ag NPs solution with a concentration of 9.00 x 10 -4 M are centrifuged, 9 / 10 of the supernatant is discarded, and the remaining components are shaken uniformly, which are the concentrated solutions of Au NPs with a concentration of 5.75 x 10 -8 M and Ag NPs with a concentration of 9.00 x 10 -3 M, and stored in a refrigerator (4°C).

[0056] (4) Preparation of Au / Ag NPs nanohybrid material: the above prepared concentrated solutions of Au NPs with a concentration of 5.75 x 10 -8 M and Ag NPs with a concentration of 9.00 x 10 -3 M are mixed uniformly at a volume ratio of 1:1 to form Au / Ag NPs nanohybrid material, which is ready for use.

[0057] The product Au / Ag NPs nanohybrid material synthesized in the above example 1 is characterized, and reference is made to Figure 1(A) is the visible spectrum of Au / Ag NPs nanometer hybrid material, (B) is TEM characterization, (C) is TEM characterization of aggregated Au / Ag NPs nanometer hybrid material.

[0058] Example 2

[0059] The manufacturing method of the folding multi-channel paper-based microfluidic chip

[0060] (1) The target pattern of filter paper is designed by using AutoCAD. The figure is an equilateral triangle with a side length of 64.30 mm. The midpoints of the three sides of the equilateral triangle are connected to form four equilateral triangle regions, i.e. four functional layers, which are color development layer, transport layer, aptamer drop layer and sample layer, and are set as hydrophobic regions, and the rest are hydrophilic regions. Three color development zones and one control zone are set on the color development layer, all of which are circular with a diameter of 6.00 mm; one circular center transport zone with a diameter of 4.00 mm, one circular transport control zone with a diameter of 6.00 mm and three transport channels each composed of a 2.00 mm wide rectangle and a 6.00 mm diameter circle and diverging from the center transport zone are set on the transport layer; three aptamer drop zones and one aptamer drop control zone are set on the aptamer drop layer, all of which are circular with a diameter of 6.00 mm; one circular center sample loading zone with a diameter of 4.00 mm and one sample loading control zone with a diameter of 6.00 mm are set on the sample layer, as shown in Figure 2 .

[0061] (2) The medium-speed filter paper is cut into A4 size and placed in the paper slot of the Xerox 8870 wax jet printer;

[0062] (3) The pattern data is loaded into the Xerox 8870 wax jet printer, and printing is selected to obtain the filter paper printed by the wax jet printer;

[0063] (4) The filter paper printed by the wax jet printer is placed in an oven at 130℃ for 30 seconds;

[0064] (5) According to Figure 3 , two equilateral triangle acrylic plates with a side length of 33.00 mm are cut as fixing layers, and a circular intermediate sample inlet with a diameter of 5.50 mm is cut in the center of each acrylic plate, and four other sample inlets with a diameter of 7.50 mm are cut near the center of each acrylic plate, and five circular magnets with a diameter of 4.00 mm and a thickness of 2.00 mm are pasted on the triangle and intermediate parts of each acrylic plate by using 502 glue, and the two acrylic plates can be pasted together by magnetic attraction force, and the sample inlets are one-to-one corresponding;

[0065] (6) The baked filter paper is cut according to the pattern, and the result is a regular triangle. Then, the color developing layer is folded according to the fold line, and the aptamer drop layer is folded to the reverse side according to the fold line. The regions of different layers are assembled in the order of the acrylic magnetic sticker plate, the filter paper treated by wax permeation, and the acrylic magnetic sticker plate, one by one, to obtain the folding type multi-channel microfluidic chip.

[0066] The structure diagram of the prepared result-visualized paper-based microfluidic chip is shown in Figure 4

[0067] Example 3

[0068] Application of the folding type multi-channel paper-based microfluidic chip in simultaneous detection of bisphenol A, 17β-estradiol and malathion in landfill leachate

[0069] The nano-mixed material in Example 1 is combined with the folding type multi-channel paper-based microfluidic chip in Example 2 to detect and analyze bisphenol A, 17β-estradiol and malathion with different concentrations. The specific implementation process is as follows:

[0070] (1) 5 μL of bisphenol A aptamer solution with a concentration of 20 μM, 5 μL of 17β-estradiol aptamer solution with a concentration of 20 μM, and 5 μL of malathion aptamer solution with a concentration of 30 μM are respectively added to the three sample adding areas of the aptamer drop layer of the folding type multi-channel paper-based microfluidic chip in Example 2, 5 μL of bisphenol A aptamer solution with a concentration of 20 μM, 5 μL of 17β-estradiol aptamer solution with a concentration of 20 μM, and 5 μL of malathion aptamer solution with a concentration of 30 μM are mixed to obtain an aptamer solution with a mixed concentration of 20 μM, which is added to the aptamer drop control area, and then dried.

[0071] (2) The folding type paper-based microfluidic chip is reversed, and 5 μL of Au / Ag NPs nano-mixed material is added to the four inlets of the color developing layer.

[0072] (3) The acrylic magnetic sticker plate is removed, the aptamer drop layer is unfolded, the sample adding layer is folded on it, and the position of the aptamer drop layer is replaced. The acrylic magnetic sticker plate is clamped and fixed. 12 μL of sample is added to the center sample adding area of the sample adding layer, and 5 μL of distilled water is added to the sample control area, and then dried.

[0073] (4) 12 μL of sodium chloride solution with a concentration of 0.5 M is added to the center sample adding area of the sample adding layer, and 5 μL of sodium chloride solution with a concentration of 0.5 M is added to the sample control area of the sample adding layer. After drying for 3-5 min, the color developing result of the color developing layer is photographed by using a smart phone, and the result is analyzed by using Image J.

[0074] The method is used for detecting and analyzing bisphenol A, 17β-estradiol and malathion with different concentrations, and the linear range and detection limit results are shown in Table 1 as follows.​

[0075] Table 1

[0076]

[0077] Example 4

[0078] Detection of mixed solution of different concentrations of environmental endocrine disruptors

[0079] Four mixed solutions of bisphenol A, 17β-estradiol and malathion with the same concentration were configured, and the concentrations of bisphenol A, 17β-estradiol and malathion were 1.00 μg / ml, 1.50 μg / ml, 2.00 μg / ml and 5.00 μg / ml. The three pollutants aptamers, nano mixed probes, mixed solution samples with different concentrations (Fig. A is 1.00 μg / mL, Fig. B is 1.50 μg / mL, Fig. C is 2.00 μg / mL, and Fig. D is 5.00 μg / mL) and sodium chloride solution with a concentration of 0.5 M were added to a plurality of folding paper-based microfluidic chips in order, and the observation results were observed. Figure 5

[0080] The color development degree of the color development zone showed a certain regularity with the content of the pollutants, and the color development results were as shown in Figure 5 According to Figs. A and B, in the mixed solution with a specific concentration, some pollutants can be visually observed to change color, which are bisphenol A and malathion with a lower detection concentration, and the ΔR / B of the two substances is greater than 0.25, which can be determined as basic color change, and the content exceeds the minimum detection concentration. 17β-estradiol does not change color, and the ΔR / B is less than 0.25, which does not exceed the detection concentration (1.6 μg / ml). According to Figs. C and D, in the mixed solution with a high concentration, the three pollutants can be visually observed to change color, and the ΔR / B is greater than 0.25, which can be determined as basic color change, and the content of the pollutants in the solution exceeds the minimum detection concentration.

[0081] Example 5

[0082] The pretreatment step of the landfill leachate specifically includes the following steps:

[0083] 1) The untreated leachate is introduced into a Buchner funnel, and the filtered leachate enters a suction filter bottle, and then the liquid in the suction filter bottle is collected. Then, the filtered leachate is loaded into a 10 mL centrifuge tube and centrifuged for 10 minutes (12000 rpm), and the supernatant is collected. At this time, the liquid has removed most of the suspended solids and impurities;

[0084] 2) The pH of the centrifuged leachate is adjusted to 3-4, and an appropriate amount of EDTA (0.2 g / L) is added and stirred uniformly;

[0085] ​3) Add pure water and methanol to the SAX column in turn, then add the weak acid eluent, and after entering the SAX column, continue to extract for 30 minutes, then add methanol to the extraction column, and collect the impurity-removed eluent;

[0086] 4) The collected concentrated liquid is subjected to nitrogen blowing treatment, blown to near dryness, dissolved with water, and then diluted to a constant volume to obtain a concentrated eluent for later detection.

[0087] Example 6

[0088] Detection of environmental endocrine disruptors in spiked landfill leachate

[0089] In this example, the Au / Ag NPs nanocomposite material synthesized in Example 1 and the paper-based microfluidic chip prepared in Example 2 are used to realize efficient detection and analysis of three environmental endocrine disruptors (bisphenol A, 17β-estradiol and malathion) in pretreated landfill leachate. The specific steps are the same as those in Example 3, and the data results are shown in Table 2 below. (Sample 1 is organic perishable landfill leachate spiked with bisphenol A, sample 2 is domestic landfill leachate spiked with bisphenol A, sample 3 is organic perishable landfill leachate spiked with 17β-estradiol, sample 4 is domestic landfill leachate spiked with 17β-estradiol, sample 5 is organic perishable landfill leachate spiked with malathion, and sample 6 is domestic landfill leachate spiked with malathion).

[0090] Table 2

[0091]

[0092]

[0093] In summary, the present application proposes a new type of folding multi-channel paper-based microfluidic chip, which can realize simultaneous detection of multiple environmental endocrine disruptors. The present application also proposes mixing Au NPs and Ag NPs in equal proportions to obtain a new Au / Ag NPs nanocomposite material, which can be used as a colorimetric detection probe in combination with specific aptamers. The present application first proposes the application of the paper-based microfluidic chip combined with the Au / Ag NPs nanocomposite material to environmental endocrine disruptors (bisphenol A, 17β-estradiol and malathion) in landfill leachate, successfully developing a visual system for recognizing environmental endocrine disruptors (bisphenol A, 17β-estradiol and malathion) in actual landfill leachate, which has the characteristics of rapidness, simplicity and low cost. In addition, corresponding aptamers can be selected and applied according to the structure of the target analyte, providing a new platform for the analysis of other environmental endocrine disruptors or other environmental pollutants. The present application has great significance in the fields of pollutant monitoring and environmental protection.

[0094] The scope of protection of the present application is not limited to the above embodiments. Variations and improvements based on the concept of the present application that can be conceived by those skilled in the art are included in the present application and are protected by the scope of the claims attached herewith.

Claims

1. A folded multi-channel paper-based microfluidic chip, characterized in that, The microfluidic chip comprises two acrylic magnetic plates and a filter paper treated with wax penetration; the filter paper has a pattern of an equilateral triangle, and the midpoint of the three sides of the equilateral triangle is connected as a fold line to form four equilateral triangle regions, i.e., four functional layers, which are a color development layer, a transport layer, an aptamer drop layer and a sample injection layer; the color development layer comprises three color development zones and one control zone; the transport layer comprises a central transport zone, a circular transport control zone and three transport channels of the same specification and diverging from the central transport zone, each transport channel being composed of a circular transport zone and a rectangle; the aptamer drop layer comprises three aptamer drop zones and one aptamer drop control zone; the sample injection layer comprises a central sample injection zone and a sample injection control zone; wherein: the central sample injection zone is located at the center of the sample injection layer, and the sample flows through the zone to the central transport zone of the transport layer, and then flows to the three color development zones of the color development layer after passing through the three transport channels to the end point; the sample injection control zone is located in the region from the center to the edge of the sample injection layer, and is used for the drop of the sample, i.e., distilled water, as a control zone, and the sample flows through the zone to the corresponding circular transport control zone of the transport layer, and then flows to the control zone of the color development layer; the paper base is a medium-speed filter paper; the color development zone, the control zone, the aptamer drop zone, the aptamer drop control zone, the sample injection control zone, the circular transport zone and the circular transport control zone are all circular shapes of the same size, and the central sample injection zone and the central transport zone are circular shapes of the same size; the transport channel is composed of the circular transport zone and the rectangle, and the rectangle connects the central transport zone and the circular transport zone; the two acrylic magnetic plates are of the same size and shape, i.e., equilateral triangles, and are provided with five circular holes, the positions of the circular holes corresponding to one sample injection zone, three color development zones and one control zone, the diameter of the central circular hole being 1.50 mm larger than that of the central sample injection zone, and the diameters of the four surrounding circular holes being 1.50 mm larger than those of the color development zones; three corners and the middle region of each of the two acrylic magnetic plates are provided with a circular magnet with a diameter of 4.00 mm and a thickness of 2.00 mm; the folding type multi-channel paper-based microfluidic chip is obtained by pressing and assembling the acrylic magnetic plate, the color development layer, the transport layer, the aptamer drop layer and the acrylic magnetic plate in this order, and the sample injection layer is not folded and pressed.

2. The method for preparing the paper-based microfluidic chip according to claim 1, characterized in that, The preparation method comprises the following steps: 1) using AutoCAD software to design the configuration of the paper-based microfluidic chip, so that the color development zone, the control zone, the aptamer drop zone, the aptamer drop control zone, the central sample injection zone, the sample injection control zone, the circular transport zone, the circular transport control zone and the central transport zone are hydrophilic regions, and the rest are hydrophobic regions; 2) cutting an A4-sized medium-speed filter paper and printing the filter paper using a Xerox 8870 wax jet printer; 3) placing the printed filter paper in an oven at 130 degrees Celsius for 30 seconds to allow the hydrophobic paraffin to completely penetrate the reverse side, and cutting the treated filter paper into an equilateral triangle according to the pattern; connecting the midpoints of the three sides of the equilateral triangle as a fold line to form four equilateral triangle regions, i.e., four functional layers, which are a color development layer, a transport layer, an aptamer drop layer and a sample injection layer. 4) three color development zones and one control zone are arranged in the color development layer; one center transport zone, three transport channels with the same size and diverging from the center transport zone, and one circular transport control zone are arranged in the transport layer; three aptamer dropping zones and one aptamer dropping control zone are arranged in the aptamer dropping layer; one center sample adding zone and one sample adding control zone are arranged in the sample adding layer; 5) cut the acrylic plate into a designed equilateral triangle, and then stick five circular magnetic stickers with a diameter of 4.00 mm and a thickness of 2.00 mm at three corners and a middle region of the acrylic plate by using 502 glue; 6) fold the color development layer according to the fold lines, and fold the aptamer dropping layer to the reverse side according to the fold lines, so that the regions of different layers correspond to each other, and finally clamp the acrylic magnetic sticker plates, wherein the circular holes of the acrylic magnetic sticker plates correspond to the color development zones and the control zone of the color development layer, the aptamer dropping zones and the aptamer dropping control zone of the aptamer dropping layer, and the center sample adding zone and the sample adding control zone of the sample adding layer.

3. Application of the paper-based microfluidic chip of claim 1 in detection and analysis of bisphenol A, 17β-estradiol and malathion in landfill leachate.

4. The use according to claim 3, wherein the compound is ###0002### The landfill leachate is a pretreated landfill leachate sample, and the pretreatment includes the following steps: 1) The treated leachate is subjected to suction filtration and centrifugation to remove most of the suspended solids and impurities; 2) adjust the pH to 3-4, add 2 g / L EDTA, and stir uniformly; 3) use SAX small column for solid phase extraction to obtain concentrated leachate; 4) collect the concentrated liquid, treat it by nitrogen blowing, blow it to near dryness, add water, completely dissolve it in an ultrasonic machine, and then dilute it to the volume, which is the pretreated landfill leachate sample.

5. The use according to claim 3, wherein the compound is ###0002### Specifically includes the following steps: 1) Preparation of Au NPs solution with concentration of 5.75 x 10 -9 M and Ag NPs solution with concentration of 9.00 x 10 -4 M, both solutions were centrifuged, 9 / 10 of the supernatant was discarded, and the remaining components were shaken evenly, which were the concentrated solutions of Au NPs with concentration of 5.75 x 10 -8 M and Ag NPs with concentration of 9.00 x 10 -3 M; two kinds of concentrated solutions were mixed evenly in a volume ratio of 1:1 to form Au / Ag NPs nanocomposite material, ready for use; 2) prepare bisphenol A aptamer solution with a concentration of 60 μM, 17β-estradiol aptamer solution with a concentration of 60 μM, and malathion aptamer solution with a concentration of 90 μM, and mix the bisphenol A, 17β-estradiol and malathion aptamer solutions in equal volumes to prepare mixed aptamer solution with concentrations of 20 μM, 20 μM and 30 μM respectively, and reserve; 3) prepare bisphenol A aptamer solution with a concentration of 20 μM, 17β-estradiol aptamer solution with a concentration of 20 μM, and malathion aptamer solution with a concentration of 30 μM, and reserve; 4) drop 5 μL of bisphenol A aptamer solution with a concentration of 20 μM, 5 μL of 17β-estradiol aptamer solution with a concentration of 20 μM, and 5 μL of malathion aptamer solution with a concentration of 30 μM into the three aptamer dropping zones of the paper-based microfluidic chip respectively, and drop 5 μL of the mixed aptamer solution into the aptamer dropping control zone, and wait for drying; 5) reverse the paper-based microfluidic chip, and drop 5 μL of Au / Ag NPs nano-mixed material into the three color development zones and one control zone of the color development layer of the paper-based microfluidic chip, and wait for drying; 6) Remove the acrylic magnetic board, fold the sample layer on it, replace the aptamer drop layer, and then clamp and fix it with the acrylic magnetic board; add 12 μL of the sample to be tested, i.e. landfill leachate, to the center sample addition area of the sample layer of the paper-based microfluidic chip, and add 5 μL of distilled water to the sample addition control area, and then wait for drying; 7) Add 12 μL of 1.5 M sodium chloride solution to the center sample addition area of the sample layer of the paper-based microfluidic chip, and add 5 μL of 1.5 M sodium chloride solution to the sample addition control area, and then wait for drying for 3-5 min, take a photo of the color development layer, and analyze the results by Image J.

6. The use according to claim 3, wherein the compound is ###0003### The detection of bisphenol A, 17β-estradiol and malathion in landfill leachate has detection limits of 0.6 μg mL -1 , 1.6 μg mL -1 and 0.9 μg mL -1 , respectively, and linear ranges of 5-20 μg mL -1 , 2-20 μg mL -1 and 1-50 μg mL -1 , respectively.

7. The use according to claim 5, wherein the compound is ###00006### The Au / Ag NPs nanohybrid material contains an electrostatic force unit.

8. The use according to claim 5, wherein the compound is ###0005### The Au / Ag NPs nanohybrid material is easy to combine with the aptamers of bisphenol A, 17β-estradiol and malathion, and the material and the aptamers have an electrostatic force unit.

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