A three-dimensional paper chip, a preparation method thereof and application thereof in nitrite detection

Abstract

The application belongs to the technical field of food detection, and discloses a three-dimensional paper chip, a preparation method thereof and application of the three-dimensional paper chip in nitrite detection. The three-dimensional paper chip is obtained by sequentially laminating and compounding pretreatment layer one, channel layer one, pretreatment layer two, channel layer two and reaction layer. The pretreatment layer one and the pretreatment layer two are provided with hydrophilic channels filled with pretreatment reagents, the channel layer one is provided with liquid outlets corresponding to the right ends of the hydrophilic channels in the pretreatment layer one and the pretreatment layer two, the channel layer two is provided with liquid outlets corresponding to the left ends of the hydrophilic channels in the pretreatment layer two, the reaction layer is provided with a hydrophilic channel from left to right, the left end of the hydrophilic channel corresponds to the liquid outlet of the channel layer two, and the right end is connected to detection area one and detection area two through a shunt channel one and a shunt channel two respectively, the detection area one and the detection area two are both filled with Griess reagent, and the shunt channel two is filled with a reducing agent. The three-dimensional paper chip can be used for detecting the content of nitrite in food.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food detection, and particularly relates to a three-dimensional paper chip, a preparation method thereof and application of the three-dimensional paper chip in nitrite detection. BACKGROUND

[0002] Nitrate ions (NO3 - ) and nitrite ions (NO2 - ) are common water-soluble inorganic anions, and excessive nitrate ions and nitrite ions (NO2 - ) can adversely affect human health. For example, nitrate ions can be converted into nitrite ions (NO2 - ) in the human body, and then combine with hemoglobin to form nitrite hemoglobin, affecting oxygen transport and hematopoietic function; in addition, nitrite ions can also react with amine substances to generate nitrosamine compounds with carcinogenicity. Meat products are one of the important sources of protein in human daily diet, but are also high-risk foods of nitrate ion pollution. Nitrate ions in meat products mainly come from two aspects: one is that animals ingest water and feed containing nitrate ions during feeding; the other is that preservatives or pigments containing nitrate and nitrite are added during the processing and storage of meat products. The content of nitrate ions in meat products not only affects the quality and safety of the meat products, but also relates to the health of consumers. Therefore, it is of great significance to establish a rapid, accurate, convenient and low-cost method for detecting the content of nitrate ions in meat products to ensure food safety and public health. At present, the commonly used methods for detecting the content of nitrate ions in meat products include ultraviolet-visible spectrophotometry, electrochemical method, chromatography, fluorescence method and the like. Although these methods have high sensitivity and accuracy, they also have some shortcomings, such as expensive instruments, complex operation, long time consumption, cumbersome sample processing, and much waste liquid discharge. Therefore, it is a hot and difficult point in current research to develop a new detection method to overcome the shortcomings of the above methods.

[0003] In recent years, paper-based microfluidic chip as a new bioanalysis platform was first proposed by WHITESIDE research group of Harvard University in 2007, and has received extensive attention and research due to its advantages of on-site rapid detection, convenience, low cost and so on. Paper-based microfluidic chip is a micro device that uses paper as a substrate material to prepare micron or millimeter level flow channel network on it, to realize the automatic transportation and mixing of liquid samples and reagents on the paper through capillary action, and to complete various biochemical analysis or diagnosis tasks by combining with appropriate signal detection methods. So far, most of the literature has reported various ways to prepare two-dimensional paper-based microfluidic chip. Two-dimensional paper-based microfluidic chip has simple structure and convenient manufacturing method. For example, a paper-based microfluidic chip for multi-parameter analysis of water quality detection as recorded in patent CN 111715312 A is directly cut into the required shape by using an embosser. By pre-loading various reaction reagents such as indicators and activators, the multiple reactions of conventional analysis and detection methods can be integrated on a microfluidic chip. However, the use effect of conventional two-dimensional paper-based microfluidic chip may have some defects, such as liquid remaining in the capillary tube or volatilizing during the flow process, which can cause low transmission efficiency, and the viscous force can seriously affect the flow of liquid in the microchannel, thereby affecting the detection effect. Three-dimensional paper-based microfluidic chip refers to the use of multiple papers to make a three-dimensional structure, which can make the fluid distribute in the vertical and horizontal directions. Based on this three-dimensional structure, multiple detection and functional integration can be realized, making the on-site detection process easier and without the need for special and expensive instruments, with the advantages of low cost, simplicity and flexibility. Generally, the manufacturing method of three-dimensional paper chip is to expand the processing technology of two-dimensional chip by adopting stacking method, paper folding method and 3D printing to form a multi-layer overlapping structure.

[0004] As disclosed in patent CN 114798021 A, a three-dimensional paper-based microfluidic chip for rapid detection of nitrite in water body is provided. The three-dimensional paper-based microfluidic chip has a four-layer structure, which is glass paper cover plate, quincunx paper chip, circular color developing layer and glass paper cushion layer in sequence. Based on the capillary action of the paper-based microfluidic chip, the chemical experimental operation steps such as sample injection, splitting and detection can be completed for the sample to be tested. However, the three-dimensional paper-based microfluidic chip does not construct a specific hydrophilic channel for liquid flow, and it still has the problems of low transmission efficiency caused by volatilization of liquid during the flow process, and serious influence of viscous force on the flow of liquid in the microchannel. In addition, the three-dimensional paper-based microfluidic chip cannot pretreat the test sample by removing impurities and resisting interference, which affects the accuracy and sensitivity of the detection. Finally, the three-dimensional paper-based microfluidic chip analyzes the results by colorimetric card and PS image processing technology based on the color of the color developing layer, which has the problems of complex processing and low quantitative accuracy.

[0005] Patent CN 111013679 A discloses a paper-based microfluidic chip preparation method, the preparation method is, using a laser engraving machine to carry out laser treatment on the hydrophobic paper-based surface, the area treated by the laser engraving machine forms a hydrophilic area, the area not treated by the laser forms a hydrophobic area, the surface of the hydrophilic area is smeared with a chemical suspension to form a paper-based microfluidic chip. The prior art uses laser to modify the hydrophobic paper-based surface, and needs to cooperate with the use of silica suspension to make the surface liquid have fluidity, so as to construct a specific hydrophilic channel for liquid flow. Its liquid is easy to evaporate during flow, resulting in low transmission efficiency, and it cannot pretreat the detection sample to remove impurities and resist interference, affecting the accuracy and sensitivity of detection. Moreover, it generally analyzes the results by color change of the reaction area, which has the problems of low accuracy and narrow range of reagents.

[0006] As can be seen, the paper-based microfluidic chip scheme of the prior art uses staples to adhere each layer in the manufacturing method, resulting in loose connection between each layer, which may cause liquid leakage, oxidation of the detection reagent, and evaporation of the sample solution during detection, resulting in inaccurate colorimetric reading. The structure is simple and does not integrate many functional units, which may make the sample pretreatment operation cumbersome. In the detection method, the gray value is calculated by PS colorimetric method after photographing, which is a tedious process, the on-site detection result cannot be obtained in time, and the deviation may occur due to different models and parameters of the photographing equipment.

[0007] Therefore, it has significant application value and commercial value to construct a three-dimensional paper-based microfluidic chip capable of simply and accurately detecting nitrite ions and nitrate ions. SUMMARY

[0008] In view of the shortcomings and deficiencies of the prior art, the primary purpose of the present application is to provide a preparation method of a three-dimensional paper chip.

[0009] Another purpose of the present application is to provide a three-dimensional paper chip prepared by the above method.

[0010] Still another purpose of the present application is to provide the application of the above three-dimensional paper chip in the detection of nitrite in food.

[0011] The present application constructs a three-dimensional paper-based microfluidic chip for detecting nitrite ions and nitrate ions in food such as meat products by stacking method without the need of electronic equipment. By integrating multiple functional units, the above problems are solved and the operation is simple, only the sample solution needs to be injected, and then the detection result is obtained by comparing the signal distance with the reference card, which greatly improves the efficiency of on-site detection and effectively reduces the detection cost.

[0012] The purpose of the present application is achieved by the following technical solutions:

[0013] A preparation method of a three-dimensional paper chip, comprising the following preparation steps:

[0014] (1) Preparation of pretreatment layer one: take filter paper and set up hydrophilic channels from left to right, the rest part is hydrophobic region after hydrophobic treatment, and the hydrophilic channels are filled with pretreatment reagent 1;

[0015] (2) Preparation of channel layer one: take filter paper and set up liquid outlet at the right end, the rest part is hydrophobic region after hydrophobic treatment, and the liquid outlet corresponds to the right end position of the hydrophilic channels in the pretreatment layer one;

[0016] (3) Preparation of pretreatment layer two: take filter paper and set up hydrophilic channels from left to right, the rest part is hydrophobic region after hydrophobic treatment, and the right end of the hydrophilic channels corresponds to the liquid outlet of the channel layer one, and the hydrophilic channels are filled with pretreatment reagent 2;

[0017] (4) Preparation of channel layer two: take filter paper and set up liquid outlet at the left end, the rest part is hydrophobic region after hydrophobic treatment, and the liquid outlet corresponds to the left end position of the hydrophilic channels in the pretreatment layer two;

[0018] (5) Preparation of reaction layer: take filter paper and set up hydrophilic channels from left to right, the left end of the hydrophilic channels corresponds to the liquid outlet position of the channel layer two, and the right end of the hydrophilic channels is connected to detection area one and detection area two through shunt channel one and shunt channel two respectively, and both the detection area one and the detection area two are filled with Griess reagent; the shunt channel two is filled with reducing agent; the rest part of the reaction layer except the hydrophilic channels, the shunt channel one, the shunt channel two, the detection area one and the detection area two is hydrophobic region after hydrophobic treatment;

[0019] (6) Stack and composite the pretreatment layer one, the channel layer one, the pretreatment layer two, the channel layer two and the reaction layer in sequence to obtain a three-dimensional paper chip.

[0020] Further, the filter paper in steps (1)-(5) is preferably slow qualitative filter paper.

[0021] Further, the hydrophobic treatment in steps (1)-(5) means that polylactic acid solution is printed on the hydrophobic region of the filter paper, and then dried. The mass concentration of the polylactic acid solution is 8%-12%, preferably 9%-10%, and more preferably 10%.

[0022] Further, the length of the hydrophilic channel in steps (1) and (3) is 5-15 mm, preferably 8-12 mm, and more preferably 10 mm; and the width of the hydrophilic channel is 2-6 mm, preferably 4-5 mm, and more preferably 4 mm.

[0023] Further, the liquid outlet in steps (2)-(4) is a circular hole with a diameter of 2-6 mm; preferably a circular hole with a diameter of 4-5 mm; more preferably a circular hole with a diameter of 4 mm.

[0024] Further, the pretreatment reagent 1 in step (1) and the pretreatment reagent 2 in step (3) are potassium ferrocyanide and zinc acetate; and the method for filling the hydrophilic channel with the pretreatment reagent 1 and the pretreatment reagent 2 is as follows: coating a potassium ferrocyanide solution onto the hydrophilic channel, naturally drying, then coating a zinc acetate solution, and naturally drying to obtain the hydrophilic channel filled with the pretreatment reagent 1 and the pretreatment reagent 2.

[0025] The present application sets a pretreatment layer containing potassium ferrocyanide and zinc acetate, and the zinc ferrocyanide precipitate is formed in the hydrophilic channel by the potassium ferrocyanide solution and the zinc acetate solution, which can co-precipitate with the protein in the sample solution, and the sample solution flowing through the area is adsorbed by the zinc ferrocyanide precipitate filled in the channel, so as to remove the protein in the sample solution. Since the food such as meat product contains a large amount of soluble basic protein such as actin, the two-layer pretreatment channel can ensure the excess of the pretreatment reagent, so as to completely remove the protein, and the structure of the three-dimensional paper chip is more compact and the area is smaller.

[0026] Further, the length of the hydrophilic channel in step (5) is 4-8 mm, and the width is 2-6 mm; the length of the shunt channel one and the shunt channel two is 8-12 mm, and the width is 2-3 mm; and the length of the detection area one and the detection area two is 20-40 mm, and the width is 2-6 mm.

[0027] Further, the reducing agent in step (5) includes hydrazine sulfate and copper sulfate; and the method for filling the reducing agent is as follows: coating a hydrazine sulfate solution and a copper sulfate solution onto the shunt channel two, and naturally drying to obtain the shunt channel two filled with the reducing agent. The shunt channel can reduce the nitrate to nitrite, and the content of the nitrite can be detected by the Griess reagent in the detection area two.

[0028] Further, the Griess reagent in step (5) is a mixed solution containing N-(1-naphthyl)ethylenediamine dihydrochloride with a mass concentration of 0.2%-0.8% and sulfanilamide with a mass concentration of 2%-8%; and the method for filling the Griess reagent is as follows: coating the Griess reagent onto the detection area one and the detection area two, so as to completely wet the detection area. The color developing principle of the Griess reagent and the nitrite is as follows: the nitrite is diazotized with the sulfanilamide, and then coupled with the N-(1-naphthyl)ethylenediamine dihydrochloride to form a red dye and develop color.

[0029] Further, the connection between the first shunt channel and the first detection area and the connection between the second shunt channel and the second detection area in step (5) are further provided with a perchloroethylene resin fence, and the width of the perchloroethylene resin fence is 1-2 mm. The greater the width of the perchloroethylene resin fence, the better the interception effect, but it will cause the sample solution to penetrate into the detection area slowly or be retained in front of the fence, affecting the fluidity of the liquid. The perchloroethylene resin fence can be obtained by screen printing a perchloroethylene solution with a mass concentration of 2-6% onto the corresponding position, preferably a perchloroethylene solution with a mass concentration of 4-5%, and more preferably a 4% perchloroethylene solution.

[0030] The present application sets a perchloroethylene resin fence, and the perchloroethylene resin has a good selective permeation effect. Nitrite ions can form a solvation shell with the perchloroethylene resin to enable them to permeate the voids of the perchloroethylene resin. Water molecules are the solvent of nitrite ions, and the hydration radius is relatively large. Although water molecules can also pass through the perchloroethylene resin fence, the concentration of water molecules will be greatly reduced. By using the selective permeation characteristics of perchloroethylene resin for nitrite ions, impurities can be effectively intercepted and nitrite ions can be enriched, thereby improving the accuracy and sensitivity of detection.

[0031] Further, the upper surface of the pretreatment layer one of the three-dimensional paper chip is covered with a transparent overmolding film printed with a size scale, and the size scale on the transparent overmolding film corresponds to the positions of the first detection area and the second detection area on the reaction layer. A sample addition port is provided at the left end of the transparent overmolding film, and the sample addition port corresponds to the left end position of the hydrophilic channel in the pretreatment layer one.

[0032] The transparent overmolding film provided on the upper surface of the pretreatment layer one can enhance the stability and convenience of sample addition. The size scale corresponding to the positions of the first detection area and the second detection area on the reaction layer can facilitate accurate reading of the detection results.

[0033] Further, the lower surface of the reaction layer of the three-dimensional paper chip is provided with a transparent overmolding film.

[0034] The provision of the above-mentioned transparent overmolding film can prevent liquid evaporation and reagent oxidation by air, making the detection results visualized and enhancing the detection stability.

[0035] A three-dimensional paper chip prepared by the above-mentioned method.

[0036] The above-mentioned three-dimensional paper chip is used for nitrite detection in food.

[0037] Further, the application method is as follows: a syringe or other pressure-providing sample introduction device is used to inject the pretreated food sample solution from the sample addition port into the three-dimensional paper chip, and the concentrations of nitrite ions and nitrate ions are calculated by the color development length of the first detection area and the second detection area in the reaction layer.

[0038] Compared with the prior art, the application has the beneficial effects that:

[0039] (1) The three-dimensional paper chip of the application can complete the chemical experimental operation steps of sample injection, pretreatment, splitting, enrichment and detection based on the capillary action of the paper-based microfluidic chip and the pressure action of the injection device such as the injection tube, and achieve the effect of low cost, high speed and precise on-site detection.

[0040] (2) The three-dimensional paper chip of the application can establish the relationship between the signal distance generated by the detection area and the concentration of nitrite ions, and can directly obtain the concentration value of the sample by referring to the distance signal card, without the need for photographing software processing by naked eye observation, and has the advantages of simple result processing, high accuracy and wide application range.

[0041] (3) The three-dimensional paper chip of the application can further construct a vinylidene chloride resin fence module with selective permeation function to achieve enrichment and separation, thereby improving the sensitivity and accuracy of the chip. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 It is a schematic diagram of the overall laminated structure of a three-dimensional paper chip in Example 1 of the application.

[0043] Figures 2 to 8 It is a schematic diagram of the plane structure of the sample adding layer, the pretreatment layer one, the channel layer one, the pretreatment layer two, the channel layer two, the reaction layer and the protection layer of the three-dimensional paper chip in Example 1 of the application.

[0044] The numbering in the figure is as follows: 1-sample adding layer, 1-1-sample port, 1-2-size scale; 2-pretreatment layer one, 2-1-hydrophilic channel, 2-2-hydrophobic area; 3-channel layer one, 3-1-liquid outlet, 3-2-hydrophobic area; 4-pretreatment layer two, 4-1-hydrophilic channel, 4-2-hydrophobic area; 5-channel layer two, 5-1-liquid outlet, 5-2-hydrophobic area; 6-reaction layer, 6-1-hydrophilic channel, 6-2-splitting channel one, 6-3-splitting channel two, 6-4-vinylidene chloride resin fence, 6-5-detection area one, 6-6-detection area two; 7-protection layer;

[0045] Figures 9 to 12 It is a time relationship diagram of each scale liquid reaching under different width conditions of different filter papers in Example 1 of the application.

[0046] Figure 13 It is a result diagram of observing the resolution and contact angle size after printing different concentrations of polylactic acid solution on slow filter paper and drying in Example 1 of the application.

[0047] Figure 14The standard curve of nitrite ion concentration and signal distance in Example 2 of the present application.

[0048] Figure 15 The length signal card corresponding to the signal distance and nitrite ion concentration obtained according to the standard curve in Example 2 of the present application.

[0049] Figure 16 The stability test result graph of the three-dimensional paper chip in Example 2 of the present application.

[0050] Figure 17 The test result comparison graph of the three-dimensional paper chip of the present application and the ultraviolet spectrophotometer.

[0051] Figure 18 The flowchart of the three-dimensional paper chip in Example 3 of the present application for detecting nitrite ions in food such as meat products. DETAILED DESCRIPTION

[0052] The present application will be further described in detail below with reference to the examples and the accompanying drawings, but the embodiments of the present application are not limited thereto.

[0053] Example 1

[0054] The preparation method of a three-dimensional paper chip in this embodiment includes the following preparation steps:

[0055] (1) Preparation of pretreatment layer one: take filter paper and set up a hydrophilic channel 2-1 from left to right, and the rest is a hydrophobic area 2-2 after coating with polylactic acid solution. The left and right ends of the hydrophilic channel are semicircular, and the middle is rectangular; the length of the hydrophilic channel can be set to 5-15 mm as needed, preferably 8-12 mm, and more preferably 10 mm; the width of the hydrophilic channel can be set to 2-6 mm as needed, preferably 4-5 mm, and more preferably 4 mm. The hydrophilic channel is filled with pretreatment reagent 1; the filling method is to uniformly apply 5 μl of 0.5% w / v potassium ferrocyanide reagent on the hydrophilic channel, dry and repeat 4 times; then uniformly apply 5 μl of 10% w / v zinc acetate reagent on the hydrophilic channel, dry and repeat 4 times.

[0056] (2) Preparation of channel layer one: take filter paper and set up a liquid outlet 3-1 at the right end, and the rest is a hydrophobic area 3-2 after coating with polylactic acid solution. The liquid outlet can be designed as a circular hole with a diameter of 2-6 mm; preferably a circular hole with a diameter of 4-5 mm; more preferably a circular hole with a diameter of 4 mm. The liquid outlet corresponds to the right end position of the hydrophilic channel in the pretreatment layer one.

[0057] (3) Preparation of pretreatment layer two: Take filter paper and set hydrophilic channel 4-1 from left to right, and the rest is hydrophobic region 4-2 after being coated with polylactic acid solution. The shape and size of the hydrophilic channel are exactly the same as those in the pretreatment layer one, and the right end of the hydrophilic channel corresponds to the liquid outlet of the channel layer one. The hydrophilic channel is filled with pretreatment reagent 2; the filling method is to uniformly apply 5 μl of 0.5% w / v potassium ferrocyanide reagent on the hydrophilic channel, and repeat 4 times after drying; then uniformly apply 5 μl of 10% w / v zinc acetate reagent on the hydrophilic channel, and repeat 4 times after drying.

[0058] (4) Preparation of channel layer two: Take filter paper and set liquid outlet 5-1 at the left end, and the rest is hydrophobic region 5-2 after being coated with polylactic acid solution. The shape and size of the liquid outlet are exactly the same as those of the liquid outlet of the channel layer one. The liquid outlet corresponds to the left end position of the hydrophilic channel in the pretreatment layer two.

[0059] (5) Preparation of reaction layer: Take filter paper and set hydrophilic channel 6-1 from left to right, the length of the hydrophilic channel is 4-8 mm, and the width is 2-6 mm; the left end of the hydrophilic channel is semicircular corresponding to the liquid outlet position of the channel layer two, and the right end of the hydrophilic channel is connected to detection area one 6-5 and detection area two 6-6 through shunt channel one 6-2 and shunt channel two 6-3 respectively. The length of the shunt channel one and the shunt channel two is 8-12 mm, and the width is 2-3 mm; the length of the detection area one and the detection area two is 20-40 mm, and the width is 2-6 mm. Both the detection area one and the detection area two are filled with Griess reagent (the filling method is to add 35 μl of 0.5% Griess reagent to the detection area one and the detection area two respectively by pipette, and repeat 4 times after natural drying); the shunt channel two is filled with reducing agent (the filling method is to take 5 μl of 1 g / L sulfuric acid solution, uniformly apply it on the shunt channel two as a reducing area, repeat 4 times after drying, then take 10 μl of 2.65×10 -4 mol / L copper sulfate solution and uniformly apply it in the reducing area, and repeat 4 times after natural drying); the connection between the shunt channel one and the detection area one and the connection between the shunt channel two and the detection area two are also provided with perchloroethylene resin fence 6-4. The width of the perchloroethylene resin fence is 1-2 mm. The perchloroethylene resin fence is obtained by silk screen printing 4% filtered ethylene resin solution to the corresponding position and drying. The rest of the reaction layer except the hydrophilic channel, the shunt channel one, the shunt channel two, the detection area one and the detection area two is hydrophobic region after being coated with polylactic acid solution.

[0060] (6) The pretreatment layer one, the channel layer one, the pretreatment layer two, the channel layer two and the reaction layer are sequentially laminated and bonded to form a composite, and a transparent overmolding film printed with a size scale 1-2 is covered on the upper surface of the pretreatment layer one as a sample adding layer 1, the size scale on the transparent overmolding film corresponds to the positions of the detection area one and the detection area two on the reaction layer; a sample adding port 1-1 is provided at the left end of the sample adding layer, which corresponds to the left end position of the hydrophilic channel in the pretreatment layer one. A transparent overmolding film is covered below the reaction layer as a protection layer 7. The obtained laminated structure is hot-pressed and placed in an oven at 100°C for 30 min to obtain a three-dimensional paper chip.

[0061] The schematic diagram of the overall laminated structure of the three-dimensional paper chip obtained in the embodiment is shown in Figure 1 The schematic diagram of the planar structure of the sample adding layer, the pretreatment layer one, the channel layer one, the pretreatment layer two, the channel layer two, the reaction layer and the protection layer is shown in Figures 2 to 8

[0062] The parameter conditions of the three-dimensional paper chip in the embodiment are optimized as follows:

[0063] (1) Selection of filter paper type and hydrophilic channel width:

[0064] The control of fluid in the microfluidic chip is the key to the whole chip system, and the driving force of fluid in the hydrophilic channel completely depends on the action of capillary force and moves in a laminar flow state under microscale. The length and width of the hydrophilic channel determine the flow rate and volume of the liquid, and too high flow rate will cause the drift phenomenon of the embedded reaction reagent, and too slow flow rate will cause too much liquid residue and evaporation, thereby affecting the accuracy and sensitivity of the detection result. In order to determine the most suitable length and width of the hydrophilic channel size, five groups of rectangular paper strips with a length of 5 cm and widths of 2 mm, 3 mm, 4 mm, 5 mm and 6 mm were designed in three types of filter paper (slow qualitative filter paper, medium-speed qualitative filter paper and fast qualitative filter paper, Hangzhou Wohua Filter Paper Co., Ltd.), the middle and sample adding port of the paper strip were embedded with reaction reagent, and the time for each scale liquid to reach was recorded by vertically inserting the paper strip into 2 mm deep water, and whether each group of embedded reagent had drift phenomenon was recorded. The relationship between the time for each scale liquid to reach under different width conditions of different filter papers is shown in Figures 9 to 12 ​The flow rate of the whole filter paper is the fastest, and the flow rates of the slow and medium speed filter papers are similar. However, the whole deviation of the fast filter paper and the medium speed filter paper is large, and the whole deviation of the slow filter paper is the smallest, indicating that the uniformity is the best. From the graph of the slow filter paper, it can be seen that with the increase of the diameter of the capillary, the flow rate of the fluid shows a trend of first increasing and then decreasing, which may be because when the width of the paper strip is wider, there are more pores formed by the interweaving of fibers in the channel, thereby providing stronger capillary force. However, due to the increase of the diameter of the paper strip, the gravity and viscosity of the liquid also increase, hindering the climbing of the liquid. The flow rate of the 4mm size is similar to that of the other sizes, and the filled reagent does not drift, so the slow filter paper and the 4mm width are selected as the most suitable paper base material and hydrophilic channel size.

[0065] (2) Concentration selection of hydrophobic treated reagent polylactic acid solution:

[0066] The concentration of the polylactic acid solution determines the hydrophobic performance and pattern resolution of the microfluidic chip, thereby affecting the directional movement of the liquid. To determine the most suitable concentration of the polylactic acid solution, 0.08g, 0.09g, 0.1g, 0.11g and 0.12g of polylactic acid particles were weighed into a measured dichloromethane solution, heated to dissolve in a 40°C water bath, and vortexed for 1 min after complete dissolution. Polylactic acid solutions of 8% w / w, 9% w / w, 10% w / w, 11% w / w and 12% w / w were prepared. The resolution and contact angle of the polylactic acid solution with different concentrations were observed after printing and drying on the slow filter paper, and the results are shown in Figure 13 From the graph, it can be seen that as the concentration of the polylactic acid solution increases, the contact angle shows an upward trend, but when the concentration exceeds 10%, the hydrophobic performance shows a downward trend. This phenomenon indicates that when the concentration of the polylactic acid solution is 10%, the combination of the polylactic acid solution and the filter paper reaches saturation, and the drying speed of the polylactic acid solution with a concentration exceeding 10% is too fast to completely combine with the paper to form a hydrophobic structure. After printing, it was found that the resolution of the 10% polylactic acid solution and the 9% polylactic acid solution was close, and the 10% polylactic acid solution had better hydrophobic effect, so the 10% concentration of the polylactic acid solution was selected as the most suitable concentration.

[0067] Example 2

[0068] Performance test of the three-dimensional paper chip of Example 1 in the application of nitrite ion detection.

[0069] (1) Drawing of standard curve and length signal card:

[0070] First, a series of concentration gradient of sodium nitrite standard solution is prepared, and the concentrations are 0 mg / L, 1 mg / L, 1.5 mg / L, 5 mg / L, 10 mg / L, and 20 mg / L, respectively. Then, a series of concentration gradient of nitrate + nitrite standard solution is prepared, and the concentrations are 0 mg / L, 1 mg / L + 1 mg / L, 1.5 mg / L + 1.5 mg / L, 2 mg / L + 2 mg / L, 5 mg / L + 5 mg / L, and 10 mg / L + 10 mg / L, respectively. The samples are loaded by using a syringe, and then react with the Griess reagent under the driving of pressure. After the reaction is completed, the color development length of the detection zone is recorded. Finally, the data of signal distance and nitrite ion concentration are processed and analyzed. The standard curve of nitrite ion concentration and signal distance is shown in Figure 14 According to the standard curve, the length signal card corresponding to the signal distance and the nitrite ion concentration is shown in Figure 15 The nitrate content = the reading of the nitrate detection zone (detection zone two) - the reading of the nitrite detection zone (detection zone one).

[0071] (2) Stability test:

[0072] The assembled three-dimensional paper-based microfluidic chip product is subjected to an accelerated stability experiment. Under the set storage environment, it is confirmed that the effective period will not change, so as to ensure the quality of the product. The accelerated stability test can be used to estimate the shelf life. According to the Arrhenius formula, the relationship between them is shown in the following table:

[0073] Table 1 Relationship between accelerated stability and real-time stability

[0074]

[0075] Test method: The assembled three-dimensional paper-based microfluidic chip product is placed at 50℃ for storage, and 2 mg / L nitrite standard solution is taken on the 0th, 7th, 28th, and 34th days for detection of the three-dimensional paper-based microfluidic chip and recording of the results. The results are shown in Figure 16 According to the calculation according to the formula, the effective period of use of the three-dimensional paper-based microfluidic chip of the application is one year.

[0076] (3) Accuracy test:

[0077] The test sample is taken and tested by using an ultraviolet spectrophotometer and the three-dimensional paper-based microfluidic chip of the application, and the comparison of the test results is shown in Figure 17 There is no obvious difference between the results of the ultraviolet spectrophotometer and the three-dimensional paper-based microfluidic chip of the application.

[0078] Example 3

[0079] Application of the three-dimensional paper chip of Example 1 in the detection of nitrite ions in food:

[0080] (1) In the Guangzhou halogen meat stall, buy halogen meat products, bacon products, put them into the centrifuge tube containing saturated borax solution after cutting them with scissors, shake 10 times, and stand for 5 min.

[0081] (2) 0.1 mL of supernatant in the centrifuge tube after standing is sucked by a syringe.

[0082] (3) The syringe is injected into the sample inlet in the three-dimensional paper-based microfluidic chip, and the color development result is waited after the injection is completed.

[0083] (4) The concentration of the sample to be measured is obtained according to the color development result and the length signal card.

[0084] The flowchart of the detection of the embodiment is shown in Figure 18 .

[0085] The above embodiment is a preferred embodiment of the present application, but the embodiment of the present application is not limited by the above embodiment, and any change, modification, substitution, combination, simplification made without departing from the spirit and principle of the present application should be an equivalent replacement mode, which is included in the protection scope of the present application.

Claims

1. A method for preparing a three-dimensional paper chip, characterized in that, The preparation steps include the following: (1) Preparation of pretreatment layer 1: Take filter paper and set hydrophilic channels from left to right. The remaining part is hydrophobic after hydrophobic treatment. The hydrophilic channels are filled with pretreatment reagent 1. (2) Preparation of channel layer one: Take filter paper and open a liquid outlet at the right end. The remaining part is hydrophobic after hydrophobic treatment. The liquid outlet corresponds to the right end of the hydrophilic channel in the pretreatment layer one. (3) Preparation of pretreatment layer 2: Take filter paper and set up hydrophilic channels from left to right. The remaining part is hydrophobic after hydrophobic treatment. The right end of the hydrophilic channel corresponds to the liquid outlet of channel layer 1. The hydrophilic channel is filled with pretreatment reagent 2. (4) Preparation of channel layer two: Take filter paper and open a liquid outlet at the left end. The remaining part is hydrophobic after hydrophobic treatment. The liquid outlet corresponds to the left end position of the hydrophilic channel in the pretreatment layer two. (5) Preparation of the reaction layer: Take filter paper and set up hydrophilic channels from left to right. The left end of the hydrophilic channel corresponds to the liquid outlet position of channel layer two. The right end of the hydrophilic channel is connected to detection zone one and detection zone two through diversion channel one and diversion channel two, respectively. Detection zone one and detection zone two are filled with Griess reagent; diversion channel two is filled with reducing agent; the rest of the reaction layer except for the hydrophilic channel, diversion channel one, diversion channel two, detection zone one and detection zone two are hydrophobic regions after hydrophobic treatment. (6) The pretreatment layer 1, channel layer 1, pretreatment layer 2, channel layer 2 and reaction layer are stacked sequentially to obtain a three-dimensional paper chip.

2. The method for preparing a three-dimensional paper chip according to claim 1, characterized in that, The filter paper mentioned in steps (1) to (5) is a slow-speed qualitative filter paper; the hydrophobic treatment refers to printing polylactic acid solution onto the hydrophobic area of ​​the filter paper and then drying it. The polylactic acid solution has a mass concentration of 8% to 12%.

3. The method for preparing a three-dimensional paper chip according to claim 1, characterized in that, The length of the hydrophilic channel in steps (1) and (3) is 5-15 mm and the width is 2-6 mm; the liquid outlet in steps (2) to (4) is a round hole with a diameter of 2-6 mm.

4. The method for preparing a three-dimensional paper chip according to claim 1, characterized in that, The pretreatment reagent 1 mentioned in step (1) and the pretreatment reagent 2 mentioned in step (3) are potassium ferrocyanide and zinc acetate; the method for filling the hydrophilic channel with pretreatment reagent 1 and pretreatment reagent 2 is as follows: apply potassium ferrocyanide solution to the hydrophilic channel, let it dry naturally, then apply zinc acetate solution, let it dry naturally, and obtain the hydrophilic channel filled with pretreatment reagent 1 and pretreatment reagent 2.

5. The method for preparing a three-dimensional paper chip according to claim 1, characterized in that, The hydrophilic channel in step (5) has a length of 4-8 mm and a width of 2-6 mm; the first and second diversion channels have a length of 8-12 mm and a width of 2-3 mm; the first and second detection areas have a length of 20-40 mm and a width of 2-6 mm.

6. The method for preparing a three-dimensional paper chip according to claim 1, characterized in that, The reducing agent mentioned in step (5) includes hydrazine sulfate and copper sulfate; the method of embedding the reducing agent is: coating the hydrazine sulfate solution and copper sulfate solution onto the second diversion channel and allowing it to dry naturally to obtain the second diversion channel filled with the reducing agent; the Griess reagent is a mixed solution containing 0.2% to 0.8% N-(1-naphthyl)ethylenediamine dihydrochloride and 2% to 8% sulfonamide by mass concentration; the method of embedding the Griess reagent is: coating the Griess reagent onto detection area one and detection area two to completely wet the entire detection area.

7. The method for preparing a three-dimensional paper chip according to claim 1, characterized in that, In step (5), the connection between the first diversion channel and the first detection area and the connection between the second diversion channel and the second detection area are also provided with a chlorinated polyvinyl chloride resin fence. The width of the chlorinated polyvinyl chloride resin fence is 1-2 mm. The chlorinated polyvinyl chloride resin fence is obtained by screen printing a chlorinated polyvinyl chloride solution with a mass concentration of 2%-6% onto the corresponding position and then drying it.

8. A method for preparing a three-dimensional paper chip according to any one of claims 1 to 7, characterized in that, The upper surface of the pretreatment layer of the three-dimensional paper chip is covered with a transparent overmolded film with size markings printed on it. The size markings on the transparent overmolded film correspond to the positions of detection area one and detection area two on the reaction layer. A sample dispensing port is provided at the left end of the transparent overmolded film, which corresponds to the left end position of the hydrophilic channel in the pretreatment layer. A transparent overmolded film is provided on the lower surface of the reaction layer of the three-dimensional paper chip.

9. A three-dimensional paper chip, characterized in that, It is prepared by the method described in any one of claims 1 to 8.

10. The application of the three-dimensional paper chip according to claim 9 in the detection of nitrite in food, characterized in that, The application method is as follows: the pretreated food test solution is injected into the three-dimensional paper chip through the sample dispensing port using a syringe, and the concentrations of nitrite ions and nitrate ions are calculated by the color development lengths of detection zone one and detection zone two in the reaction layer.

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