Programmable paper chip and preparation method thereof, microfluidic analysis chip

Abstract

The present invention discloses a programmable paper chip and a preparation method thereof, and a microfluidic analysis chip, which belongs to the field of paper-based microfluidic technology. The programmable chip is a laminated structure and includes a lower film layer, a paper layer, and an upper film layer from bottom to top; there is adhesion between the lower film layer and the upper film layer and the paper layer; the paper layer includes: a paper sheet made of a paper-based material; a flow channel boundary drawn with oil-based ink is provided on the paper sheet, and the area within the flow channel boundary is a fluid channel; the fluid channel is separated by a timing valve, and the timing valve is an area impregnated with water-based ink; a sample addition hole connected to the fluid channel on one side of the timing valve is provided on the upper film layer; wherein the oil-based ink and the water-based ink are both hydrophobic inks. The preparation of the timing valve specifically includes: pre-cutting the boundary of the preset area and then impregnating it with water-based ink to a saturated state. The present invention can accurately, quickly, and low-costly achieve fluid control without introducing substances outside the system and without relying on external large-scale equipment.

Description

Technical Field

[0001] The present invention belongs to the field of paper-based microfluidics technology, and more specifically, relates to a programmable paper chip and a preparation method thereof, and a microfluidics analysis chip. Background Art

[0002] Microfluidics has emerged as a promising alternative for biochemical analysis, offering advantages such as rapid mass and heat transfer, high-throughput detection, reduced sample consumption, high automation, and robust integration. Currently, PDMS-based soft lithography microfluidic chips are the mainstream choice, characterized by high fabrication precision, excellent optical transparency, and biocompatibility. However, their complex manufacturing process, high cost, and cleanroom requirements limit their application in point-of-care (POCT) testing.

[0003] For over a decade, paper-based microfluidic devices have provided a way to enable point-of-care (POCT) testing using inexpensive, portable devices. Paper, as a common material, is readily available, low-cost, and easily mass-produced. As early as the 1930s and 1940s, researchers began exploring the use of paper to construct multi-well assay plates and fluidic systems for chemical analysis. The first paper-based diagnostic device for semi-quantitative detection of glucose in urine was demonstrated in the 1950s. This achievement was further developed into an immunoassay paper test device and commercialized. The paper-based continuous flow microfluidic (p-CMF) device was first invented by Muller and Clegg in 1949. In 2007, the Whitesides group published a research report, and the concept of paper-based microfluidic analysis devices (μPADs) for point-of-care testing (POCT) began to emerge. Paper-based microfluidic devices have gradually become the research focus of many scholars and have begun to develop rapidly. The most essential feature of the current advanced paper-based microfluidic devices is the ability to be programmed to transport fluid samples to the target location within a set time. Programmable paper-based microfluidic devices can automatically transport fluids in sequence to complete the entire detection process. Therefore, they can be used in a wide range of analytical applications, especially multi-step detection processes. In summary, paper-based microfluidic technology has great application potential in many aspects such as home, medical, resource-scarce and extreme conditions.

[0004] The main research direction of paper-based microfluidics is the preparation of paper-based microfluidic devices, which includes two aspects. The first is the construction of fluid channels. Currently, most research has successfully constructed fluid channels on paper-based materials through the use of various hydrophobic materials. Among them, the more popular hydrophobic materials include photoresist, wax, polydimethylsiloxane, etc. Channels can be constructed on paper-based materials through methods such as inkjet printing / etching, plasma / laser processing, or stamping. On the other hand, how to achieve fluid control on paper-based materials is the main bottleneck for improving the practicality of paper-based materials in analytical devices. Valves in paper-based microfluidic devices are crucial for controlling fluid flow and facilitating multi-step analytical processes.

[0005] Valves in paper-based microfluidic devices can be divided into active valves and passive valves. With active valves, users can adjust fluid flow and manipulate reagent release through practical operations such as folding, sliding, or rotating. Passive valves include soluble substance control valves, absorption expansion control valves, and electronic control valves, which can complete fluid control without user intervention. However, these fluid control methods may be subject to various limitations. For example, soluble substance control valves often introduce substances outside the system, absorption expansion control valves involve complex structures, and electronic control valves require external equipment. Summary of the Invention

[0006] In response to the defects of the existing technology and the need for improvement, the present invention provides a programmable paper chip and its preparation method, and a microfluidic analysis chip. Its purpose is to enable paper-based microfluidic devices to achieve fluid control accurately, quickly and at low cost without introducing extraneous substances or relying on external large-scale equipment.

[0007] To achieve the above objectives, according to one aspect of the present invention, a programmable paper chip is provided, which has a laminated structure and includes, from bottom to top, a lower film layer, a paper layer, and an upper film layer; there is adhesion between the lower film layer and the upper film layer and the paper layer;

[0008] The paper layer includes: a paper sheet made of a paper base material; a flow channel boundary drawn with oil-based ink is provided on the paper sheet, and the area within the flow channel boundary is a fluid channel; the fluid channel is separated by a timing valve, and the timing valve is an area soaked with water-based ink;

[0009] The upper membrane layer is provided with a sample addition hole connected to the fluid channel on one side of the timing valve;

[0010] Among them, oil-based ink and water-based ink are both hydrophobic inks.

[0011] According to another aspect of the present invention, there is provided a method for preparing the programmable paper chip, comprising:

[0012] Step S1: using oil-based ink to draw a flow channel boundary on the upper surface of a paper sheet made of a paper-based material; the area within the flow channel boundary is the fluid channel;

[0013] Step S2: using aqueous ink to impregnate a predetermined area of ​​the fluid channel that blocks the fluid channel, so that a timing valve formed in the aqueous ink-impregnated area blocks the fluid channel; a paper sheet with a flow channel boundary drawn and a timing valve set thereon forms a paper layer;

[0014] Step S3: After covering the lower and upper surfaces of the paper layer with a lower film layer and an upper film layer, respectively, the lower film layer, the paper layer, and the upper film layer are laminated to obtain a programmable paper chip; the lower film layer and the upper film layer are both adhered to the paper layer, and the upper film layer is provided with a sample injection hole connected to the fluid channel on one side of the timing valve;

[0015] Among them, oil-based ink and water-based ink are both hydrophobic inks.

[0016] Furthermore, before step S1, the process further includes: determining preparation parameters of the programmable paper chip according to a desired delay time, so that after the fluid is added through the sample injection hole and impregnates the fluid channel on one side of the timing valve, it passes through the timing valve and enters the fluid channel on the other side after the desired delay time. The preparation parameters include:

[0017] The fiber orientation of the paper base material, the hydrophobicity of the water-based ink, the width of the timing valve, the number of timing valves, the lamination temperature and the lamination pressure.

[0018] Furthermore, step S2 includes:

[0019] After pre-cutting the boundaries of a preset area, the area is impregnated with water-based ink. After the paper-based material in the area absorbs the water-based ink to a saturated state, a timing valve is formed.

[0020] According to yet another aspect of the present invention, there is provided a microfluidic analysis chip comprising: M fluid control modules;

[0021] Each fluid control module includes the programmable paper chip provided by the present invention and a reaction zone; the reaction zone is connected to the fluid channel in the programmable paper chip, and the side where the reaction zone is connected to the fluid channel and the side where the sample addition hole is connected to the fluid channel are located on different sides of the timing valve;

[0022] Wherein, M is a preset positive integer, and when M>1, the reaction areas of the M fluid control modules are connected, and the timing valves of the programmable paper chips in the M fluid control modules have different delay times, which are used to control the order in which the reagents added to the programmable paper chips in different fluid control modules through the sample addition holes are released into the reaction areas.

[0023] In some optional embodiments, M>1, and each fluid control module further comprises: a housing for accommodating the programmable paper chip and the reaction zone; in the housing, the upper and lower surfaces of the portion where the reaction zone is located are convex on one side and concave on the other side, forming a connecting portion between the fluid control modules; a drainage hole is provided at the center of the connecting portion, and the drainage hole is filled with a paper-based material;

[0024] Before assembly, each fluid control module is relatively independent; after assembly, adjacent fluid control modules are installed by interlocking the connecting parts, and the drainage holes of all fluid control modules overlap, so that the reaction areas in each fluid control module are connected through the paper-based material filled in the drainage holes.

[0025] Furthermore, the upper and lower surfaces of the connecting portion in the fluid control module are both circular.

[0026] According to another aspect of the present invention, a high-throughput microfluidic analysis device is provided, comprising: a water absorption layer and a plurality of the above-mentioned microfluidic analysis chips, wherein each microfluidic analysis chip is arranged around the water absorption layer, and the reaction area of ​​each microfluidic analysis chip is connected to the water absorption layer.

[0027] According to another aspect of the present invention, another microfluidic analysis chip is provided, comprising a plurality of detection modules; the detection modules comprise the programmable paper chip provided by the present invention and a reaction zone; the reaction zone is connected to a fluid channel in the programmable paper chip, and the side where the reaction zone is connected to the fluid channel and the side where the sample loading hole is connected to the fluid channel are located on different sides of the timing valve;

[0028] In each detection module, the sample addition hole and the fluid channel on one side connected to the sample addition hole form the sample addition area of ​​the detection module, and the sample addition areas of each detection module overlap.

[0029] In some optional embodiments, the microfluidic analysis chip provided by the present invention is used for pesticide residue detection, which includes two detection modules; the timing valves of the programmable paper-based chips in the two detection modules have the same delay time, and the reaction area of ​​one detection module is provided with a pesticide residue detection test paper based on a DTNB reaction system, and the reaction area of ​​the other detection module is provided with a pesticide residue detection test paper based on a MnO2 reaction system.

[0030] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects:

[0031] (1) The programmable paper chip provided by the present invention uses oil-based ink to draw the flow channel boundary and uses water-based ink to draw the timing valve. Since the oil-based ink is highly hydrophobic, it is not easy to separate from the upper and lower film layers under the infiltration of the fluid, and the bonding force between the upper and lower film layers is strong, which can form a stable flow channel boundary; since the water-based ink is less hydrophobic, it is easy to separate from the upper and lower film layers after a certain period of time under the infiltration of the fluid, so that the fluid continues to flow forward through the gap formed by the separation after a corresponding period of time, thereby realizing the timing function. The materials required for the entire programmable paper chip only include the upper and lower film layer materials, paper, and ink. The production is simple, fast and low-cost. At the same time, the timing function is accurately realized by means of the bonding force between the water-based ink and the upper and lower film layers, without the need for external equipment and without generating any substances outside the system.

[0032] (2) The present invention comprehensively explores the relevant factors that affect the delay time of the timing valve and takes them as key parameters. These key parameters are determined before the programmable chip is manufactured, ensuring that the delay time of the timing valve is accurately controllable.

[0033] (3) The method for preparing the programmable paper chip provided by the present invention, in its preferred embodiment, adopts the method of pre-cutting and local impregnation to make the timing valve, that is, after pre-cutting the boundary of the area where the timing valve is located, the area is impregnated with water-based ink, and the timing valve is formed after the paper-based material in the area absorbs the water-based ink to a saturated state. The timing valve made in this way has a smoother boundary, and the parameters and functional stability of the timing valve are significantly improved. In addition, the production method can be carried out simultaneously on multiple layers of paper to achieve batch processing, thereby improving the production efficiency of programmable paper chips.

[0034] (4) The microfluidic analysis chip provided by the present invention may include one or more fluid control modules depending on the specific analysis reaction. Each fluid control module includes the programmable timing valve provided by the present invention, which can accurately control the time when the fluid enters the reaction zone, thereby realizing accurate microfluidic analysis.

[0035] (5) The microfluidic analysis chip provided by the present invention, in its preferred embodiment, includes multiple fluid control modules, and a housing is designed for each fluid control module. The portion of the housing where the reaction zone is located is designed with a connecting portion with one side raised and one side recessed, so that the multiple fluid control modules are three-dimensionally stacked and assembled through the connecting portion. After assembly, the entire microfluidic analysis chip is fan-shaped. The modular design makes the microfluidic analysis chip flexible in structure, highly scalable, and easy to prepare and assemble. In a further preferred embodiment, the upper and lower surfaces of the connecting portion are circular, so that after assembly, the fluid control module can rotate with the rotating portion as the axis, so that the angle between adjacent fluid control modules can be adjusted according to the number of fluid control modules, thereby improving space utilization.

[0036] (6) The high-throughput microfluidic analysis device provided by the present invention utilizes a water-absorbing layer to aggregate multiple microfluidic analysis chips together, thereby enabling multiple analytical reactions to be performed simultaneously, thereby improving the analytical throughput. At the same time, the water-absorbing effect of the water-absorbing layer can promote the flow of fluids in each microfluidic analysis chip and accelerate the analytical reaction.

[0037] (7) Another microfluidic analysis chip provided by the present invention includes multiple detection modules, each of which includes the programmable timing valve provided by the present invention, and the sample addition areas of each detection module overlap. Based on this structural design, different detection reactions can be performed on the same sample at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the programmable paper-based chip provided by the present invention;

[0039] Figure 2 Schematic diagram of the principle of creating a timing valve using hydrophobic ink provided by the present invention; (a) is a schematic diagram of paper impregnated with two types of hydrophobic ink (three colors) and bonded to tape; (b) is a photograph of the contact angles of paper impregnated with different inks and ultrapure water; (c) is a comparison of the adhesive properties of the tape; (d) is a schematic diagram of the valve delay principle; and (e) is a microscopic image of paper treated with different inks and bonded to tape under an ultra-depth-of-field microscope.

[0040] Figure 3 Schematic diagrams of the preparation methods using direct writing methods provided by the present invention;

[0041] Figure 4 Comparison of fluid characteristics under different fiber distribution directions of paper-based materials provided by the present invention; wherein (a) is the characteristics of fluid under longitudinal distribution, and (b) is the characteristics of fluid under transverse distribution;

[0042] Figure 5 Figures 1 and 2 show factors influencing the time delay of the timing valve provided by the present invention; (a) is a schematic diagram illustrating the effect of lamination temperature on valve time delay; (b) is a diagram illustrating the physical state of paper in a laminated structure when the processing temperature is varied; (c) is a graph showing the relationship between void ratio and processing temperature; (d) is a graph showing the relationship between time delay and processing temperature for a 0.5 mm valve; (e) is a dynamic diagram showing the position of the fluid front over time at different processing temperatures; and (f) is a diagram illustrating the time delay of valves prepared with different inks.

[0043] Figure 6Schematic diagram showing the effect of the width and number of timing valves on the time delay of the timing valves provided by the present invention; (a) is a schematic diagram of the fluid flow when the width and number of valves are changed, (b) is a real-life diagram of the fluid flow when the width and number of delay valves are changed using pigment verification, (c) is a graph showing the relationship between time delay and valve width (number of experimental repetitions N = 5), and (d) is a graph showing the relationship between time delay and number of valves (N = 5).

[0044] Figure 7 A schematic diagram of the preparation method using pre-cutting and local impregnation provided by the present invention;

[0045] Figure 8 Performance comparison charts of programmable paper chips prepared by the two preparation methods provided by the present invention; (a) is a comparison chart of the distribution of the grayscale values ​​of the timing valve color, (b) is a comparison chart of the degree of dispersion of the grayscale values ​​of the straight line color, (c) is a comparison chart of the degree of dispersion of the valve width (N=8), and (d) is a comparison chart of the degree of dispersion of the delay of the 0.5mm valve (N=10);

[0046] Figure 9 Schematic diagram of the microfluidic analysis chip provided by the present invention; wherein (a) is a schematic diagram of a microfluidic analysis chip in which the fluid release sequence is controlled by controlling the width of the timing valves, (b) is a schematic diagram of a microfluidic analysis chip in which the fluid release sequence is controlled by controlling the number of timing valves, (c) is the dynamic grayscale data of each timing valve area in (a) read using ImageJ, and (d) is the dynamic grayscale data of each timing valve area in (b) read using ImageJ;

[0047] Figure 10 Schematic diagram of another microfluidic analysis chip provided by the present invention; wherein (a) is a schematic diagram of a fluid control module, (b) is a schematic diagram of a microfluidic analysis chip integrated with two fluid control modules that can achieve timed release of two reagents, and (c) is a physical image of the pigment verification of the chip shown in (b);

[0048] Figure 11 Schematic diagram of another microfluidic analysis chip provided by the present invention; wherein (a) is a schematic diagram of a microfluidic analysis chip integrated with three fluid control modules that can achieve timed release of three reagents, (b) is a diagram of the actual structure of a microfluidic analysis chip integrated with three fluid control modules that can achieve timed release of three reagents, and (c) is a physical diagram of the pigment verification of the chip shown in (a);

[0049] Figure 12Schematic diagram of the pesticide residue detection chip provided by the present invention; wherein (a) is a schematic diagram of the reaction principle of the DTNB (CA) pesticide residue detection system; (b) is a schematic diagram of the test paper manufacturing process for pesticide residue detection; (c) is a schematic diagram of the detection process of a single timed valve chip used in the CA system; (d) is a color-enhanced image of the inside and outside of the CA system chip; (e) is a graph of the normalized color intensity data inside and outside the CA system chip at various pesticide concentrations (N=3);

[0050] Figure 13 Schematic diagram of another pesticide residue detection chip provided by the present invention; wherein (a) is a schematic diagram of the reaction principle of the MnO2 (CE) pesticide residue detection system, (b) is an electron microscope image of MnO2 in the CE system in the presence and absence of pesticides, (c) is a schematic diagram of the detection process of the single timing valve chip used in the CE system, and (d) is a schematic diagram comparing the color change differences between the CA and CE reaction systems;

[0051] Figure 14 A schematic diagram of another pesticide residue detection chip provided by the present invention;

[0052] Figure 15 The high-throughput microfluidic analysis device provided by the present invention; wherein (a) is a schematic diagram of the high-throughput microfluidic analysis device structure, (b) is a color enhancement image of the inside and outside of the CE system chip, and (c) is a normalized color intensity data diagram of the inside and outside of the CE system chip at various pesticide concentrations (N=3);

[0053] Figure 16 Schematic diagram of another pesticide residue detection chip provided by the present invention; wherein, (a) is a schematic diagram of the sample detection process, (b) is a physical diagram of the dual-mode chip functional verification, (c) is a color-enhanced image of 20 positive samples and 20 negative samples, (d) is a heat map and bar chart based on the grayscale values ​​of the samples, (e) is a box plot of positive and negative samples in the CA system and CE system, (f) is the ROC curve of the CA system, CE system, and CA+CE dual system, and (g) is a confusion matrix reflecting the classification performance of CA and CE mode samples and comparing the consistency of the results of the CA and CE systems. DETAILED DESCRIPTION

[0054] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0055] In the present invention, the terms "first", "second", etc. (if any) in the present invention and the drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence.

[0056] To address the problem that passive valves in existing paper-based microfluidic analysis devices rely on external equipment or introduce extraneous substances while achieving fluid control, the present invention provides a programmable paper chip, a method for preparing the same, and a microfluidic analysis chip. The chip structure is based on the discovery that the different binding forces between inks of varying hydrophobicity and membrane materials enable the realization of a timing valve function. Based on this structural design, the present invention also improves the preparation method of the related structure. This is described in detail below with reference to the following examples.

[0057] Example 1:

[0058] A programmable paper chip, such as Figure 1 As shown, it is a laminated structure and includes a lower film layer, a paper layer and an upper film layer from bottom to top; there is adhesion between the lower film layer and the upper film layer and the paper layer;

[0059] The paper layer includes: a paper sheet made of a paper base material; a flow channel boundary drawn with oil-based ink is provided on the paper sheet, and the area within the flow channel boundary is a fluid channel; the fluid channel is separated by a timing valve, and the timing valve is an area soaked with water-based ink;

[0060] The upper membrane layer is provided with a sample addition hole connected to the fluid channel on one side of the timing valve;

[0061] Among them, oil-based ink and water-based ink are both hydrophobic inks.

[0062] The following combination Figure 2 The principle of using water-based ink to make a timing valve to achieve time delay is explained in this embodiment. First, different hydrophobic inks (oil-based and water-based) are selected, such as Figure 2 As shown in (a), by measuring the contact angles of the paper after being soaked with different inks, it was found that the contact angles of the paper treated with water-based green ink and water-based black ink were 99° and 109° respectively, which were both smaller than the 121° after being treated with metallic oil-based ink. Figure 2 As shown in (b) in the figure. Further measurements were made on the adhesion of these ink-treated papers to the tape when fully soaked. It was observed that the more hydrophobic the ink, the harder it was to completely separate. The papers treated with green and black water-based inks separated from the tape after 30 and 60 seconds of soaking, respectively. Therefore, green and black inks are called layerable inks. The paper treated with metallic inks was difficult to separate from the tape. Therefore, metallic oil-based inks are called non-layerable inks. Figure 2 As shown in (c) in .

[0063] On the other hand, at the microscopic level, paper is composed of fibers and has a rough surface. Under the tape-paper-tape laminated structure, the raised parts of the fibers adhere to the tape to form adhesion points with a blocking effect, while the concave parts form flow channel cavities. When water-based ink is impregnated, the rough structure of the paper is retained and the adhesion points are broken through one by one. However, it is more difficult to break through when using oil-based ink. Figure 2 As shown in (d) in the figure, subsequent tests of fluid flow in laminated structures with different fiber orientations also confirmed this view. Under an ultra-depth microscope, it was observed that the laminated structure retained a rougher surface after being stained with water-based hydrophobic marker ink. In contrast, the rough surface structure of the paper was retained after treatment with water-based ink, while the surface of the paper treated with metallic oil-based ink was relatively smoother and had smaller gaps. Specifically, due to its special composition, the metallic oil-based marker will be deposited on the surface of the paper after drying, reducing the surface roughness of the paper and forming a patch with good hydrophobicity, such as Figure 2 As shown in (e) in the figure, the contact area between them and the tape is increased and their adhesion to the tape is enhanced to avoid delamination.

[0064] Therefore, in this embodiment, the fluid control function is mainly the hydrophobic ink. Since the oil-based ink is highly hydrophobic and has a strong bonding force with the upper and lower film layers, it is not easy to separate from the upper and lower film layers under the infiltration of the fluid, and a stable flow channel boundary can be formed; since the water-based ink is less hydrophobic and has a weak bonding force with the upper and lower film layers, it will separate from the upper and lower film layers after a certain period of time under the infiltration of the fluid, so that the fluid continues to flow forward after a corresponding period of time, thereby realizing the timing function.

[0065] Optionally, in this embodiment, the paper-based material is specifically Kimberly-Clark 34155 dust-free wiping paper, which has a relatively neat fiber distribution; the upper film layer and the lower film layer are specifically transparent tape, and specifically Scotch heavy-duty transport packaging tape.

[0066] Optionally, in this embodiment, the oil-based ink used to draw the flow channel boundary is specifically a Sharpie metallic permanent marker, and the width of the drawn flow channel boundary is 5 mm; the water-based ink used to make the timing valve is specifically a Sharpie ultra-fine marker, and the width of the constructed timing valve is determined by the required delay time.

[0067] The programmable paper chip provided in this embodiment requires only tape, paper, and ink, and is simple, fast, and low-cost to manufacture. Furthermore, the timing function is accurately achieved by utilizing the bonding force between the aqueous ink and the upper and lower film layers, without the need for external equipment and without generating any extra-system substances.

[0068] Example 2:

[0069] A method for preparing a programmable paper chip, for preparing the programmable paper chip provided in Example 1 above, such as Figure 3 Shown, including:

[0070] Step S1: using oil-based ink to draw a flow channel boundary on the upper surface of a paper sheet made of a paper-based material; the area within the flow channel boundary is the fluid channel;

[0071] Step S2: using aqueous ink to impregnate a predetermined area of ​​the fluid channel that blocks the fluid channel, so that a timing valve formed in the aqueous ink-impregnated area blocks the fluid channel; a paper sheet with a flow channel boundary drawn and a timing valve set thereon forms a paper layer;

[0072] Step S3: After covering the lower and upper surfaces of the paper layer with a lower film layer and an upper film layer, respectively, the lower film layer, the paper layer, and the upper film layer are laminated to obtain a programmable paper chip; the lower film layer and the upper film layer are both adhered to the paper layer, and the upper film layer is provided with a sample injection hole connected to the fluid channel on one side of the timing valve;

[0073] Among them, oil-based ink and water-based ink are both hydrophobic inks.

[0074] Directly drawing with a marker is the easiest way to imprint ink, which is also called direct writing. In this embodiment, in step S1, the flow channel boundary is drawn with oil-based ink, and in step S2, the timing valve is made with water-based ink, which adopts the direct writing method.

[0075] Accurately controlling the delay time is the key to manufacturing a timing valve. This embodiment conducts a comprehensive exploration of the factors that affect the delay time of the timing valve.

[0076] First, the effects of different fiber distributions on the fluid characteristics of paper were analyzed. After analysis, this embodiment found that when the paper fibers are distributed longitudinally (i.e., the paper fiber distribution direction is consistent with the fluid flow direction), using inks with different hydrophobicities to impregnate is equivalent to constructing capillaries with different wall hydrophobicities in the fiber gaps, and the fluid flows through the gaps. Figure 4 When the fluid shown in (a) passes through, a short delay will occur. When distributed horizontally (i.e., the paper fiber distribution direction is perpendicular to the fluid flow direction), the layering properties of the two types of inks can be used to print the difficult-to-layer fluid channel and the timing valve area to be layered using water-based ink on the paper respectively. Finally, by bonding the upper and lower layers of tape, the timing valve channel can be quickly and cost-effectively prepared. It can form a relatively consistent flow at the fluid front and can produce a relatively long delay, such as Figure 4 From this, we can see that the fiber distribution direction of the paper will affect the flow characteristics of the fluid.

[0077] Further analyze other processing conditions and parameters that affect the timing valve delay time.

[0078] In this embodiment, the tape, paper, and adhesive tape are ultimately bonded together through lamination. The lamination temperature is a significant factor influencing the timer valve delay. Different processing temperatures affect the adhesion between the paper and the tape, which in turn affects the ability of the ink valve to separate from the tape, thereby affecting the time delay. Increasing the processing temperature promotes the melting of the adhesive polymer in the tape, increasing the contact area between the polymer and the raised portions of the paper fibers (i.e., the adhesion point area), generating a stronger adhesive force, which in turn affects the time delay. Figure 5 As shown in (a) of Figure 1, this example uses image processing to obtain the porosity of the paper surface under adhesive coating to reflect the area of ​​the adhesion point: first, the structure of the laminated paper is photographed using an ultra-depth-of-field microscope, and then the image is processed using ImageJ software to obtain the porosity of the paper in the laminated structure under different processing temperature conditions. This method compares the porosity data under different processing temperature conditions with the corresponding delay (valve width: 0.5mm), and a good correlation is obtained: as the temperature increases, the porosity decreases and the delay increases, as shown in Figure 1. Figure 5 As shown in (b), (c) and (d).

[0079] This example also investigates the flow rate of the fluid on the paper after processing at different temperatures. According to the Lucas-Washburn equation, it can be deduced that the density of the paper in the laminated structure increases with increasing temperature, such as Figure 5 As shown in (e) in FIG, as the processing temperature increases, the fluid front at the same time point moves further back, which means a greater flow resistance.

[0080] In addition, timing valves made with inks of different hydrophobicity also have different time delays. Black and green are both water-based inks, and both can be used as raw materials for making time delay valves. However, because black ink is more hydrophobic than green, it exhibits a longer time delay. When the valve width is fixed at 0.5mm, the time delay of the valve made with green ink is about 150s, while that of the valve made with black ink is about 300s. Figure 5 As shown in (f) in .

[0081] This embodiment further explores the relationship between the structural parameters of the timing valve and the delay time of the timing valve. The study found that with a wider valve width and a larger number of valves, the delay will increase accordingly as the number of fibers covered by ink increases. Figure 6 As shown in (a) in .

[0082] The delay time of different widths (0.5mm-3.0mm) was preliminarily verified using pigments, such as Figure 6 As shown in (b), it can be observed that the smaller the width of the valve, the shorter the time it takes to break through. The time delay data of different width timing valves were obtained by independent repeated tests, as shown in Figure 6 As shown in (c) in the figure, within the test range, as the valve width increases, the valve delay also increases, and shows a good linear trend. On the other hand, a good linear relationship can also be observed with the number of different valves (0-3 1mm wide timing valves) and the delay, as shown in Figure 6 Based on this research result, it can be seen that controllable delay change can be achieved by changing the ink immersion width and the number of valves.

[0083] Based on the above analysis, the fiber orientation of the paper-based material, the hydrophobicity of the water-based ink, the width of the timing valve, the number of timing valves, the lamination temperature, and the lamination pressure all affect the timing valve delay time, and a specific quantitative relationship can be derived through experiments. In order to accurately control the timing valve delay time, this embodiment also includes, before step S1, determining the preparation parameters of the programmable paper chip based on the desired delay time, so that after the fluid is added through the sample injection hole and impregnates the fluid channel on one side of the timing valve, it will break through the timing valve and enter the fluid channel on the other side after the desired delay time. The preparation parameters include:

[0084] The fiber orientation of the paper base material, the hydrophobicity of the water-based ink, the width of the timing valve, the number of timing valves, the lamination temperature and the lamination pressure.

[0085] In this embodiment, the selection of ink and paper may refer to the description in the above embodiment 1.

[0086] Example 3:

[0087] A method for preparing a programmable paper chip is used to prepare the programmable paper chip provided in the above embodiment 1. This embodiment is similar to the above embodiment 2, except that: Figure 7 As shown, in step S2 of this embodiment, when using water-based ink to make the timing valve, a direct writing method is not adopted, but a pre-cutting and local impregnation method is adopted. Specifically, after pre-cutting the boundary of the preset area, the area is impregnated with water-based ink. After the paper-based material in the area absorbs the water-based ink to a saturated state, the timing valve is formed.

[0088] This embodiment further improves the manufacturing method of the timing valve. This is because although it is easy to manufacture the timing valve using direct writing, direct writing with a marker has the following limitations in actual use: (1) The width of the line is limited by the thickness of the pen tip; (2) The thickness of the line and the color depth of the imprint are affected by the manufacturer's force and drawing speed, which limits the functional stability of the valve; and (3) Batch production depends on machines.

[0089] Compared with the direct writing method, the pre-cutting and local dipping method adopted in this embodiment is slightly more complicated in the production process, but the pre-cutting and local dipping processes can be carried out simultaneously on multiple layers of paper to achieve batch processing, which in the direct writing method largely requires reliance on machines such as writing robots. In terms of the minimum processing width, the pre-cutting and local dipping production methods will pre-cut and isolate the area where the timing valve is expected to be constructed, so the minimum width depends on the cutting accuracy. The accuracy of the mold made using a laser engraving machine can be as low as 100μm, which is less than the 500μm limit of the pen tip during direct writing. In addition, after the dyeing step is added, the paper absorbs the ink to saturation, and presents a consistent processing effect on all dyed areas. This can be confirmed in the grayscale value-position diagram of a straight line printed by the two methods at the same time, such as Figure 8 As shown in (a) and (b) in .

[0090] In addition, after pre-cutting, the timing valve boundary is smoother, while the direct writing method is affected by the speed and force of the line drawing, resulting in a very rough boundary. We used an ultra-depth of field microscope to test the valve width data prepared by the two methods under the same conditions for verification. The results showed that the coefficient of variation of the pre-cutting and localized impregnation methods was reduced by 4.82% (7.68%-2.86%) compared with the direct writing method. Figure 8 This example further tests the corresponding timing valve data (valve width 0.5mm) of the two methods, and the coefficient of variation of the delay of the pre-cutting and local dipping methods is reduced from 74.4% to 24.1%. Figure 8 As shown in (d), this fully demonstrates the significant improvement in valve parameters and functional stability after the introduction of the pre-cutting and dipping process.

[0091] A comparison of different timing valve manufacturing methods is shown in Table 1.

[0092] Table 1 Comparison of properties between pre-cutting and direct writing

[0093]

[0094] In general, the pre-cutting and partial impregnation timing valve manufacturing method proposed in this embodiment has a timing valve with smoother boundaries, and the parameters and functional stability of the timing valve are significantly improved. In addition, this manufacturing method can be carried out simultaneously on multiple layers of paper to achieve batch processing, thereby improving the manufacturing efficiency of programmable paper chips.

[0095] Example 4:

[0096] A microfluidic analysis chip, such as Figure 9As shown, it includes: 3 fluid control modules; each fluid control module includes the programmable paper chip and a reaction area provided in the above embodiment 1; the reaction area is connected to the fluid channel in the programmable paper chip, and the side where the reaction area is connected to the fluid channel and the side where the sample addition hole is connected to the fluid channel are located on different sides of the timing valve;

[0097] In this embodiment, the reaction areas of the three fluid control modules are connected, and the timing valves of the programmable paper chips in the three fluid control modules have different delay times, which are used to control the order in which the reagents added to the programmable paper chips in different fluid control modules through the sample addition holes are released into the reaction areas.

[0098] In some optional embodiments, such as Figure 9 As shown in (a), the timing valves of the programmable paper chip in the three fluid control modules achieve different delay times by using different timing valve widths. From left to right, the widths of the three timing valves are 3.0 mm, 0, and 1.0 mm, respectively. In other optional embodiments, such as Figure 9 As shown in (b), the timing valves of the programmable paper chips in the three fluid control modules achieve different delay times by using different numbers of timing valves. The width of a single timing valve is fixed at 1.0 mm. From left to right, the number of timing valves in the three modules is 2, 0, and 1, respectively.

[0099] Different colors of pigments are added to the three fluid control modules, and the grayscale value changes of the corresponding timing valve areas are read. Figure 9 As shown in (c) and (d) in .

[0100] according to Figure 9 The results show that the three pigments are released sequentially, and the release order is consistent with the delay time of the timing valve.

[0101] Example 5:

[0102] A microfluidic analysis chip, such as Figure 10 As shown, it includes two fluid control modules. The structure of the fluid control module in this embodiment is similar to that of the fluid control module in the above-mentioned embodiment 4. The difference is that in this embodiment, a modular structural design method is adopted, and the programmable paper chip containing the timing valve is made into a modular structure.

[0103] In this modular structural design, in addition to the programmable paper chip and the reaction area, each fluid control module also includes a shell for accommodating the programmable paper chip and the reaction area, such as Figure 10Optionally, in this embodiment, the housing is laminated with PMMA sheets. Each layer is designed using AutoCAD software, and then a laser engraving machine is used to cut 0.3 mm thick PMMA sheets to obtain the corresponding structure. Double-sided tape is used to adhere each layer to encapsulate the paper-based device in the housing.

[0104] To facilitate assembly between fluid control modules, the upper and lower surfaces of the reaction zone portion of the fluid control module housing are convex on one side and concave on the other side to form a connection between the fluid control modules; a drainage hole is provided in the center of the connection, and the drainage hole is filled with a paper-based material;

[0105] Before assembly, each fluid control module is relatively independent. By stacking the fluid control modules in three dimensions through connecting parts, the entire microfluidic analysis chip can be assembled. After assembly, adjacent fluid control modules are installed by interlocking the connecting parts, and the drainage holes of all fluid control modules overlap, so that the reaction areas in each fluid control module are connected through the paper-based material filled in the drainage holes.

[0106] As a preferred embodiment, the upper and lower surfaces of the connecting portion are both circular, so that after assembly, the fluid control module can rotate about the rotating portion as an axis.

[0107] In this embodiment, one of the two fluid control modules has no timing valve and the other includes a 1.0 mm timing valve. Figure 10 As shown in (b) of Figure 2, the process is visualized by different pigments, such as Figure 10 As shown in (c), the results show that in the fluid control module without a timing valve, the pigment first flows to the reaction zone, and after a certain delay, the pigment in the other fluid control module is also displayed in the reaction zone, which indicates that this embodiment achieves the sequential release of the two reagents.

[0108] Example 6:

[0109] A microfluidic analysis chip, such as Figure 11 As shown, this embodiment is similar to the above embodiment 5, except that this embodiment includes three fluid control modules, and from left to right, the timing valves in the three fluid control modules are 1.0mm timing valve, 0.5mm timing valve and no timing valve, as shown in FIG. Figure 11 As shown in (a) and (b) of Figure 1, the reaction zone is visualized using different colored pigments, showing a concentric ring pattern. From the outside inward, the colors of the concentric circles are the same as the pigments added to the rightmost module, the middle module, and the leftmost module, respectively. This also indicates that the three reagents are released sequentially in the expected order.

[0110] It is easy to understand that in a microfluidic analysis chip, the number of fluid control modules can be set according to the types of reagents actually involved in the reaction. Especially when a modular structure design is adopted, different analytical reactions can be achieved directly by stacking the fluid control modules. The entire microfluidic analysis chip structure is flexible and highly scalable. In addition, Figure 11 As shown, when there are multiple fluid control modules, the entire microfluidic analysis chip is fan-shaped after assembly, and the angles between the fluid control modules can be controlled by rotating the fluid control modules to adapt to different numbers of fluid control modules.

[0111] Example 7:

[0112] A microfluidic analysis chip for pesticide residue detection. Figure 12 As shown, this embodiment includes only one fluid control module, and in this embodiment, the structure of the fluid control module is the same as that of the fluid control module in the above-mentioned embodiment 4.

[0113] Pesticide residues are a very popular detection direction in the field of food safety. The current mainstream detection method is enzyme inhibition, which uses the inhibitory effect of organophosphorus pesticides on cholinesterase to reduce the activity of cholinesterase in promoting subsequent reactions. The principle of the common kits on the market is to use the thiol group in thiocholine (TCh) generated by thioacetylcholine (ATCh) under the catalysis of cholinesterase to react specifically with 5,5-dithio-2,2-dinitrobenzoic acid (DTNB) to generate yellow 5-mercapto-2-nitrobenzoic acid (TNB). When the pesticide content increases, the activity of the enzyme is more strongly inhibited, and the final yellow substance produced is also reduced accordingly, and the color becomes lighter. Figure 12 As shown in (a) in the figure, the chip based on this detection principle can be called a color attenuation system (CA).

[0114] This embodiment is a color reduction system. To achieve pesticide residue detection, in this embodiment, the reaction area of ​​the fluid control module is provided with a DTNB test paper for detecting pesticide residues. Figure 12 As shown in (b), in this embodiment, the preparation method of the DTNB test paper includes:

[0115] A filter paper with a diameter of 0.5 mm was used as the test paper substrate. 1 μL of ATCh solution and 1 μL of DTNB solution were added to the substrate and dried at 4°C to prepare a DTNB reaction system pesticide residue detection test paper.

[0116] This embodiment effectively controls the reaction process by combining a programmable paper chip with a timing valve and DTNB test paper. Based on this reaction system, the test paper is combined with a single-valve chip to create a complete pesticide residue detection device. Each chip is smaller than a thumb, and can detect more than 10 samples on an area the size of a bank card. Phoxim solutions of varying concentrations are added to the programmable paper chip through the sample injection port. Simultaneously, a corresponding control experiment is performed outside the chip, and images are uniformly captured at 25 minutes. Figure 12 As shown in (c) in the figure, the final chip internal and external detection results are as follows Figure 12 As shown in (d) in the figure, it can be seen that the experimental trends on the chip and off-chip are basically the same, which shows the effectiveness of this embodiment. The grayscale value was further measured and normalized using ImageJ software, and the data of the on-chip and off-chip results were plotted. The concentration with a significant difference between the normalized intensity and the value of 0 was used as the detection sensitivity, as shown in Figure 2. Figure 12 As shown in (e), the detection sensitivity on the chip is 6.45×10 -2 In addition, the color change on the chip is greater than that off the chip in the range of 0–12.9 μmol / mL, demonstrating the higher sensitivity of this embodiment in the low concentration range.

[0117] Example 8:

[0118] A microfluidic analysis chip for pesticide residue detection, such as Figure 13 This embodiment is similar to the above embodiment 7, except that the detection test paper provided in the reaction area of ​​this embodiment is different from that in embodiment 7.

[0119] Another reaction system for pesticide residue detection is based on MnO2 nanoparticles, which can undergo redox reactions with (thio)choline and are also widely used. Among them, 3,3',5,5'-tetramethylbenzidine (TMB) reacts with MnO2 to generate oxidized TMB, which turns purple and is used in the colorimetric detection of pesticide residues. When the pesticide content increases, the activity of cholinesterase is inhibited, and the generated thiocholine decreases, resulting in a decrease in the amount of manganese dioxide that ultimately participates in the reaction. The larger the residual amount, the darker the purple color. Figure 13 As shown in (a) and (b) in the figure, this reaction system is called the color enhancement system (CE). The change trend of this reaction system is exactly opposite to that of the above-mentioned color enhancement system.

[0120] This embodiment is a color enhancement system. To detect pesticide residues, in this embodiment, the reaction area of ​​the fluid control module is provided with a MnO2 test paper for detecting pesticide residues. Figure 12 As shown in (b), in this embodiment, the preparation method of MnO2 test paper includes:

[0121] A filter paper with a diameter of 5 mm was used as the test paper substrate. 5 μL of 0.2 mg / mL MnO2 solution was added to the substrate and dried at 60°C. Then, 2 μL of ATCh solution was added and dried at 4°C to prepare the MnO2 test paper.

[0122] This embodiment can effectively control the reaction process by combining a programmable paper chip with a timing valve and a MnO2 test paper. Its effectiveness can be verified in preliminary single-channel chip testing, such as Figure 13 As shown in (c) in the figure, the color-enhancing system is colorless in the absence of pesticides and increases in color in the presence of pesticides. Compared with the subtractive system, it is more conducive to naked eye recognition, such as Figure 13 As shown in (d) in .

[0123] Example 9:

[0124] A microfluidic analysis chip for pesticide residue detection, such as Figure 14 As shown. This embodiment is also a detection and analysis chip based on color enhancement reaction, and its detection principle is the same as that of the above-mentioned embodiment 8. Structurally, the difference between this embodiment and the above-mentioned embodiment 8 is that the present embodiment includes two fluid control modules, the reaction areas of the two fluid control modules are connected, and the reaction areas are provided with MnO2 test paper. Among the two fluid reaction modules, one is used to add mixed reaction reagents, and the delay time of its timing valve is 5 minutes, during which the enzyme inhibition process is completed; the other is used to add TMB, and the delay time of its timing valve is 15 minutes (enzyme inhibition time 5 minutes + manganese dioxide reaction time 10 minutes). In this embodiment, the timed addition of TMB is completed by adding a timing valve to achieve a more complete sample-in-result-out detection.

[0125] During the test, the test solution is mixed with the enzyme solution and incubated for 5 minutes before flowing into the reaction zone to react with iodinated thioacetylcholine and manganese dioxide. After about 10 minutes of reaction, the delay valve of the other channel opens and TMB flows into the reaction zone to complete the color enhancement.

[0126] Example 10:

[0127] A high-throughput microfluidic analysis device, such as Figure 15 Shown, including:

[0128] The water absorption layer and the plurality of microfluidic analysis chips provided in the above embodiment 9 are arranged around the water absorption layer, and the reaction areas of the microfluidic analysis chips are connected to the water absorption layer.

[0129] This embodiment utilizes a water-absorbing layer to aggregate multiple microfluidic analysis chips together, thereby enabling multiple analytical reactions to be performed simultaneously, improving analytical throughput, and optimizing chip processing efficiency. Furthermore, the water-absorbing effect of the water-absorbing layer can promote the flow of fluids in each microfluidic analysis chip and accelerate the analytical reactions.

[0130] Since the structure of the dual-valve chip in the above-mentioned embodiment 9 is relatively complex, the two-dimensional preparation method process is very complicated. In order to optimize the chip processing efficiency and realize high-throughput parallel detection, this embodiment proposes a disc-type paper chip, which contacts each other in a stacked manner in a three-dimensional manner. Finally, the entire chip includes a three-dimensional stacked drainage layer, a color enhancement layer and a water absorption layer. In each microfluidic analysis chip, the two fluid control modules of the microfluidic analysis chip are located in the channel layer (below the drainage layer), and the reaction area of ​​the microfluidic analysis chip is located in the color enhancement layer (above the drainage layer), thereby realizing stacking on the three-dimensional level. The stacked structure is supported by a PMMA plate engraved with a specific pattern, such as Figure 15 Finally, seven concentrations from 0 to 129 μM were used for on-chip and off-chip comparative tests. The visual test results are shown in Figure 15 As shown in (b) in the figure, the detection sensitivity on the chip and off the chip is calculated to be 0.645 μM, which is basically consistent. At this concentration, the result on the chip shows a greater color change, which is more conducive to user identification, as shown in Figure 2. Figure 15 As shown in (c) in .

[0131] It is easy to understand that, in addition to the microfluidic analysis chip in Example 9, other microfluidic analysis chips composed of a single or multiple fluid control modules can be integrated into a high-channel microfluidic analysis device in the manner of this embodiment, and will not be listed one by one here.

[0132] Example 11:

[0133] A microfluidic analysis chip, such as Figure 16 As shown, it includes multiple detection modules; the detection modules include the programmable paper chip and the reaction area provided in the above embodiment 1; the reaction area is connected to the fluid channel in the programmable paper chip, and the side where the reaction area is connected to the fluid channel and the side where the sample addition hole is connected to the fluid channel are located on different sides of the timing valve;

[0134] In each detection module, the sample addition hole and the fluid channel on one side connected to the sample addition hole form the sample addition area of ​​the detection module, and the sample addition areas of each detection module overlap.

[0135] This embodiment can simultaneously perform different detection reactions for the same reagent. Specifically, this embodiment is used for pesticide residue detection and specifically includes two detection modules, such as Figure 16As shown in (a) and (b), the two detection modules are respectively a subtractive system and an enhancer system, thereby realizing a dual-system detection chip. Accordingly, in this embodiment, the programmable timing valves in the two detection modules have the same delay time, and the two timing valves are symmetrically arranged about the sample application area. In addition, the reaction area of ​​one detection module is provided with a pesticide residue detection test strip based on the DTNB reaction system (subtractive), and the reaction area of ​​the other detection module is provided with a pesticide residue detection test strip based on the MnO2 reaction system (enhancement).

[0136] In order to verify the performance of the microfluidic analysis chip provided in this embodiment, in this embodiment, a tape with a 1cm×1cm square hole is attached to the surface of a clean apple to construct a sampling area, 20μL of high-concentration pesticide is dripped into the area, and after it is completely dried, 200μL of eluent is used to blow repeatedly three times in the hole, and the eluent after blowing is recovered to obtain a positive sample solution. The untreated area is extracted according to the above method to obtain a negative sample solution. The dual-mode chip is obtained by combining a paper-based chip with two symmetrically distributed valves and two color enhancement test papers. During the test, a mixed solution of 36μL of sample and enzyme (5:1) is added to the sample wells of the two detection modules. Finally, at the end of the reaction, the color enhancement area data can be obtained by taking a photo with a smartphone (2μLTMB needs to be added to the manganese dioxide system). Afterwards, positive and negative group samples are prepared by manual sampling, such as Figure 16 As shown in (a) in .

[0137] When the sample solution is added, the fluid can reach the two timing valves at the same time, and after the same delay, it will synchronously enter the color enhancement area of ​​the two detection systems to obtain the detection results of the two systems, such as Figure 16 As shown in (b) in .

[0138] The dual-system mode detection chip provided in this embodiment was used to test 20 positive samples and 20 negative samples. The color enhancement area data was obtained by taking photos with a smartphone at 25 minutes. Figure 16 As shown in (c), the color data of the two color enhancement test strips are read as features for analysis. Data graphs such as heat maps and bar graphs based on the grayscale values ​​of the sample color enhancement well reflect the color enhancement differences between positive and negative samples, as shown in Figure 2. Figure 16 As shown in (d) and (e).

[0139] The ROC (Receiver Operating Characteristic Curve) curves were further drawn for the data of the subtractive (CA) system and the color enhancement (CE) system, and their AUC (Area Under Curve) values ​​were obtained to be 0.99 and 1.00, respectively. Figure 16As shown in (f) in the figure, the data has good classification characteristics. Specifically, for a total of 40 samples, the detection results of the two systems showed 100% sensitivity and higher than 95% and 97.5% specificity and accuracy. Only one sample showed a difference in the detection results of the two systems, as shown in Figure 2. Figure 16 This is shown in (g) in the figure, which demonstrates the consistency of the test results. Through the mutual verification of the two systems, testers can be promptly reminded to conduct further testing and verification on samples with discrepancies, thereby reducing the risk of incorrect test results.

[0140] It is easy to understand that in some other embodiments of the present invention, other numbers and types of detection modules can be set according to the needs of analysis and detection to achieve analysis of different properties of the same reagent, or to use different means to perform comparative analysis on the same property of the same reagent.

[0141] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A programmable paper chip, characterized in that: It is a laminated structure and includes, from bottom to top, a lower film layer, a paper layer and an upper film layer; there is adhesion between the lower film layer and the upper film layer and the paper layer; The paper layer includes: a paper sheet made of a paper-based material; a flow channel boundary drawn with oil-based ink is provided on the paper sheet, and the area within the flow channel boundary is a fluid channel; the fluid channel is separated by a timing valve, and the timing valve is an area soaked with water-based ink; The upper membrane layer is provided with a sample addition hole connected to the fluid channel on one side of the timing valve; Wherein, the oil-based ink and the water-based ink are both hydrophobic inks.

2. The method for preparing the programmable paper chip according to claim 1, characterized in that: include: Step S1: using oil-based ink to draw a flow channel boundary on the upper surface of a paper sheet made of a paper-based material; the area within the flow channel boundary is the fluid channel; Step S2: using water-based ink to impregnate a predetermined area in the fluid channel that isolates the fluid channel, so that a timing valve formed in the water-based ink-impregnated area isolates the fluid channel; a paper sheet with a flow channel boundary drawn and a timing valve set thereon forms a paper layer; Step S3: After covering the lower and upper surfaces of the paper layer with a lower film layer and an upper film layer, respectively, laminating the lower film layer, the paper layer, and the upper film layer to obtain the programmable paper chip; the lower film layer and the upper film layer are both adhered to the paper layer, and the upper film layer is provided with a sample addition hole connected to the fluid channel on one side of the timing valve; Wherein, the oil-based ink and the water-based ink are both hydrophobic inks.

3. The preparation method according to claim 2, wherein Before step S1, the method further includes: determining preparation parameters of the programmable paper chip according to a desired delay time, so that after the fluid is added through the sample injection hole and impregnates the fluid channel on one side of the timing valve, it breaks through the timing valve and enters the fluid channel on the other side after the desired delay time; the preparation parameters include: The fiber orientation of the paper-based material, the hydrophobicity of the aqueous ink, the width of the timing valve, the number of the timing valves, the lamination temperature and the lamination pressure.

4. The preparation method according to claim 3, wherein The step S2 comprises: After the boundary of the preset area is pre-cut, the area is impregnated with the water-based ink, and the timing valve is formed after the paper-based material in the area absorbs the water-based ink to a saturated state.

5. A microfluidic analysis chip, characterized in that: include: M fluid control modules; Each of the fluid control modules comprises the programmable paper chip of claim 1 and a reaction zone; the reaction zone is connected to the fluid channel in the programmable paper chip, and the side where the reaction zone is connected to the fluid channel and the side where the sample loading hole is connected to the fluid channel are located on different sides of the timing valve; Wherein, M is a preset positive integer, and when M>1, the reaction areas of the M fluid control modules are connected, and the timing valves of the programmable paper chips in the M fluid control modules have different delay times, which are used to control the order in which the reagents added to the programmable paper chips in different fluid control modules through the sample addition holes are released into the reaction areas.

6. The microfluidic analysis chip according to claim 5, characterized in that: M>1, and each of the fluid control modules further comprises: a housing for accommodating the programmable paper chip and the reaction zone; in the housing, the upper and lower surfaces of the portion where the reaction zone is located are convex on one side and concave on the other side, forming a connecting portion between the fluid control modules; a drainage hole is provided at the center of the connecting portion, and the drainage hole is filled with a paper-based material; After assembly, adjacent fluid control modules are installed by interlocking the connecting parts, and the drainage holes of all fluid control modules overlap, so that the reaction areas in each fluid control module are connected through the paper-based material filled in the drainage holes.

7. The microfluidic analysis chip according to claim 6, characterized in that: The upper and lower surfaces of the connecting portion of the fluid control module are both circular.

8. A high-throughput microfluidic analysis device, characterized in that: include: A water absorption layer and a plurality of microfluidic analysis chips according to any one of claims 5 to 7, wherein each microfluidic analysis chip is arranged around the water absorption layer, and the reaction area of ​​each microfluidic analysis chip is connected to the water absorption layer.

9. A microfluidic analysis chip, characterized in that: The device comprises a plurality of detection modules; the detection modules comprise the programmable paper chip according to claim 1 and a reaction zone; the reaction zone is connected to a fluid channel in the programmable paper chip, and the side where the reaction zone is connected to the fluid channel and the side where the sample loading hole is connected to the fluid channel are located on different sides of the timing valve; In each detection module, the sample addition hole and the fluid channel on one side connected to the sample addition hole form the sample addition area of ​​the detection module, and the sample addition areas of each detection module overlap.

10. The microfluidic analysis chip according to claim 9, characterized in that: Used for pesticide residue detection, it includes two detection modules; the timing valves of the programmable paper-based chips in the two detection modules have the same delay time, and the reaction area of ​​one detection module is set with a pesticide residue detection test paper based on a DTNB reaction system, and the reaction area of ​​the other detection module is set with a pesticide residue detection test paper based on a MnO2 reaction system.

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