Paper-based microfluidic chips, microfluidic detection systems, liquid detection methods, and uses thereof

Abstract

The present invention discloses a paper-based microfluidic chip, a microfluidic detection system, a liquid detection method, and their uses. The chip includes a paper substrate layer (2), and a detection cell (21) is provided on the paper substrate layer (2). The detection cell (21) has a first contact angle region (211) and a second contact angle region (212) therein, and the contact angle of the liquid in the detection cell (21) at the first contact angle region (211) is larger than the contact angle at the second contact angle region (212). Based on the mechanism of chromaticity transfer on the paper chip, the surface tension of the liquid is adjusted and controlled to provide a driving force toward the inside of the droplet, thereby causing the droplet to shrink from the first contact angle region (211) to the second contact angle region (212) with a smaller contact angle, and the chromaticity is transferred to the second contact angle region (212) and concentrated, thereby improving the chromaticity and uniformity per unit area under the premise that the concentration of the object to be measured is the same, improving the detection sensitivity, lowering the detection limit, and widening the spot collection range, thereby improving the handling of spot collection and the detection reproducibility.

Description

Detailed Description of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the rights and benefit of Chinese Patent Applications Nos. 202211379392.8, 202222939918.5, 202222939919.X, and 202211379390.9, filed on November 4, 2022, the contents of which are incorporated herein by reference. [Technical Field]

[0002] The present invention relates to microfluidic detection technology, particularly to a paper-based microfluidic chip. Based on this, the present invention also relates to a microfluidic detection system including the paper-based microfluidic chip, and a liquid detection method using the paper-based microfluidic chip. Furthermore, the present invention also relates to the use of the paper-based microfluidic chip, the microfluidic detection system, and the liquid detection method. [Background technology]

[0003] Microfluidic technology has the advantages of fast mass and heat transfer, high analytical efficiency, low reagent consumption, low analytical cost, environmental friendliness, and easy integration. It can also be easily adapted to small, portable detection and analysis devices. Therefore, it is expected to be widely used in fields such as water quality detection, environmental detection, and food medicine. While microfluidic technology offers new development directions for detection and analysis devices, such as portable water quality detectors, new challenges remain for the portability of these devices due to the additional fluid control requirements and the increased volume of the detection devices.

[0004] Paper-based microfluidic chips can effectively solve these challenges in terms of fluid control. Paper-based microfluidic chips, known as "paper chips," are a microfluidic analysis technology platform that relies on capillary forces to achieve self-actuation. Compared to microfluidic chips using other substrates, paper chips are characterized by low cost and excellent portability, making them promising for applications in fields such as water quality detection, environmental detection, and food medicine. With the ongoing development and advancement of smartphone imaging technology and software capabilities, it is now possible to use a mobile phone to capture images of the detection cells on a paper chip, allowing colorimetric identification and colorimetric analysis to be performed through built-in software, eliminating the need for additional signal analysis equipment. Combining paper chips with mobile phone imaging and analysis can further promote the portability of detection equipment.

[0005] However, due to limitations in reagent loading, the non-uniformity of paper materials, and problems such as insufficient color development due to capillary action (such as the coffee ring effect), poor uniformity, and poor reproducibility, detection using paper chips has low sensitivity, low detection limits, and insufficient precision and reproducibility, which are common problems that limit the development of paper chip detection technology.

[0006] To improve the sensitivity and accuracy of paper chip detection, researchers have attempted various approaches, such as designing a bidirectional liquid inlet channel, pre-positioning reagents on both sides of the detection cell, and driving the reagents into the detection cell from both sides by the target liquid, thereby reducing the diffusion of color to the edges of the detection cell and improving the color uniformity of the detection cell. Another example is the coffee ring effect, which can be used to extract color from the coffee ring produced by a color reaction, thereby lowering the detection limit. Prior art has also proposed utilizing the adsorption effect of target substances on precious metal nanoparticles or carbon quantum dots, loading these adsorbents onto paper chips to concentrate the target, thereby improving detection sensitivity. However, these methods still suffer from the following drawbacks: While improving color uniformity can achieve a uniform distribution of color, it does not improve detection sensitivity or lower the detection limit. When quantifying by coffee ring concentration, the randomness and variability of coffee ring formation make it impossible to fix the color extraction position. Furthermore, the different color extraction positions result in large color differences, which results in inaccurate detection, reproducibility, and ease of use. Summary of the Invention [Problem to be solved by the invention]

[0007] The object of the present invention is to provide a paper-based microfluidic chip, a microfluidic detection system, and a liquid detection method to solve the problems of the colorimetric quantification paper chip of the prior art, which has insufficient color and uneven color distribution, resulting in insufficient detection limit and detection sensitivity, and low detection accuracy and reproducibility, and the paper-based microfluidic chip and liquid detection method have high detection accuracy and detection sensitivity, can effectively lower the detection limit of liquid detection, and are easy to handle and have good reproducibility. [Means for solving the problem]

[0008] In order to achieve the above object, one aspect of the present invention provides a paper-based microfluidic chip including a paper substrate layer, a detection cell provided in the paper substrate layer, the detection cell having a first contact angle region and a second contact angle region therein, and configured such that the contact angle of a liquid in the detection cell at the first contact angle region is larger than the contact angle at the second contact angle region.

[0009] A second aspect of the present invention provides a microfluidic detection system including the paper-based microfluidic chip described above, wherein the microfluidic detection system further includes an adjustment and control unit for adjusting and controlling one or more of the environmental temperature, air flow rate, humidity, and vacuum level of the area in which the detection cell is located.

[0010] A third aspect of the present invention provides a liquid detection method, comprising step S1 of introducing a liquid to be measured into a detection cell of the above-mentioned paper-based microfluidic chip, step S2 of leaving the paper-based microfluidic chip stationary for a predetermined period of time, and step S3 of performing chromaticity discrimination and / or colorimetric analysis on a predetermined region in the detection cell.

[0011] A fourth aspect of the present invention provides the use of the above paper-based microfluidic chip, microfluidic detection system, or liquid detection method in water quality detection, environmental detection, and food medicine. [Effects of the Invention]

[0012] According to the above technical solution, the present invention is based on the chromaticity movement mechanism on a paper chip, and provides a first contact angle region and a second contact angle region with different contact angles in a detection cell. The surface tension of the liquid is adjusted and controlled to provide a driving force toward the inside of the droplet, thereby causing the first contact angle region to shrink to the second contact angle region with a smaller contact angle, and the chromaticity to move to the second contact angle region and become concentrated. This improves the chromaticity and uniformity per unit area under the premise that the concentration of the object to be measured is the same, improves the detection sensitivity, lowers the detection limit, and also widens the spot sampling range, improving the ease of handling spot sampling and the detection reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 is a schematic diagram of a paper-based microfluidic chip according to a preferred embodiment of the present invention. FIG. 2 is a detection effect diagram of the paper-based microfluidic chip according to a preferred embodiment of the present invention. FIG. 3 is a schematic diagram of the gradient distribution of each contact angle region of the detection cell of the paper-based microfluidic chip according to a preferred embodiment of the present invention. FIG. 4 is an exploded schematic diagram of a paper-based microfluidic chip according to another preferred embodiment of the present invention. FIG. 5 is a scatter diagram showing the relationship between the color deepening effect and the hole diameter of the ventilation hole. FIG. 6 is a schematic diagram of a paper-based microfluidic chip according to another preferred embodiment of the present invention. FIG. 7 is a diagram showing the detection effect of the paper-based microfluidic chip in FIG. Figure 8 compares the color-enhancing effects of various paper-based microfluidic chips. FIG. 9 is a schematic diagram of a paper-based microfluidic chip according to another preferred embodiment of the present invention. FIG. 10 is a distribution diagram of detection cells in a paper-based microfluidic chip according to another preferred embodiment of the present invention. FIG. 11 is a scatter plot of the relationship between color deepening effect and detection time at various environmental temperatures. FIG. 12 is a schematic diagram of an exploded view of the paper-based microfluidic chip according to Example 1 of the present invention. FIG. 13 is a schematic diagram of the color deepening effect of the paper-based microfluidic chip in FIG. FIG. 14 is a structural schematic diagram of a paper-based microfluidic chip according to Example 2 of the present invention. FIG. 15 is a schematic diagram of the color deepening effect of the paper-based microfluidic chip in FIG. 16 and 17 are quantitative curves of the paper-based microfluidic chip in Example 3 of the present invention, respectively. [Mode for Carrying Out the Invention]

[0014] Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings. It should be understood that the embodiments of the present invention described in this specification are for the purpose of explaining and interpreting the present invention, and do not limit the present invention.

[0015] In the present invention, the endpoints of the disclosed ranges and any values ​​should be understood not to be limited to the precise ranges or values, but to include values ​​close to these ranges or values. In the case of numerical ranges, the endpoints of each range, the endpoints of each range and individual dot values, and the individual dot values ​​can be combined to obtain one or more new numerical ranges, and these numerical ranges are considered to be specifically disclosed herein.

[0016] As shown in FIG. 1 , a first aspect of the present invention provides a paper-based microfluidic chip. The paper-based microfluidic chip includes a paper substrate layer 2. The paper substrate layer 2 may be made of a paper / paper-like material such as filter paper or a cellulose filtration membrane. For example, the paper-based microfluidic chip functions as a microfluidic analytical technology platform for liquid detection. Compared to conventional microfluidic chips, the paper substrate layer 2 has advantages such as low cost, no need for external drive units, and excellent biocompatibility and portability. The paper-based microfluidic chip may include one or more paper substrate layers 2, and the thickness of the paper substrate layer 2 is generally not particularly limited. In each embodiment shown in the drawings of the present invention, only a single-layer paper substrate layer 2 is shown to clearly illustrate the relevant structures (see FIGS. 4 and 9 ).

[0017] A detection cell 21 is formed on the paper substrate layer 2 of the paper-based microfluidic chip, and a liquid to be detected (e.g., wastewater, food, or pharmaceutical solution) may be introduced into the detection cell 21 to detect / analyze information such as components and concentration in the liquid by means of colorimetric identification, colorimetric analysis, etc., as described below. In the present invention, as shown in Fig. 1, the detection cell 21 has a first contact angle region 211 and a second contact angle region 212 therein, and the contact angle of the liquid to be detected introduced into the detection cell 21 at the first contact angle region 211 is larger than the contact angle at the second contact angle region 212.

[0018] Therefore, when the liquid to be detected enters the detection cell 21, the contact angle regions in the detection cell 21 adjust and control the surface tension of the liquid to be detected, providing a driving force toward the inside of the droplet, causing the liquid to shrink from the first contact angle region 211 to the second contact angle region 212 with a smaller contact angle, and shifting the chromaticity to the second contact angle region for concentration. By providing regions with different contact angles, the paper-based microfluidic chip of the present invention can improve the chromaticity and uniformity per unit area, improve detection sensitivity, lower the detection limit, and widen the spot sampling range, improving the ease of handling spot sampling and detection reproducibility, provided that the concentration of the analyte is the same.

[0019] It should be understood that the contact angle of the liquid to be detected in each region in the detection cell 21 mainly refers to the contact angle of these regions with water or a hydrophilic liquid. Therefore, although the paper-based microfluidic chip of the present invention itself does not contain the liquid to be detected, the same liquid to be detected has different wettability and therefore different contact angles with respect to different contact angle regions of the detection cell 21. For example, the water contact angle of the first contact angle region 211 may be set to be greater than 90°, so that water or a hydrophilic liquid does not easily wet the region. On the other hand, the water contact angle of the second contact angle region 212 may be set to less than 30°. Therefore, when the target liquid enters the detection cell 21, the large contact angle of the first contact angle region 211 generates a driving force toward the second contact angle region 212 during the evaporation process of the target liquid in the detection cell 21, causing the droplet to shrink toward the second contact angle region 212. As the water in the target liquid evaporates, the chromaticity moves toward the second contact angle region 212 due to this shrinkage process. Therefore, the chromaticity is concentrated in the second contact angle region 212 in the detection cell 21, forming a high-chromaticity spot, facilitating chromaticity identification and colorimetric analysis using a portable detection device (e.g., a smartphone with embedded software). Figure 2 shows the detection effect of the paper-based microfluidic chip of the present invention. The first contact angle region 211 surrounds the second contact angle region 212 and has a water contact angle of 91°. The second contact angle region 212 is circular and has a diameter of 2 mm. Therefore, by providing areas with different contact angles, the color concentration effect within the detection cell can be improved, the detection sensitivity can be increased, the detection limit can be lowered, and the point sampling range can be expanded, improving the ease of handling point sampling and detection reproducibility, making color discrimination and colorimetric analysis easier.

[0020] In the present invention, the first contact angle region 211 and the second contact angle region 212 may be formed in the detection cell 21 by various methods. For example, the first contact angle region 211 may be formed by laying, depositing, or impregnating a hydrophobic material in a portion close to the outer periphery of the detection cell 21, and / or the second contact angle region 212 may be formed by laying, depositing, or impregnating a hydrophilic material in a central portion of the detection cell 21. Specifically, the first contact angle region 211 with a large contact angle may be obtained by laying a polytetrafluoroethylene film in a portion close to the outer periphery of the detection cell 21, or by depositing or impregnating a hydrophobic material such as a silanization reagent or a fluorine-containing material on the surface of that portion, thereby separating the solution to be detected from the substrate of the paper substrate layer or hydrophobically modifying the substrate of the paper substrate layer. In the central portion of the detection cell 21, the hydrophilic second contact angle region 212 is formed without any treatment using a material such as filter paper or a cellulose filtration membrane made of the paper substrate layer itself. Alternatively, the surface of this portion is plasma-treated and then coated with a bovine serum albumin (BSA) solution or the like to reduce the contact angle through surface modification, forming the second contact angle region 212 with a small contact angle. In a preferred embodiment, the contact angle of the entire detection cell 21 is increased by laying a polytetrafluoroethylene film inside the detection cell 21 or depositing or impregnating a hydrophobic material such as a silanized reagent or a fluorine-containing material inside the detection cell 21, thereby forming a hydrophobic layer with a relatively large contact angle. Then, in the central portion of the detection cell 21, a hydrophilic material (e.g., laying filter paper or applying BSA) is laid, deposited, or impregnated on top of the hydrophobic layer, forming the second contact angle region 212 with a relatively small contact angle in the central portion of the detection cell 21. In this case, the remaining portions of the detection cell 21 become the first contact angle region 211.

[0021] In the detection cell 21 of the paper-based microfluidic chip according to the present invention, the first contact angle region 211 may be configured to have a water contact angle greater than 60°, preferably greater than 90°, and more preferably greater than 120°, and the second contact angle region 212 may be configured to have a water contact angle less than 30°, or even closer to 0. As a result, the solution in the detection cell 21 gathers in the second contact angle region 212 as evaporation progresses, thereby concentrating the color in the second contact angle region 212 and forming a spot. In this way, low concentrations of the target liquid can be effectively detected, the detection sensitivity can be improved, and the detection limit can be lowered.

[0022] As described above, in the present invention, by providing regions with different contact angles within the detection cell 21, the color intensity is increased in a specific region, thereby allowing the detection point collection position to be determined as needed. This point collection position depends on the position of the second contact angle region 212 within the detection cell 21. In the preferred embodiment shown, the detection cell 21 is formed in a circular shape with a diameter of 2 mm to 8 mm, and the second contact angle region 212 is located at the center of the detection cell 21. This allows the liquid to be detected that enters the detection cell 21 to be uniformly collected at the center from all directions, which contributes to improved detection accuracy and precision. In an alternative embodiment, the second contact angle region 212 may be located at another position of the detection cell 21, for example, at another central portion offset from the center, while the first contact angle region 211 surrounds the second contact angle region 212. Alternatively, the detection cell 21 may be formed in the shape of a regular polygon with a circumscribing circle diameter of 2 mm to 8 mm, and the second contact angle region 212 may be disposed at the center of the regular polygon.

[0023] Based on the chromaticity transfer mechanism within the detection cell 21, the size and intensity of the spot formed by the chromaticity concentration greatly depend on the size of the second contact angle region 212. That is, when the second contact angle region 212 is small, the chromaticity concentration is high and the spot formed is small, which contributes to the detection of low-concentration solutions. Therefore, the size of the detection cell 21 may be determined according to the size of the second contact angle region 212 so that the ratio of the area of ​​the second contact angle region 212 to the area of ​​the detection cell 21 is 50% or less, preferably 30% or less. In the case of a general detection cell (a circle with a diameter of 2 mm to 8 mm, or a regular polygon with a circumscribed circle diameter of 2 mm to 8 mm), the second contact angle region 212 may be configured as a circular region with a diameter of 0.5 mm to 5 mm (preferably 1 mm to 3 mm) or a regular polygon with a circumscribed circle diameter of 0.5 mm to 5 mm (preferably 1 mm to 3 mm).

[0024] 1 and 3 , in order to enhance the effect of enriching and uniformly distributing the color, the paper-based microfluidic chip according to the present invention may further include a multi-step contact angle region in the detection cell 21, whereby the contact angle gradually increases from the center to the periphery of the detection cell 21, forming a plurality of contact angle regions distributed in a gradient. Specifically, the first contact angle region 211 may include a first gradient region 2111 near the outer edge of the detection cell 21 and a second gradient region 2112 near the second contact angle region 212, where the contact angle of the second gradient region 2112 is smaller than that of the first gradient region 2111. Therefore, during the evaporation process of the liquid, the driving force gradually decreases along the direction from the edge of the detection cell 21 toward the second contact angle region 212, thereby gradually slowing down the acceleration of the liquid moving to the second contact angle region 212, which contributes to the uniform distribution of the color in the second contact angle region 212. 3 shows a schematic diagram of the gradient distribution of each contact angle region of the detection cell. It should be understood that the first contact angle region 211 including the two-step gradient region shown is merely exemplary, and that in the paper-based microfluidic chip of the present invention, multiple gradient regions may be provided in the detection cell 21.

[0025] In the paper-based microfluidic chip according to the present invention, the detection cell 21 may be configured to be open to the outside, and the volatile components in the liquid to be detected may be directly evaporated through the upper opening of the detection cell 21 until the chromaticity is concentrated in the second contact angle region 212. Based on this, in the present invention, the liquid to be detected is volatilized only in a specific region, thereby controlling the direction and speed of chromaticity movement and further improving the chromaticity concentration effect in the detection cell, which will be described in detail below.

[0026] 4, a paper-based microfluidic chip according to another preferred embodiment of the present invention includes the above-mentioned paper substrate layer 2 having detection cells (not shown) provided therein, and an upper layer 3 and a lower layer 1 provided respectively on the upper and lower sides of the paper substrate layer 2. The lower layer 1 may be covered by the lower side (first side) of the paper substrate layer 2 by adhesive or the like, and the upper layer 3 may be covered by the upper side (second side) of the paper substrate layer 2 by adhesive or the like. The upper layer 3 and the lower layer 1 may cover all or only a part of the detection cells to form water-impermeable and air-impermeable portions. For example, the upper layer 3 and the lower layer 1 may be made entirely of a water-impermeable and air-impermeable material, and the portions covering the detection cells may be made of a water-impermeable and air-impermeable material. Specifically, the non-permeable and non-breathable material may be polyvinyl chloride, polyethylene, polypropylene, polystyrene, silicone, polytetrafluoroethylene, etc. The upper layer 3 and lower layer 1 made of a water-permeable material may be hydrophobized by, for example, applying wax to filter paper and heating it to impregnate the wax into the filter paper, or by immersing the filter paper in a plastic solution dissolved in an organic solvent and drying it. The lower layer 1 and upper layer 3 may or may not contact each other, and their areas and shapes may not be the same. The area may be smaller than the area of ​​the paper substrate layer 2, but must cover the entire detection cell. The lower layer 1 and upper layer 3 may be made of either transparent or opaque materials, with transparent materials being preferred.

[0027] An air vent 31 is formed in the portion of the upper layer 3 covering the detection cell, allowing the liquid 4 in the detection cell to volatilize in a specific region through the air vent 31. The air vent 31 can guide the liquid 4 in the detection cell so that it gathers at the position of the air vent 31 during the evaporation process and concentrates the chromaticity at that position. Specifically, the upper layer 3 covers the edge of the detection cell to prevent the liquid 4 from evaporating from the edge of the detection cell, and the air vent 31 in the upper layer 3 connects the detection cell to the outside space, allowing the liquid in the detection cell to evaporate to the outside only through the air vent 31. Therefore, during the evaporation process, the evaporation rate of water is faster at a position close to the air vent 31 in the detection cell, and the water at the edge position is replenished to a position close to the air vent 31. Thus, the chromaticity moves to that position, forming a spot with concentrated chromaticity.

[0028] In this case, the position of the ventilation hole 31 relative to the detection cell may be set so that the position where the ventilation hole 31 is located corresponds to the position of the second contact angle region 212 within the detection cell (is on the same vertical line), thereby further enhancing the chromaticity enhancement effect within the detection cell, lowering the detection limit, and improving the detection sensitivity.

[0029] Therefore, the vent hole 31 may have the same shape and dimensions as the second contact angle region 212, and may be configured so that both the area and the hole diameter are smaller than those of the second contact angle region 212. For example, the vent hole 31 may be a regular polygon or a circle whose diameter or circumscribed circle diameter may be 0.5 mm to 5 mm, preferably 1 mm to 3 mm. The vent hole 31 may be provided above the center of the detection cell to uniformly increase the chromaticity during the evaporation process of the liquid in the detection cell.

[0030] Furthermore, the diameter of the air vent 31 significantly affects the chromaticity enhancement effect. As shown in FIG. 5, within a certain range (before the chromaticity becomes saturated), the smaller the diameter of the air vent, the higher the chromaticity of the spot that is ultimately formed. For example, if the diameter of the air vent is 3 mm, the chromaticity (distance) after enhancement is only about 120, while if the diameter of the air vent is 1.5 mm, the chromaticity (distance) after enhancement reaches 200 or more. Therefore, to achieve a higher chromaticity enhancement effect, the diameter or circumscribed circle diameter of the air vent 31 may be set to 1 mm to 3 mm, but this may result in a longer enhancement time. For this reason, the evaporation of the liquid may be accelerated by adjusting and controlling the environmental temperature, humidity, and vacuum level in the area where the detection cell is located, as will be described in detail below.

[0031] FIG. 6 illustrates an improved embodiment of the paper-based microfluidic chip of the present invention, providing a chip structure with beneficial effects. Specifically, in this paper-based microfluidic chip, the paper substrate layer 2 is provided with a liquid storage cell 24 surrounding the detection cell 21. Therefore, if the liquid in the detection cell 21 evaporates too quickly to move all of the chromaticity to the vicinity of the vent 31, a colorless liquid can be added to the liquid storage cell 24 to replenish the liquid that should evaporate in the detection cell 21, thereby allowing the chromaticity to continue moving to the vicinity of the vent 31. The liquid supply channel may include one or more openable liquid supply holes in the upper layer 3 covering the liquid storage cell 204. The liquid supply holes may be opened when liquid needs to be replenished and closed after the liquid supply is complete. In actual detection, the colorless liquid added to the liquid storage cell 24 may be water or a mixture of different solvents. However, the added liquid must be able to dissolve / carry chromaticity and not interact with other parts of the chip. The detection effect of the paper-based microfluidic chip with the addition of the storage cell 24 is shown in Figure 7. Figure 8 shows a comparison of the color deepening effect of the paper-based microfluidic chip with and without the storage cell 24. As can be seen from this figure, adding a make-up liquid into the storage cell 24 can significantly improve the degree of color deepening near the air vent. Under the same conditions, the chip without the storage cell (chip 2) deepens the color from 48.2 to 190.3, while the chip with the storage cell (chip 1) deepens the color from 49.4 to 257.4.

[0032] In some embodiments of the present invention, the liquid to be detected may be added directly into the detection cell 21 from above; for example, the above-mentioned vent hole 31 may function as a sample addition hole. In other embodiments, the paper substrate layer 2 may have a sample addition region 22 at a position spaced apart from the detection cell 21, and the sample addition region 22 may be connected to the detection cell 21 via a diffusion channel 23. In this case, the upper layer 3 may have a sample addition hole 32 at a position corresponding to the sample addition region 22, as shown in FIG. 9 . As a result, the liquid to be detected may be injected into the sample addition region 22 through the sample addition hole 32 and enter the detection cell 21 via the diffusion channel 23 by a self-actuated action, and then undergo the subsequent color concentration and detection process. In some embodiments of the present invention, as shown in FIG. 10 , a plurality of the detection cells 21 may be provided, and the number of the detection cells may be determined depending on the number of samples to be detected and / or the number of parameters to be detected. It is preferable that each detection cell 21 is spaced from the sample application region 22 by the same distance, i.e., each diffusion channel 23 has the same length. The detection cell 21 has seven sample application regions 22 surrounding the center, so that when parallel detection or testing of multiple samples needs to be repeated multiple times, the chip can be used to complete the detection in one go, thus improving the detection throughput and further reducing the error between parallel tests.

[0033] To facilitate detection, a color reagent may be pre-placed in the detection cell 21 or the above-mentioned diffusion channel 23, which can cause the liquid in the detection cell 21 to develop a distinct color through a chemical reaction, etc., thereby facilitating color discrimination and colorimetric analysis, etc.

[0034] A second aspect of the present invention provides a microfluidic detection system comprising the above-mentioned paper-based microfluidic chip, and the microfluidic detection system includes associated equipment, such as a chip holder and a camera, that is used in combination with the above-mentioned paper-based microfluidic chip.

[0035] In particular, in a paper-based microfluidic chip with ventilation holes, the microfluidic detection system may be provided with an adjustment and control unit for adjusting and controlling one or more of the environmental temperature, air flow rate, humidity, and vacuum level of the area where the detection cell 21 is located. By adjusting these environmental factors, the evaporation rate of the liquid in the detection cell 21 can be controlled, thereby improving the color enrichment effect. For example, a heating plate may be provided for heating the environmental temperature of the area where the detection cell 21 is located, and the heating plate maintains the environmental temperature of the area where the detection cell 21 is located within a predetermined temperature range between 35°C and 45°C. FIG. 11 shows the relationship between the chromaticity distance and the detection time at environmental temperatures of 25°C, 35°C, and 45°C. Here, when the color enrichment rate at 35°C and 45°C is much higher than that at 25°C, the final enrichment degree is approximately the same. Therefore, by providing an appropriate adjustment and control unit, the detection efficiency can be effectively improved.

[0036] The regulation and control unit may be configured to regulate and control other environmental factors besides the environmental temperature, and may include, for example, a ventilation device for releasing pressurized gas above the detection cell 21 or for replacing the air above the detection cell 21 to increase the air flow rate around the ventilation hole 31 or to reduce the humidity near the ventilation hole 31. Alternatively, the regulation and control unit may include a vacuum oven, and in the detection process, the paper-based microfluidic chip is placed in the vacuum oven and left there for a predetermined time to accelerate color deepening.

[0037] A third aspect of the present invention provides a method for detecting a liquid using the above-mentioned paper-based microfluidic chip, including step S1 of introducing a liquid to be measured into the detection cell 21 of the paper-based microfluidic chip, step S2 of leaving the paper-based microfluidic chip stationary for a predetermined time, and step S3 of performing color discrimination and / or colorimetric analysis on a predetermined region in the detection cell 21. The predetermined region is a color-enriched region in the detection cell 21.

[0038] As mentioned above, in order to increase the liquid evaporation rate and improve the color concentration effect, in the above step S2, one or more of the environmental temperature, humidity, and vacuum degree of the area where the detection cell 21 is located can be adjusted and controlled. For example, the environmental temperature of the area where the detection cell 21 is located can be adjusted and controlled to be within a predetermined temperature range maintained between 25°C and 60°C.

[0039] A fourth aspect of the present invention provides the use of the above-mentioned paper-based microfluidic chip, microfluidic detection system, or liquid detection method in water quality detection, environmental detection, and food medicine. For example, the above-mentioned paper-based microfluidic chip, microfluidic detection system, and liquid detection method can be used to detect the content of nickel, chromium, phosphate, etc. in water, measure various indicators in biomedicine, and standardize the measurement of various substances in food.

[0040] The paper-based microfluidic chip, microfluidic detection system, and liquid detection method according to the present invention can improve quantitative accuracy and lower the detection limit. Compared with conventional techniques, the present invention: 1) enhances the color intensity of the detection cell in colorimetric assay, improving the color response from a faint one to an almost invisible level, thereby lowering the detection limit for the analyte. 2) By adjusting and controlling the color, the color on the paper chip is improved, while the distribution uniformity is improved, allowing for a larger number of sampling locations, improving ease of handling, and further improving detection accuracy and reproducibility. 3) This can be achieved through a simple method of combining material surface modification through a three-dimensional structural design, eliminating the need for additional complex equipment, thereby ensuring the portability of paper chip detection. 4) It is applicable to most paper chips for colorimetric assays, making it highly practical and versatile.

[0041] The present invention will be described in detail below with reference to examples, in which contact angles are measured using an OCA200 fully automatic single fiber contact angle measuring device. Example 1

[0042] FIG. 12 shows a paper-based microfluidic chip according to this embodiment, which includes a bottom layer 1, a paper substrate layer 2, and a thickening layer 25. The paper substrate layer 2 includes six detection cells 21 arranged in a circular array and a blank control detection cell in the center. The thickening layer 25 is a thickening carrier attached to each detection cell 21, and the thickening carrier is located in the center (including, but not limited to, the center) of the detection cell 21. The detection cells 21 are made of a material with a large contact angle (e.g., filter paper modified with a fluorinated silane reagent or a plastic sheet with a large contact angle itself), and the thickening carrier is made of a material with a small contact angle (e.g., filter paper, cellulose filtration membrane, or other material that has been plasma-treated or surface-modified). The thickening carrier is smaller than the detection cells 21 and forms a second contact angle region with a small contact angle. The thickening carrier is generally preferably circular or a regular polygon with a circumscribed circle diameter of 1 mm to 3 mm. The remaining portion within the detection cell 21 is a first contact angle region having a large contact angle. The condensed carrier, the detection cell, and the underlayer are adhered to each other by adhesive or other methods.

[0043] After the reaction, the colored solution (e.g., a 0.1% dye solution, or a solution obtained by mixing a solution of the analyte with a specific reagent at various concentrations) was dropped into each of the detection cells arranged in a circular array, with the central detection cell used as a blank control. The color intensity effect is shown in Figure 13. After color intensity enhancement under specific conditions (e.g., 35°C, 10 min), the detection cells were photographed under natural light conditions, and the RGB values ​​of the photograph were analyzed to calculate the chromaticity distance D. The chromaticity of a 0.1% red dye solution after enhancement is shown in the table below.

[0044] [Table 1] Example 2

[0045] In this example, a paper chip with a three-layer structure is used as the base chip with enhanced color, and an aqueous solution with added dye is used as the sample, demonstrating that the present invention can enhance the color of all color-based solutions, and that color enhancement occurs in any small area that is connected to air and does not need to be coaxial with the sample addition hole.

[0046] The paper chip was constructed as shown in Figure 14. The upper and lower sides of the paper substrate layer, prepared by cutting, were covered with a transparent, non-breathable film. Air holes were left in the upper film (upper layer) at positions corresponding to the detection cells. The sample addition holes were located at the same distance as each detection cell. A dye solution was injected through the sample addition holes, which distributed along the hydrophilic diffusion channel and reached each of the detection cells 1 to 7. When the paper chip was left in a natural environment (ambient temperature 26°C, humidity 70%), after a predetermined time (10 to 60 minutes), the chromaticity of the vent hole area of ​​the detection cell became darker. Over time, the contrast between the chromaticity of the darkened area and the chromaticity of the other areas became more pronounced. The RGB values ​​of the chromaticities of multiple areas within different detection cells and within the same detection cell were read, and the results are shown in the table below and Figure 15.

[0047] [Table 2]

[0048] [Table 3]

[0049] As can be seen from each of the above tables, the R, G, and B values ​​are similar between different detection cells and between different regions of the same detection cell, and the standard deviations of the R, G, and B values ​​between different detection cells and between different regions of the same detection cell are all less than 3%, which suggests that the chromaticity proposed in the present invention is enhanced and the uniformity and reproducibility of the liquid detection method are excellent. Example 3

[0050] The example illustrates the lowering of the detection limit for nickel in water by color-enhancing structures.

[0051] In the paper-based microfluidic chip used in this example, the lower layer is a transparent, non-breathable membrane, the paper substrate layer is a hydrophobically modified filter paper (the sample application area, diffusion channel, and detection cell are hydrophilic, while the remaining area is hydrophobically modified, and the detection cell is pre-filled with a complex reagent whose main component is dimethylglyoxime and which can undergo a specific color reaction with nickel), and the upper layer is a transparent, non-breathable membrane, which is provided with a sample application hole communicating with the sample application area and an air vent communicating with the detection cell. The detection cell has a diameter of 4 mm, and the air vent hole has a diameter of 2 mm.

[0052] During detection, a nickel-containing water sample is injected through the sample addition hole. The sample flows along the diffusion channel into the detection cell and reacts with the composite reagent, producing a pink substance. After the chip is left for a predetermined period of time, the water vapor evaporates along the upper air vents. The color-producing components diffuse and collect in the color-enhanced region due to evaporation, gradually darkening the color. After the reaction is complete, the color-enhanced region on the paper chip is imaged, and the color information of this region is extracted using MATLAB, allowing for quantitative calculations.

[0053] As can be seen from Figures 16 and 17, the normal paper chip has a high detection limit for nickel in water, but when the color is increased, the detection limit for nickel in water drops to 0.1 mg / L, which is a significant reduction in the detection limit. Fitting the standard curves for each of the nickel solutions in the linear range showed a significant improvement in the linearity of the fitted lines. Example 4

[0054] The example illustrates the reduction of the detection limit for chromium in water due to color-enhancing structures.

[0055] In the paper-based microfluidic chip used in this example, the lower layer is a transparent, non-permeable membrane, the middle layer is hydrophobically modified filter paper (the sample application area, diffusion channel, and detection cell are hydrophilic, while the remaining area is hydrophobically modified, and the detection cell is pre-filled with a complex reagent whose main component is diphenylcarbazide and which can undergo a specific color reaction with chromium), and the upper layer is a transparent, non-permeable membrane, which is provided with a sample application hole communicating with the sample application area and an air vent communicating with the detection cell. The detection cell has a diameter of 4 mm, and the air vent hole has a diameter of 2.5 mm.

[0056] During detection, a chromium-containing water sample is injected into the sample application area, and the sample flows along the diffusion channel into the detection cell, reacting with the complex reagent to produce a pink substance. After the chip is left for a certain period of time, the water vapor evaporates along the upper air vent, and the color-producing component diffuses and gathers in the color-enhancing area due to evaporation, gradually darkening the color.

[0057] As a result, before color deepening, the detection limit of chromium in a normal paper chip is 0.05 mg / L, but in the paper chip of this example, color development is enhanced and the detection limit drops to 0.03 mg / L. Example 5

[0058] The example illustrates the lowering of the detection limit for nitrous acid in water by a color-enhancing structure.

[0059] In the paper-based microfluidic chip used in this example, the lower layer is a transparent, non-breathable membrane, the paper substrate layer is a hydrophobically modified filter paper (the sample application area, diffusion channel, and detection cell are hydrophilic, while the remaining area is hydrophobically modified, and the detection cell is pre-filled with Griess reagent, which can specifically react with nitrite to produce a color reaction), and the upper layer is a transparent, non-breathable membrane with a sample application hole communicating with the sample application area and a vent hole communicating with the detection cell. The detection cell has a diameter of 5 mm, and the vent hole has a diameter of 2 mm.

[0060] During detection, a chromium-containing water sample is injected into the sample application area, and the sample flows along the diffusion channel into the detection cell, where the complex reagent reacts to produce a pink substance. After the chip is left for a certain period of time, the water vapor evaporates along the upper air vent, and the color-producing components diffuse and gather in the color-enhancing area due to evaporation, gradually darkening the color. As a result, before the color intensity increases, the detection limit of chromium in a normal paper chip is 0.1 mg / L, but in the paper chip according to this embodiment, the color intensity increases and the detection limit drops to 0.05 mg / L.

[0061] Although the preferred embodiments of the present invention have been described in detail above with reference to the drawings, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, the technical concept of the present invention can be easily modified in multiple ways, including combining individual specific technical features in any appropriate manner. To avoid unnecessary repetition, various combinations of the present invention will not be described separately. However, these simple modifications and combinations should also be considered as the disclosure of the present invention, and all fall within the protection scope of the present invention. [Brief explanation of the drawings]

[0062] [Figure 1] 1 is a schematic diagram of a paper-based microfluidic chip according to a preferred embodiment of the present invention. [Figure 2] FIG. 1 is a detection effect diagram of a paper-based microfluidic chip according to a preferred embodiment of the present invention. [Figure 3] FIG. 1 is a schematic diagram of the gradient distribution of each contact angle region of the detection cell of the paper-based microfluidic chip according to a preferred embodiment of the present invention. [Figure 4] FIG. 1 is an exploded schematic diagram of a paper-based microfluidic chip according to another preferred embodiment of the present invention. [Figure 5] FIG. 10 is a scatter diagram showing the relationship between color deepening effect and the hole diameter of the ventilation hole. [Figure 6] FIG. 1 is a schematic diagram of a paper-based microfluidic chip according to another preferred embodiment of the present invention. [Figure 7]FIG. 7 shows the detection effect of the paper-based microfluidic chip in FIG. 6. [Figure 8] FIG. 10 compares the color-enhancing effect of various paper-based microfluidic chips. [Figure 9] FIG. 1 is a schematic diagram of a paper-based microfluidic chip according to another preferred embodiment of the present invention. [Figure 10] FIG. 10 is a distribution diagram of detection cells in a paper-based microfluidic chip according to another preferred embodiment of the present invention. [Figure 11] 10 is a scatter plot of the relationship between color deepening effect and detection time at various environmental temperatures. [Figure 12] FIG. 1 is a schematic disassembled view of a paper-based microfluidic chip according to Example 1 of the present invention. [Figure 13] FIG. 13 is a schematic diagram of the color-enhancing effect of the paper-based microfluidic chip in FIG. 12. [Figure 14] FIG. 1 is a structural schematic diagram of a paper-based microfluidic chip according to Example 2 of the present invention. [Figure 15] FIG. 15 is a schematic diagram of the color-enhancing effect of the paper-based microfluidic chip in FIG. 14. [Figure 16] 10 is a quantitative curve of the paper-based microfluidic chip in Example 3 of the present invention. [Figure 17] 10 is a quantitative curve of the paper-based microfluidic chip in Example 3 of the present invention.

Claims

1. A paper-based microfluidic chip comprising: The paper-based microfluidic chip includes a paper substrate layer (2), in which a detection cell (21) is provided, the detection cell (21) having a first contact angle region (211) and a second contact angle region (212) therein, and configured such that the contact angle of the liquid in the detection cell (21) at the first contact angle region (211) is larger than the contact angle at the second contact angle region (212).

2. 2. The paper-based microfluidic chip of claim 1, wherein the first contact angle region (211) is provided in the detection cell (21) with a hydrophobic material laid thereon, deposited thereon, or impregnated therewith, and / or the second contact angle region (212) is provided in the detection cell (21) with a hydrophilic material laid thereon, deposited thereon, or impregnated therewith.

3. 2. The paper-based microfluidic chip of claim 1, wherein a hydrophobic layer is formed in the detection cell (21) by laying, depositing, or impregnating a hydrophobic material in the detection cell (21), and a hydrophilic material is laid, deposited, or impregnated above the hydrophobic layer in the central portion of the detection cell (21), thereby forming the second contact angle region (212) and the first contact angle region (211) surrounding the second contact angle region (212).

4. The paper-based microfluidic chip of claim 1, wherein the second contact angle region (212) is located in a central portion of the detection cell (21), and the first contact angle region (211) surrounds the second contact angle region (212).

5. 5. The paper-based microfluidic chip according to claim 4, wherein the detection cell (21) is formed as a circle with a diameter of 2 mm to 8 mm or a regular polygon with a circumscribing circle diameter of 2 mm to 8 mm, the second contact angle region (212) is located at the center of the detection cell (21), and / or the second contact angle region (212) is a circle with a diameter of 0.5 mm to 5 mm or a regular polygon with a circumscribing circle diameter of 0.5 mm to 5 mm.

6. 5. The paper-based microfluidic chip of claim 4, wherein the first contact angle region (211) is configured such that the contact angle of the liquid in the detection cell (21) at the first contact angle region (211) increases from the second contact angle region (212) toward the edge of the detection cell (21).

7. The paper-based microfluidic chip of claim 1, wherein the water contact angle of the first contact angle region (211) is greater than 60°, preferably greater than 90°, more preferably greater than 120°.

8. The paper-based microfluidic chip according to any one of claims 1 to 7, comprising a lower layer (1) provided on a first side of the paper substrate layer (2) and an upper layer (3) provided on a second side of the paper substrate layer (2) opposite the first side, the lower layer (1) and the upper layer (3) having a water-impermeable and air-impermeable portion covering the detection cell (21), and an air vent (31) formed in the portion of the upper layer (3) covering the detection cell (21).

9. The paper-based microfluidic chip of claim 8, wherein the air vent (31) is a regular polygon or a circle, and both the area and the hole diameter are smaller than those of the second contact angle region (212), and preferably the diameter or circumscribed circle diameter of the air vent (31) is 0.5 mm to 5 mm, preferably 1 mm to 3 mm.

10. 9. The paper-based microfluidic chip of claim 8, wherein the positions of the vent holes (301) in the upper layer (3) correspond to the positions of the second contact angle regions (212) in the detection cell (201).

11. The paper substrate layer (2) is further provided with a sample application area (22) and a diffusion channel (23) connecting the sample application area (22) and the detection cell (21); 9. The paper-based microfluidic chip according to claim 8, wherein the upper layer (3) has a sample application hole (32) at a position corresponding to the sample application region (22).

12. The paper-based microfluidic chip according to claim 11, wherein a color-developing reagent is pre-disposed in the detection cell (21) and / or the diffusion channel (23).

13. 9. The paper-based microfluidic chip according to claim 8, wherein the paper substrate layer (2) is further provided with a liquid storage cell (24) arranged to surround the detection cell (21) so that the detection cell (21) can be replenished with liquid.

14. A microfluidic detection system comprising the paper-based microfluidic chip according to any one of claims 1 to 13.

15. 15. The microfluidic detection system of claim 14, further comprising an adjustment and control unit for adjusting and controlling one or more of the environmental temperature, air flow rate, humidity, and vacuum level of the area in which the detection cell (21) resides.

16. A liquid detection method comprising: Step S1: introducing a liquid to be measured into a detection cell (21) of the paper-based microfluidic chip according to any one of claims 1 to 13; Step S2 of leaving the paper-based microfluidic chip standing for a predetermined time; and step S3 of performing color discrimination and / or colorimetric analysis on a predetermined area within the detection cell (21).

17. 17. The liquid detection method according to claim 16, wherein in step S2, the environmental temperature of the area where the detection cell (21) exists is set to a temperature between 25°C and 60°C.

18. 17. The liquid detection method according to claim 16, wherein in step S2, an adjustment and control unit is used to adjust and control one or more of the environmental temperature, humidity, and vacuum degree of the area where the detection cell (21) exists.

19. Use of the paper-based microfluidic chip according to any one of claims 1 to 13, the microfluidic detection system according to claim 14 or 15, or the liquid detection method according to any one of claims 16 to 18 in water quality detection, environmental detection, food medicine.

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