A colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip and its preparation and application

Abstract

The present invention belongs to the field of microfluidic technology, and particularly relates to a colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip and its preparation and application. The three-dimensional paper-based microfluidic chip includes an upper layer, a middle layer, and a lower layer; the upper layer is provided with a sample injection area and a detection area; the middle layer is provided with a first functional area and a second functional area, and a first hydrophilic channel and a second hydrophilic channel respectively connected thereto; the lower layer is provided with a lower hydrophilic area; a colorimetric signal reagent with a target recognition element coated on its surface is contained in the first functional area, which also serves as a fluorescence quenching reagent; a fluorescence signal reagent is contained in the second functional area; and a neutral salt is contained in the lower hydrophilic area. The present invention can solve the problems of requiring professional personnel for operation and large-scale detection instruments in the past. The dual-mode detection results of the nanomaterials can be mutually verified, reducing the interference of environmental factors on the results, with high sensitivity and strong specificity. At the same time, the manufacturing method is simple and fast, and the cost is low, which is suitable for on-site real-time detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidics, and particularly relates to a colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip and its preparation and application. Background Art

[0002] Recently, dual-mode (dual-signal) detection methods have attracted more and more in-depth attention from researchers. Compared with single-mode detection methods, dual-mode detection methods can provide more information, reduce the interference of environmental factors, and the detection results can be mutually verified, greatly improving the reliability of the detection results. Common dual-mode detection methods mainly include fluorescence-SERS, fluorescence-colorimetry, fluorescence-electrochemistry, etc. Combining the high-sensitivity response characteristics of nanomaterials and the high specificity of aptamers, more and more dual-mode biosensing detection methods have gradually been applied to the detection of food hazards. Although these methods have good sensitivity and accuracy, they usually rely on expensive instruments and professional experimental operators and cannot achieve rapid detection.

[0003] Three-dimensional paper-based microfluidic chips (3D-μPADs) are an emerging point-of-care testing technology. 3D-μPADs are assembled by pasting, folding, etc. two-dimensional μPADs with two or more layers, enabling a device for liquid to flow and react between different layers of paper. Compared with two-dimensional μPADs, 3D-μPADs have a faster detection speed, more flexible mode design, and functional components can be added therein. And it can be combined with a variety of common biosensing methods to transfer traditional complex laboratory detection methods to a small paper chip for implementation. Combined with portable detection devices or smartphones, numerous complete detection systems applicable to point-of-care testing (POCT) have been developed.

[0004] However, in the prior art, dual-mode detection methods and three-dimensional paper-based microfluidic chips are used independently. Therefore, a new solution is needed to combine the two to give full play to their respective advantages. Summary of the Invention

[0005] The present invention aims to provide a colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip and its preparation and application. By combining microfluidic detection technology with dual-mode detection, it can improve the convenience of detection and has the advantages of high sensitivity and strong specificity.

[0006] According to the technical solution of the present invention, the colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip includes an upper layer, a middle layer, and a lower layer;

[0007] The upper layer includes an upper hydrophobic region and an upper hydrophilic region, and the upper hydrophilic region includes a sample injection region and a detection region;

[0008] The middle layer includes a middle hydrophobic region and a middle hydrophilic region. The middle hydrophilic region is provided with a first functional region and a second functional region corresponding to the sample injection region and the detection region respectively, and a first hydrophilic channel and a second hydrophilic channel connecting the first functional region and the second functional region respectively;

[0009] The lower layer includes a lower hydrophobic region and a lower hydrophilic region. The lower hydrophilic region is respectively connected to the first functional region and the second functional region through the first hydrophilic channel and the second hydrophilic channel;

[0010] The first functional region contains a colorimetric signal reagent, and the surface of the colorimetric signal reagent is coated with a target recognition element; the second functional region contains a fluorescence signal reagent; the lower hydrophilic region contains a neutral salt;

[0011] The colorimetric signal reagent can quench the fluorescence of the fluorescence signal reagent, and the neutral salt can react with the colorimetric signal reagent and cause a color change.

[0012] Further, the colorimetric signal reagent is silver nanoparticles (AgNPs), which also serves as a fluorescence quenching reagent;

[0013] The target recognition element is an aptamer (apt);

[0014] The fluorescence signal reagent is magnesium and nitrogen co-doped carbon dots (Mg,N-CDs);

[0015] The neutral salt is selected from one or more of sodium chloride, magnesium chloride, and potassium chloride.

[0016] Specifically, the detection principle is as follows: First, mix the yellow AgNPs solution with apt and incubate it to make apt coat on the surface of AgNPs. When there is no analyte in the system, the AgNPs protected by apt are not affected by the introduced NaCl and remain in a dispersed state showing yellow. After adding Mg,N-CDs, since the fluorescence emission peak of Mg,N-CDs overlaps well with the ultraviolet-visible absorption spectrum of AgNPs, the fluorescence of Mg,N-CDs is quenched by the inner filter effect (IFE). When there is an analyte in the system, the high affinity of apt causes it to specifically bind to the target preferentially. AgNPs lose the protection of the aptamer and aggregate under the action of NaCl, and the solution changes from yellow to colorless, resulting in a change in the colorimetric signal. After adding the synthesized Mg,N-CDs, due to the aggregation of AgNPs, the fluorescence signal does not change. That is to say, the fluorescence signal in the system increases with the increase of the analyte concentration, and the colorimetric signal decreases with the increase of the analyte concentration.

[0017] Further, the content ratio of the silver nanoparticles, the aptamer, the magnesium and nitrogen co-doped carbon dots, and NaCl is 0.3 - 0.5 mmol : 6 - 10 μmol : 12 - 20 mg / mL : 70 - 100 mmol.

[0018] Further, the magnesium and nitrogen co-doped carbon dots are obtained by heating and reacting a mixture of an organic acid, a magnesium salt, and a nitrogen source.

[0019] Further, the organic acid is selected from one or more of citric acid, malic acid, and tartaric acid, the magnesium salt is selected from Mg(OH)2 and / or MgCl, and the nitrogen source is selected from o-phenylenediamine or 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

[0020] Further, the temperature of the heating reaction is 180 - 220 °C, the time is 2 - 4 h, and the heating method can be hydrothermal.

[0021] Further, after the heating reaction, the precipitate is removed by centrifugation, the supernatant is filtered and dialyzed to obtain a purified carbon dot solution, and then freeze-dried to obtain the magnesium and nitrogen co-doped carbon dots.

[0022] The second aspect of the present invention provides a method for preparing the above colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip, including the following steps:

[0023] S1: Provide upper, middle, and lower three layers of filter paper, perform hydrophobic treatment on the upper hydrophobic area of the upper filter paper, the middle hydrophobic area of the middle filter paper, and the lower hydrophobic area of the lower filter paper, and assemble the three layers of filter paper after hydrophobic treatment to obtain a semi-finished product;

[0024] S2: Drop the colorimetric signal reagent coated with the target recognition element into the first functional area of the semi-finished product, drop the fluorescent signal reagent into the second functional area; drop neutral salt into the lower hydrophilic area to obtain the colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip.

[0025] Further, in the step S1, after printing carbon powder on the filter paper, it is heated and melted to obtain the filter paper after hydrophobic treatment.

[0026] Further, carbon powder is printed on the surface of the filter paper by carbon powder laser printing, and the temperature of heating and melting is 180 - 220 °C.

[0027] Specifically, in the step S1, by carbon powder laser printing, the upper, middle, and lower three layers of paper-based are simultaneously printed on a piece of filter paper according to the designed shape of the hydrophobic area; then heat treatment for hydrophobicity is carried out, and then the three layers of paper-based are cut and pasted and assembled.

[0028] Further, in step S2, each reagent is added dropwise in the form of a solution, and the dropwise addition amount is 20-40 μL. Among them, the concentration of silver nanoparticles is 0.3-0.5 mM, the concentration of aptamer is 6-10 μM, the concentration of magnesium and nitrogen co-doped carbon dots is 12-20 mg / mL, and the concentration of NaCl is 70-100 mM.

[0029] The third aspect of the present invention provides the application of the above-mentioned colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip in the visual detection of antibiotics, and the target recognition element is an antibiotic aptamer.

[0030] Further, the antibiotic is sulfadimethoxine (SDM), and the target recognition element is a sulfadimethoxine aptamer (SDM, sequence: 5’-C6-TCATCTATCTATGGTACATTA CTATCTGTAATGTGATATG-3’).

[0031] Further, it includes the following steps:

[0032] Add a series of concentrations of antibiotic standard solutions to the sample injection area of the three-dimensional paper-based microfluidic chip. After sufficient reaction, collect and quantitatively detect the colorimetric intensity and fluorescence intensity in the detection area, and draw the standard curves of colorimetric intensity vs. antibiotic concentration and fluorescence intensity vs. antibiotic concentration;

[0033] Add the test solution to the sample injection area of the blank three-dimensional paper-based microfluidic chip. After sufficient reaction, collect the colorimetric intensity and fluorescence intensity in the detection area, and obtain the concentration of the antibiotic in the test solution according to the standard curve.

[0034] Further, the acquisition and quantification method of the colorimetric intensity is: collect the image of the detection area under daylight conditions, extract the RGB values of the image and convert them into gray values for quantification;

[0035] The acquisition and quantification method of the fluorescence intensity is: collect the image of the detection area under ultraviolet light conditions, extract the RGB values of the image, and quantify with the B-channel value.

[0036] Further, the image of the detection area is collected by a smart phone.

[0037] Specifically, during detection, 100 μL of a series of standard solutions containing SDM was slowly dropped onto the first-layer sample injection area. The liquid flowed vertically through each layer area in the following order: upper-layer sample injection area - middle-layer first functional area (AgNPs / apt) - lower layer (NaCl) - middle-layer second functional area (Mg,N-CDs) - upper-layer detection area. After fully reacting for 15 minutes, it was air-dried naturally at room temperature and then placed in a portable detection device. The daylight light source and ultraviolet light source were turned on in sequence. When the ultraviolet light source was turned on, a filter was installed in the device, and an RGB signal on the image was collected using a smartphone. The yellow AgNPs image was related to the antibiotic concentration by converting the RGB values to grayscale values, and the blue fluorescent CDs took the B-channel value to establish a connection with the antibiotic concentration. Three points were selected in each detection area as the average value of the detection results.

[0038] The technical solution of the present invention has the following advantages compared with the prior art:

[0039] (1) The present invention designs a three-dimensional paper-based microfluidic chip prepared by a carbon powder laser printer. The paper-based substrate uses filter paper, and the preparation method is simple and low-cost. The three-dimensional paper-based microfluidic chip is small in size, consumes less sample volume, is simple and portable, and meets the immediate detection requirements for food hazards.

[0040] (2) The present invention designs a three-dimensional paper-based microfluidic chip in which liquid can flow reciprocally, taking advantage of the hydrophilicity of filter paper. During detection, the liquid starts from the upper-layer sample injection area, flows to the lower layer, and finally returns to the upper-layer detection area to display the detection result, reducing the originally required five-layer structure to three layers, saving preparation materials and simplifying the multi-layer structure.

[0041] (3) The method of the present invention combines the three-dimensional paper-based microfluidic chip detection technology with two kinds of nanomaterials and aptamers, making the detection method have better sensitivity and specificity. The reaction reagents are loaded in different areas of the three-dimensional paper-based microfluidic chip, and through step-by-step reactions in each layer structure, a new colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip detection platform is established. The detection results of the two modes can verify each other, improving the reliability of the detection results and reducing the interference of environmental factors on the detection results.

[0042] (4) The present invention combines a smartphone imaging system and a color picking APP to achieve simple, rapid, visual and quantitative detection in two modes: complementary color and fluorescence. During detection, the CMOS sensor of the smartphone is used to collect detection images of the detection area of the three-dimensional paper-based microfluidic chip under daylight and 365 nm ultraviolet light. The RGB values of the detection area are extracted through the color picking APP of the smartphone. In the colorimetric mode, the RGB values are converted into grayscale values, and in the fluorescence mode, the B value is taken as the detection result of the two modes respectively. The average value of three points is taken in the detection area as the signal intensity, and the detection result is calculated. When applied to the detection of the antibiotic sulfamethoxydiazine, detection limits as low as 0.21 ng / mL and 0.13 ng / mL are obtained in the two modes respectively. Description of the Drawings

[0043] Figure 1 It shows the preparation process (A), composition structure (B) and detection process (C) of the three-dimensional paper-based microfluidic chip prepared in Example 2.

[0044] Figure 2 It is the size design diagram of the three-dimensional paper-based microfluidic chip prepared in Example 2.

[0045] Figure 3 It is the physical diagram of the three-dimensional paper-based microfluidic chip prepared in Example 2 under natural light and ultraviolet light.

[0046] Figure 4 It is the schematic diagram of dye flow of the three-dimensional paper-based microfluidic chip prepared in Example 2.

[0047] Figure 5 It is the scanning electron microscope (SEM) image of the three-dimensional paper-based microfluidic chip prepared in Example 2 (A and B: SEM images of the upper detection area of the blank paper-based microfluidic chip at scales of 10 μm and 1 μm; C: SEM image of the upper detection area after reaction with the blank solution).

[0048] Figure 6 It is the feasibility verification diagram of the application of the three-dimensional paper-based microfluidic chip in SDM detection in Example 3, including feasibility verification in a homogeneous system and feasibility verification on the paper-based microfluidic chip (A: colorimetric mode; B: fluorescence mode).

[0049] Figure 7 It is the overlap of the absorption spectrum of AgNPs and the fluorescence emission spectrum of Mg,N-CDs in Example 3.

[0050] Figure 8 It is the fluorescence lifetime of Mg,N-CDs before and after adding AgNPs in Example 3.

[0051] Figure 9Optimization results of the concentrations of Mg,N-CDs, NaCl, and apt in Example 3, with the results presented as those on the paper-based microfluidic chip.

[0052] Figure 10 Standard curve and corresponding physical diagram of the application of the three-dimensional paper-based microfluidic chip in SDM detection in Example 3 (A: colorimetric mode; B: fluorescence mode).

[0053] Figure 11 Specificity of the application of the three-dimensional paper-based microfluidic chip in SDM detection in Example 3 (A: colorimetric mode; B: fluorescence mode). Detailed implementation manners

[0054] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0055] Example 1 Preparation of nanomaterials

[0056] 1. Preparation of Mg,N-CDs

[0057] First, dissolve 0.5 g of citric acid in 20 mL of distilled water, then add 0.2 g of Mg(OH)2 and 0.2 g of o-phenylenediamine, and ultrasonicate for 15 min to form a colorless, transparent, and homogeneous solution. Then transfer it to a 50 mL reaction kettle lined with polytetrafluoroethylene and perform hydrothermal treatment at 200 °C for 3 h. Subsequently, after cooling the obtained solution at room temperature, centrifuge it at 12,000 rpm for 20 min to remove the solid sediment, and then further filter the supernatant through a water-phase microporous membrane with a pore size of 0.22 μm. Finally, dialyze the filtrate with a dialysis bag (MW = 0.5 - 1 KD) and ultrapure water for 24 h to purify the carbon dot solution, and replace the ultrapure water every 4 h. Store the obtained liquid sample at 4 °C and obtain the carbon dot powder by freeze-drying.

[0058] 2. Preparation of AgNPs

[0059] First, add 10 mL of 1 mM AgNO3 and 1 mL of 0.03 M CTAB to a conical flask, stir in an ice bath for 20 min, then add 1 mL of 0.01 M NaBH4 and stir for another 20 min. When the solution changes from colorless to bright yellow, it indicates the formation of AgNPs.

[0060] Example 2 Preparation of a colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip based on nanomaterials

[0061] The preparation of the three-dimensional paper-based microfluidic chip (3D-μPAD) is as Figure 1As shown in Figure A. First, the planar pattern of the 3D-μPAD is designed using word software, then printed on filter paper with a toner laser printer. The toner on the paper surface is heated and melted to form a hydrophobic barrier in the paper substrate. Finally, it is cut and assembled. As Figure 1 As shown in Figure B, the 3D-μPAD is divided into upper, middle, and lower layers. The upper layer is the sample layer, which contains a square sample injection area with a side length of 5 mm and a sample detection area with a diameter of 5 mm respectively. The middle layer is the functional layer. In the upper right corner is the first functional area loaded with apt-coated AgNPs with a diameter of 4 mm, and in the lower left corner is the second functional area loaded with Mg,N-CDs with the same diameter of 4 mm. Two functional areas are respectively connected with two first hydrophilic channels and second hydrophilic channels with a length of 5 mm and a width of 1.5 mm. The two channels are not connected to each other at both ends and are only used to communicate with the hydrophilic area of the lower layer. The lower layer is the NaCl layer, which contains a hydrophilic area with a diameter of 5 mm at the center position for loading NaCl. After preparing the paper substrate, nanomaterials and reagents are respectively dropped at the corresponding reserved positions to complete the preparation of the final colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic aptasensor based on Mg,N-CDs.

[0062] The detection process of this 3D-μPAD is as Figure 1 shown in Figure C, which respectively represents the liquid flow direction from top to bottom, the states in the corresponding paper-based regions in the presence and absence of the analyte, and the cross-sectional flow schematic diagram of the 3D-μPAD. Specifically, in the first step, the analyte solution is added to the sample injection area, and the liquid flows from the sample injection area to the first functional area loaded with apt-coated AgNPs. In the presence of the analyte, apt detaches from the surface of AgNPs and preferentially binds to the analyte, and then they flow horizontally and then vertically along the first hydrophilic channel to the lower NaCl layer together. In the absence of the analyte, the apt-coated AgNPs remain unchanged and also flow to the lower layer in the order of first horizontal and then vertical with the liquid. In the second step, the AgNPs without apt coating will aggregate under the action of a certain concentration of NaCl in the lower layer, and the solution changes from yellow to colorless, while the apt-coated AgNPs are not affected by NaCl. In the third step, both states of AgNPs flow vertically back to the middle layer, and then flow horizontally along the second hydrophilic channel connecting the second functional area loaded with Mg,N-CDs to this functional area. Because of the presence of the analyte, the aggregated AgNPs lacking apt protection cannot quench the fluorescence of Mg,N-CDs, and the fluorescence intensity of Mg,N-CDs is weakened by the free AgNPs under the action of IFE. In the fourth step, all substances in the liquid flow vertically back to the circular detection area of the upper layer. A strong fluorescence signal can be observed in the upper detection area in the presence of the analyte, while the colorimetric signal is weak. The situation is opposite in the absence of the analyte.

[0063] Next, the prepared three-dimensional paper-based microfluidic chip was characterized by unfolding it on the same plane. Figure 2 This is the design drawing of its size. Figure 3 These are its physical images under visible light (upper figure) and ultraviolet light (lower figure). It can be observed that the functional area of AgNPs under visible light is bright yellow, and the functional area of Mg,N-CDs shows bright blue fluorescence under ultraviolet light. When the dye was added to the three-dimensional paper-based microfluidic chip, the flow order of the liquid was as Figure 4 shown. Then, the surface of the paper substrate was characterized by SEM. First, the upper blank detection area was photographed at scales of 10 μm and 1 μm ([[]]END]] Figure 5 A and B), and the cellulose structure of the filter paper could be clearly seen, with no impurities on the surface. Then, the prepared three-dimensional paper-based microfluidic chip was reacted with a blank solution without the analyte, and the upper detection area was also sampled and photographed. The results were as Figure 5 shown in C. At a scale of 1 μm, many nanomaterial particles of Ag NPs / CDs were observed on the surface, which not only characterized the surface morphology of the paper substrate but also verified the feasibility of the detection principle of this three-dimensional paper-based microfluidic chip.

[0064] Example 3 SDM Detection Application of Colorimetric-Fluorescent Dual-Mode Three-Dimensional Paper-Based Microfluidic Chip Based on Nanomaterials

[0065] 1. Verification of Detection Feasibility

[0066] To verify the feasibility of colorimetric-fluorescent dual-mode detection (SDM) based on nanomaterials, the signal intensities at different stages under colorimetric and fluorescent conditions were studied respectively. As Figure 6 shown in A, after adding NaCl to AgNPs (line d), the absorption peak of AgNPs at 400 nm broadened and disappeared, while the AgNPs protected by apt were more stable, and the absorbance intensity hardly changed after adding NaCl (line b). Moreover, adding Mg,N-CDs to the system had no effect on AgNPs. However, after adding the analyte to the system, the unstable AgNPs aggregated under the action of NaCl, and the absorbance decreased (line c). The images of AgNPs in the detection area of the 3D-μPAD at each reaction stage were as Figure 6 shown in the inset of A, corresponding to the four curves a, b, c, and d respectively. To ensure the consistency of the results, a was the color effect of the detection area when only AgNPs were loaded in the first functional area of the middle layer of the three-dimensional paper-based microfluidic chip, d was the detection effect when AgNPs and NaCl were loaded in the middle and lower layers of the three-dimensional paper-based microfluidic chip respectively, and b and c were the tests performed on the complete 3D-μPAD. a and b were bright yellow, while c and d were almost colorless, which was consistent with the absorbance results.

[0067] The feasibility of the fluorescence detection mode was as Figure 6As shown in Fig. B, Mg,N-CDs itself has strong fluorescence (line a). After adding dispersed AgNPs, the fluorescence is significantly quenched due to the IFE effect (line d). When there is no target in the detection system, AgNPs are protected by apt and are not affected by NaCl, and can significantly quench the fluorescence of Mg,N-CDs. When SDM exists in the system, apt detaches from the surface of AgNPs and aggregates under the action of NaCl. The aggregated AgNPs have little effect on the fluorescence signal intensity of Mg,N-CDs. Similar to the colorimetric mode, the feasibility of the fluorescence detection mode was also verified on the 3D-μPAD, and the upper detection area was selected as the verification result. a is the detection result of only loading Mg,N-CDs in the functional area, d is the detection result of only loading Ag NPs and Mg,N-CDs in the functional area, and b and c are Figure 6 The effects of the insets b and c in Fig. A under ultraviolet light. The blue fluorescence signal intensity of insets a and b is stronger, and the fluorescence of Mg,N-CDs in insets c and d is quenched, corresponding to the results in the homogeneous system. In summary, the feasibility of the method established in this chapter was verified in both homogeneous and solid-phase systems, fully demonstrating that the colorimetric-fluorescent dual-mode 3D-μPAD is feasible for detecting SDM.

[0068] At the same time, in order to prove the principle that AgNPs quench the fluorescence of Mg,N-CDs through the inner filter effect (IFE), the ultraviolet-visible absorption spectrum of AgNPs and the fluorescence emission spectrum of Mg,N-CDs were first compared. Figure 7 It shows that the absorption spectrum of AgNPs significantly overlaps with the fluorescence emission spectrum of Mg,N-CDs, proving that AgNPs can quench the fluorescence of Mg,N-CDs. To verify the fluorescence quenching mechanism, the fluorescence decay spectra of Mg,N-CDs with and without added AgNPs were measured, and the corresponding fluorescence lifetimes were calculated. As Figure 8 shown, before and after mixing with the quencher, the fluorescence lifetimes of the CDs are 6.66 ns and 6.57 ns respectively, and the fluorescence lifetime hardly decays, indicating that the quenching mechanism between AgNPs and Mg,N-CDs is the inner filter effect.

[0069] 2. Optimization of detection conditions

[0070] The detection conditions of the established colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip based on nanomaterials were optimized by optimizing the concentrations of Mg,N-CDs, SDM aptamer, and NaCl. The gray value of the detection area image under visible light was used as the colorimetric signal and the B-channel value under ultraviolet light was used as the fluorescence signal respectively.

[0071] First, the effect of the concentration of the synthesized Mg,N-CDs on the fluorescence signal intensity of the final detection system was studied. As Figure 9As shown in Figure A, when the concentration of CDs is in the range of 2.5 - 15 mg / mL, the fluorescence detection signal on the three-dimensional paper-based microfluidic chip increases with the increase of concentration. When the concentration is in the range of 15 - 25 mg / mL, the fluorescence intensity hardly changes. Therefore, 15 mg / mL is selected as the optimal concentration of Mg,N-CDs. Then, the concentration of NaCl loaded on the three-dimensional paper-based microfluidic chip is optimized, and the results are as Figure 9 shown in Figure B. When there is no apt in the system, the increase of NaCl concentration leads to an increase in the aggregation degree of AgNPs, resulting in a decrease in the colorimetric signal intensity and an increase in the fluorescence signal intensity. When the NaCl concentration reaches 80 mM, the colorimetric signal and the fluorescence signal reach their peaks and tend to be stable. Therefore, 80 mM is selected as the optimal NaCl concentration. Next, the concentration of the SDM aptamer coated on the surface of AgNPs is optimized. As the concentration of the aptamer increases, the protective effect on AgNPs is enhanced, the effect of NaCl is reduced, the colorimetric signal is enhanced, and the fluorescence signal is weakened. As Figure 9 shown in Figure C, when the aptamer concentration is in the range of 1 - 5 μM, the colorimetric signal on the 3D-μPAD gradually increases and stabilizes at 5 μM, while the fluorescence signal gradually decreases when the aptamer concentration is in the range of 1 - 7.5 μM and stabilizes at 7.5 μM. Therefore, in order to achieve the best detection effect, 7.5 μM is selected as the optimal concentration of the aptamer.

[0072] 3. Detection performance research

[0073] On the basis of optimizing the detection conditions, the detection results were used to collect images of the colorimetric intensity and fluorescence intensity of the upper detection area under natural light and 365 nm ultraviolet light using a smartphone camera, and the RGB values of the images were extracted through the smartphone color-picking APP ColorColl. For the colorimetric detection mode, the detection results are as Figure 10 shown in Figure A. Taking the color change of AgNPs as the detection signal (gray value = R×0.299 + G×0.587 + B×0.114), in order to realize visual quantitative detection based on a smartphone, the RGB values of the final detection result image are selected to be converted into gray values for quantification. The calibration curve for the SDM concentration is made using Δ gray value (Δ gray value = G - G0, where G and G0 represent the gray values of the colorimetric images with or without SDM antibiotics added). When the SDM concentration is in the range of 10 - 1000 ng / mL, there is a good linear relationship between the gray value and the logarithmic concentration value of SDM, and the linear regression equation is Δ gray value = 11.01×logC SDM - 5.23 (R 2 = 0.9955), and the corresponding limit of detection (LOD) is 0.21 ng / mL (3SD / k, n = 10). For the fluorescence detection mode, the detection results are as Figure 10As shown in Figure B, Mg,N-CDs produce fluorescence signals under the excitation of 365 nm ultraviolet light. The ΔB channel value of the fluorescence image (ΔB channel value = B - B0, where B and B0 represent the B channel values of the fluorescence images with or without SDM antibiotic added) is selected to make the standard curve of SDM. When the SDM concentration is in the range of 10 - 1000 ng / mL, the ΔB channel value also has a good linear relationship with the logarithmic concentration value of SDM. The linear regression equation is ΔB channel value = 15.12×logC SDM - 1.04 (R 2 = 0.9908), and the detection limit is 0.13 ng / mL (3SD / k, n = 10).

[0074] To study the specificity of the established three-dimensional paper-based microfluidic chip, antibiotics commonly used in other aquatic foods with a 10-fold SDM concentration were selected as interferents for testing, including sulfonamide antibiotics SMZ and SMS, which are structural analogs of SDM, and other antibiotics such as TC, OTC, CAP, and ENR. As Figure 11 shown, after adding other antibiotics, the intensity changes of the two detection signals, Δ grayscale value and ΔB channel value, are small or almost unchanged compared with the blank sample. However, when adding SDM antibiotic, both the colorimetric and fluorescence signals change significantly, which indicates the good specificity of this method.

[0075] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip, characterized in that It includes an upper layer, a middle layer and a lower layer; The upper layer includes an upper hydrophobic region and an upper hydrophilic region, and the upper hydrophilic region includes a sample injection region and a detection region; The middle layer includes a middle hydrophobic region and a middle hydrophilic region. The middle hydrophilic region includes a first functional region and a second functional region respectively corresponding to the sample injection region and the detection region, and a first hydrophilic channel and a second hydrophilic channel respectively connecting the first functional region and the second functional region; The lower layer includes a lower hydrophobic region and a lower hydrophilic region, and the lower hydrophilic region is communicated with the first functional region and the second functional region respectively through the first hydrophilic channel and the second hydrophilic channel; The first hydrophilic channel and the second hydrophilic channel are not connected to each other and are only used for communicating with the hydrophilic region of the lower layer; The first functional region contains a colorimetric signal reagent, and the surface of the colorimetric signal reagent is coated with a target recognition element; the second functional region contains a fluorescence signal reagent; the lower hydrophilic region contains a neutral salt; The colorimetric signal reagent can quench the fluorescence of the fluorescence signal reagent, and the neutral salt can react with the colorimetric signal reagent and cause a color change.

2. The colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip according to claim 1, wherein The colorimetric signal reagent is silver nanoparticles; The target recognition element is an aptamer; The fluorescence signal reagent is magnesium and nitrogen co-doped carbon dots; The neutral salt is selected from one or more of sodium chloride, magnesium chloride and potassium chloride.

3. The colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip according to claim 2, characterized in that, The content ratio of the silver nanoparticles, aptamer, magnesium and nitrogen co-doped carbon dots and NaCl is 0.3-0.5 mmol: 6-10 mmol: 12-20 mg / mL: 70-100 mmol.

4. The colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip according to claim 2, wherein The magnesium and nitrogen co-doped carbon dots are obtained by heating and reacting a mixture of an organic acid, a magnesium salt and a nitrogen source.

5. The preparation method of the colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip according to any one of claims 1-4, characterized in that, It includes the following steps S1: Provide upper, middle and lower filter papers, perform hydrophobic treatment on the upper hydrophobic region of the upper filter paper, the middle hydrophobic region of the middle filter paper and the lower hydrophobic region of the lower filter paper, and assemble the three filter papers after hydrophobic treatment to obtain a semi-finished product; S2: Drop the colorimetric signal reagent coated with the target recognition element into the first functional region of the semi-finished product, drop the fluorescence signal reagent into the second functional region; drop the neutral salt into the lower hydrophilic region to obtain the colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip.

6. The preparation method according to claim 5, characterized in that, In step S1, carbon powder is printed on the surface of the filter paper and then heated and melted to obtain the filter paper after hydrophobic treatment.

7. Application of the colorimetric-fluorescent dual-mode three-dimensional paper-based microfluidic chip in visual detection of antibiotics according to claim 1, characterized in that, The target recognition element is an antibiotic aptamer.

8. The application according to claim 7, wherein The antibiotic is sulfadimethoxine, and the target recognition element is a sulfadimethoxine aptamer.

9. The application according to claim 7, wherein It includes the following steps: Drop a series of concentration antibiotic standard solutions into the sample injection region of the three-dimensional paper-based microfluidic chip. After sufficient reaction, collect and quantitatively detect the colorimetric intensity and fluorescence intensity in the detection region, and draw the standard curves of the colorimetric intensity vs. antibiotic concentration and the fluorescence intensity vs. antibiotic concentration; Drop the test solution into the sample injection region of the blank three-dimensional paper-based microfluidic chip. After sufficient reaction, collect the colorimetric intensity and fluorescence intensity in the detection region, and obtain the concentration of the antibiotic in the test solution according to the standard curve.

10. The application according to claim 9, wherein The method for collecting and quantifying the colorimetric intensity is as follows: Collect the image of the detection area under daylight conditions, extract the RGB values of the image, and convert them into grayscale values for quantification; The method for collecting and quantifying the fluorescence intensity is as follows: Collect the image of the detection area under ultraviolet light conditions, extract the RGB values of the image, and quantify with the B-channel value.

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