A paper-based microfluidic chip for detecting organophosphorus pesticide residues and an auxiliary analysis device thereof

Abstract

The application relates to the field of detection chips, and discloses a paper-based microfluidic chip for detecting organic phosphorus pesticide residues and an auxiliary analysis device thereof. The paper-based microfluidic chip comprises a sample titration layer, a sample detection layer and a bottom plate. The sample titration layer comprises a reaction zone, and the reaction zone is fixed with fluorescent dye-labeled nucleic acid aptamers adsorbed on the surface of graphene oxide through pi-pi stacking interaction. The interaction can cause energy transfer, thereby triggering fluorescence quenching. After the addition of a to-be-detected organic phosphorus pesticide target molecule, specific binding occurs between the nucleic acid aptamers and the organic phosphorus pesticide target molecule, so that the nucleic acid aptamers are released from the surface of graphene oxide. By detecting the change of the fluorescence signal, the concentration of the organic phosphorus pesticide target molecule can be detected. The application is based on the principle of graphene oxide fluorescence quenching, greatly reduces the interference of the fluorescence signal of graphene oxide itself, and improves the detection sensitivity and accuracy.

Description

Technical Field

[0001] This invention relates to the field of detection chip technology, specifically to a paper-based microfluidic chip for detecting organophosphorus pesticide residues and its auxiliary analysis device. Background Technology

[0002] Organophosphorus pesticides are a class of pesticides widely used in agricultural production, possessing insecticidal and fungicidal effects, effectively controlling crop pests and diseases, and increasing crop yields. However, due to the use and improper application of organophosphorus pesticides during crop growth, the problem of organophosphorus pesticide residues in agricultural products is becoming increasingly serious. Therefore, the detection of organophosphorus pesticide residues in agricultural products has become an important task in the current supervision of agricultural product quality and safety. Currently, commonly used methods for detecting organophosphorus pesticide residues include gas chromatography, liquid chromatography, and mass spectrometry. These methods can rapidly and accurately detect organophosphorus pesticide residues in agricultural products, but they also have some limitations, such as expensive instruments and the need for complex sample pretreatment.

[0003] Fluorescent biosensors are optical methods that utilize fluorescence signals to perform quantitative or qualitative analysis of target molecules. They offer advantages such as high sensitivity, good selectivity, rapid response, and ease of operation, making them an important tool in the field of bioanalysis. Currently, fluorescent biosensors are widely used to detect organophosphorus pesticide residues in agricultural products, pollutants in the environment, and additives and harmful substances in food, playing a vital role in ensuring public safety and environmental health. However, the fluorescence signal of fluorescent biosensors is susceptible to background interference: in complex sample matrices, the fluorescence signal may be affected by background interference, reducing the accuracy and sensitivity of detection. Furthermore, when using fluorescent biosensors for detection, the target molecule undergoes a specific biological reaction with the biosensor, a process that requires a certain incubation time to ensure a complete reaction. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a paper-based microfluidic chip for detecting organophosphorus pesticide residues and its auxiliary analysis device, which solves the problem that the fluorescence signal is easily affected by background interference when using traditional fluorescent biosensors to detect organophosphorus pesticide residues.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a paper-based microfluidic chip for detecting organophosphorus pesticide residues, comprising:

[0006] The sample titration layer includes a titration zone and a reaction zone, which are connected by a first hydrophilic channel. The reaction zone is immobilized with a fluorescent dye-labeled nucleic acid aptamer adsorbed on the surface of graphene oxide through π-π stacking.

[0007] The sample detection layer includes a separation zone and a detection zone, which are connected by a second hydrophilic channel. The separation zone is located below the reaction zone, and the pore size of the sample detection layer is smaller than the particle size of graphene particles.

[0008] The base plate is used to fix the sample titration layer and the sample detection layer into a whole.

[0009] Preferably, the sample titration layer is made of absorbent filter paper.

[0010] Preferably, the sample detection layer is made of nitrocellulose membrane.

[0011] Preferably, the sample titration layer has a first hydrophobic region, forming a structural pattern of a titration region, a first hydrophilic channel, and a reaction region.

[0012] Preferably, the sample detection layer is provided with a second hydrophobic region, a forming separation region, a second hydrophilic channel, and a detection region.

[0013] This invention provides a method for preparing the paper-based microfluidic chip for detecting organophosphorus pesticide residues, comprising the following steps:

[0014] S1. Mix the nucleic acid aptamer solution with the graphene oxide solution and incubate with shaking at room temperature to obtain a "closed" fluorescent probe;

[0015] S2. Using a laser printer, print the drawn structural pattern onto the filter paper of the sample titration layer and sample detection layer;

[0016] S3. Place the printed filter paper in an oven for heat curing.

[0017] S4. Add and dry the fluorescent probe in the reaction zone, repeating the process multiple times to complete the fabrication of the paper-based microfluidic chip.

[0018] This invention provides an auxiliary analytical device for the paper-based microfluidic chip used in detecting organophosphorus pesticide residues, comprising:

[0019] The upper housing is used to house the detection device with a camera and has an opening that matches the position of the camera;

[0020] The lower housing, together with the upper housing, forms the outer shell of the device;

[0021] A fluorescence detection unit is disposed within the lower housing. The fluorescence detection unit can accommodate the paper-based microfluidic chip to be detected and provide an excitation light source to excite the fluorescent markers in the paper-based microfluidic chip, and can be captured by the camera of the detection device.

[0022] The power interface unit is used to connect to a power source and supply power to the auxiliary analysis device.

[0023] Preferably, the device further includes an adjustment mechanism for supporting and fixing the fluorescence detection unit, which compensates for the positional deviation of cameras in different types of detection devices through two-dimensional adjustment.

[0024] Preferably, the fluorescence detection unit includes:

[0025] The base is used to connect and fix the fluorescence detection unit;

[0026] The outer casing is fixedly connected to the base. A detection hole is provided on the top of the outer casing, and an LED light source is provided on the bottom side of the outer casing. The detection hole and the opening on the upper casing are both located on the LED light source rays.

[0027] A chip holder is inserted into one side of the housing. A square groove for placing a paper-based microfluidic chip is provided on one side of the chip holder. A round hole is provided on one side of the square groove. The round hole coincides with the detection area of ​​the paper-based microfluidic chip and is located on the LED light source beam.

[0028] Preferably, the fluorescence detection unit further includes a first bandpass filter module for limiting the bandwidth of the LED light source and reducing the interference of excitation light on the measured fluorescence signal. The first bandpass filter module includes a first filter and a first fixing bracket. The first filter is fixedly connected to one side of the first fixing bracket. The first fixing bracket is inserted into one side of the housing and disposed between the chip bracket and the LED light source. The first filter is disposed on the LED light source beam.

[0029] Preferably, the fluorescence detection unit further includes a second bandpass filter module for further reducing the influence of excitation light and filtering out interference from other stray light. The second bandpass filter module includes a second filter and a second fixing bracket. The second filter is fixedly connected to one side of the second fixing bracket. The second fixing bracket is inserted into one side of the housing and disposed between the chip holder and the detection hole. The second filter is disposed on the LED light source beam.

[0030] This invention provides a paper-based microfluidic chip for detecting organophosphorus pesticide residues and its auxiliary analytical device. It has the following beneficial effects:

[0031] 1. Based on the principle of fluorescence quenching of graphene oxide, this invention designs a three-layer paper-based microfluidic chip. Compared with traditional fluorescence biosensing methods based on graphene oxide, this microfluidic chip uses a sample separation region to separate graphene oxide, while the sample solution to be tested flows to the detection region, which greatly reduces the interference of the fluorescence signal of graphene oxide itself and improves the detection sensitivity and accuracy.

[0032] 2. The paper-based microfluidic chip-assisted analysis device of the present invention adopts a combined filter module, which can effectively avoid interference from excitation light and other stray light; at the same time, the device is simple, portable, easy to operate, and can be matched with different models of smartphones, making it suitable for non-professionals and different occasions, and can realize rapid on-site detection of organophosphorus pesticide residues. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the paper-based microfluidic chip structure of the present invention;

[0034] Figure 2 This is a schematic diagram of the explosion of the paper-based microfluidic chip of the present invention;

[0035] Figure 3 This is a schematic diagram of the auxiliary analysis device of the present invention;

[0036] Figure 4 This is a schematic diagram of the fluorescence detection unit structure of the present invention;

[0037] Figure 5 This is a schematic diagram of the structure of the first bandpass filter module and the second bandpass filter module of the present invention;

[0038] Figure 6 This is a schematic diagram of the chip support structure of the present invention;

[0039] Figure 7 This is a schematic diagram of the adjustment mechanism structure of the present invention;

[0040] Figure 8 This is a schematic diagram of fluorescence images of five samples with different concentrations in Example 1 of the present invention;

[0041] Figure 9 The fluorescence spectra of five samples with different concentrations in Example 1 of the present invention;

[0042] Figure 10 This is a schematic diagram of Embodiment 2 of the present invention, where a is a fluorescence image obtained by a conventional fluorescence biosensing method based on graphene oxide, b is an image of the paper-based microfluidic chip of the present application without a sample, and c is a fluorescence image of the paper-based microfluidic chip of the present application after a sample is added.

[0043] Figure 11This is a schematic diagram of Embodiment 3 of the present invention, where a is a fluorescence image with the filter removed and b is a fluorescence image with the filter added.

[0044] Among them, 11. Sample titration layer; 111. Titration area; 112. First hydrophilic channel; 113. Reaction area; 114. First hydrophobic area; 12. Sample detection layer; 121. Separation area; 122. Second hydrophilic channel; 123. Detection area; 124. Second hydrophobic area; 13. Base plate; 31. Upper shell; 32. Lower shell; 33. Fluorescence detection unit; 331. Outer shell; 332. Base; 333. LED light source; 334. First bandpass filter module; 3341, first filter; 3342, first fixing bracket; 335, chip bracket; 3351, square slot; 3352, round hole; 336, second bandpass filter module; 3361, second filter; 3362, second fixing bracket; 34, adjustment mechanism; 341, horizontal adjustment knob; 342, vertical adjustment knob; 343, fixing panel; 3431, screw hole; 35, power interface unit. Detailed Implementation

[0045] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Please see the appendix Figure 1 - Appendix Figure 2 This invention provides a paper-based microfluidic chip for detecting organophosphorus pesticide residues, comprising a sample titration layer 11, a sample detection layer 12, and a base plate 13. The sample titration layer 11 and the sample detection layer 12 partially overlap, and the base plate 13 bonds the sample titration layer 11 and the sample detection layer 12 together to form a whole.

[0047] Specifically, the sample titration layer 11, the sample detection layer 12, and the base plate 13 are composed as follows:

[0048] The sample titration layer 11 includes a titration region 111, a first hydrophilic channel 112, a reaction region 113, and a first hydrophobic region 114. The titration region 111 and the reaction region 113 are connected through the first hydrophilic channel 112, allowing liquid to flow from the titration region 111 to the reaction region 113. The reaction region 113 is immobilized with a fluorescent dye-labeled nucleic acid aptamer adsorbed on the surface of graphene oxide through π-π stacking. The first hydrophobic region 114 forms the structural pattern of the titration region 111, the first hydrophilic channel 112, and the reaction region 113.

[0049] The sample detection layer 12 includes a separation region 121, a second hydrophilic channel 122, a detection region 123, and a second hydrophobic region 124. The separation region 121 and the detection region 123 are connected through the second hydrophilic channel 122, allowing liquid to flow from the separation region 121 to the detection region 123. The separation region 121 is located below the reaction region 113, which is the overlapping part of the sample titration layer 11 and the sample detection layer 12. The second hydrophobic region 124 forms the structural pattern of the separation region 121, the second hydrophilic channel 122, and the detection region 123.

[0050] The base plate 13 is used to fix the sample titration layer 11 and the sample detection layer 12 into a whole, forming a complete paper-based microfluidic chip.

[0051] Among them, the sample titration layer 11 is made of absorbent filter paper with a large pore size, such as an absorbent pad; the sample detection layer 12 is made of nitrocellulose membrane with a small pore size (5 < micrometers); and the base plate 13 is made of polyvinyl chloride material.

[0052] The main principle of this fluorescence sensing invention lies in the following: Fluorescent dye-labeled nucleic acid aptamers adsorb onto the surface of graphene oxide through π-π stacking interactions. This interaction leads to energy transfer, thereby triggering fluorescence quenching. However, when the target molecule of the organophosphorus pesticide is added, a specific binding occurs between the nucleic acid aptamer and the target molecule, causing the nucleic acid aptamer to be released from the graphene oxide surface, thus restoring fluorescence. By detecting changes in the fluorescence signal, the concentration of the organophosphorus pesticide target molecule can be detected.

[0053] In one embodiment of the present invention, when using the paper microfluidic chip, the sample solution of the organophosphorus pesticide residue to be tested is dropped into the sample titration area 111. Under capillary action, the sample solution flows through the hydrophilic channel to the sample reaction area 113. In the sample reaction area 113, the organophosphorus pesticide molecules in the sample specifically bind to the nucleic acid aptamers, thereby releasing the dye-modified nucleic acid aptamers from the surface of graphene oxide. The sample reaction process requires incubation for 5 minutes. After the sample reaction is complete, it permeates into the lower separation area 121. Because the graphene particle size is larger than the pore size of the sample detection layer 12, the graphene oxide is fixed in the separation area 121, while the dye-modified nucleic acid aptamers released from the surface of graphene oxide flow with the sample solution through the hydrophilic channel to the sample detection area 123. The intensity of the fluorescent signal of the fluorescent dye in the sample detection area 123 depends on the concentration of the aptamers released from the graphene oxide, and the aptamer concentration depends on the concentration of the organophosphorus pesticide target molecules. Therefore, the concentration of the target molecule of the organophosphorus pesticide can be determined by the change in fluorescence signal in the sample detection area 123.

[0054] In the paper microfluidic chip, the separation region 121 serves to separate the graphene oxide, while the sample solution flows to the detection region 123. Since graphene oxide can also emit fluorescence signals under the excitation light source, the fluorescence signal in the detection region 123 is only the fluorescence signal of the sample, avoiding interference from the fluorescence signal of graphene oxide.

[0055] In one embodiment of the present invention, the microfluidic chip has a size of 19*5mm and a thickness of 1mm. The sample titration layer 11 has a size of 10.8*5mm and a thickness of 0.25mm; the sample detection layer 12 has a size of 10*5mm and a thickness of 0.25mm; and the base plate 13 has a size of 19*5mm and a thickness of 0.5mm.

[0056] As one embodiment of the present invention, the method for preparing the paper-based microfluidic chip includes the following steps:

[0057] S1: Mix 10 μL of 1M nucleic acid aptamer solution with 10 μL of 2 mg / ml graphene oxide solution, and incubate with shaking at room temperature for 2 minutes to obtain a "closed" fluorescent probe.

[0058] S2: Place the sample titration layer absorbent filter paper (such as glass fiber filter paper) and the sample detection layer absorbent filter paper (such as nitrocellulose membrane) into the laser printer respectively, ensuring that the paper is in the correct position. Then, print the pattern designed in step 2 onto the surface of the sample titration layer and the sample detection layer absorbent filter paper.

[0059] S3: Place the printed sample titration layer and sample detection layer absorbent filter paper into a drying oven and heat cycle at 200℃ for 60 minutes to form a hydrophobic barrier on the surface of the test paper.

[0060] S4: Cut the sample titration layer absorbent filter paper, the sample detection layer absorbent filter paper, and the base plate into sizes of 10.8*5mm, 10*5mm, and 19*5mm respectively, and assemble them into microfluidic test paper.

[0061] S5: Add 5 μL of fluorescent probe to the reaction area of ​​the microfluidic test paper, then dry it at room temperature. Repeat this process 4 times to complete the fabrication of the paper-based microfluidic chip.

[0062] Please see the appendix Figure 3 - Appendix Figure 7 To further facilitate the use of paper-based microfluidic chips, the present invention also provides an auxiliary analysis device for paper-based microfluidic chips, including an upper housing 31, a lower housing 32, a fluorescence detection unit 33, an adjustment mechanism 34, and a power interface unit 35.

[0063] Specifically, the upper housing 31, lower housing 32, fluorescence detection unit 33, adjustment mechanism 34, and power interface unit 35 are composed as follows:

[0064] The upper housing 31 is used to house a detection device with a camera and has an opening that matches the position of the camera to capture fluorescence images; the detection device can be a smartphone or other device with camera function;

[0065] The lower housing 32, together with the upper housing 31, forms the outer shell 331 of the device;

[0066] The fluorescence detection unit 33 is disposed in the lower housing 32. The fluorescence detection unit 33 can accommodate the paper-based microfluidic chip to be detected and provide an excitation light source to excite the fluorescent marker in the paper-based microfluidic chip. It can also be captured by the camera of the detection device. In this way, the fluorescence signal can be recorded and analyzed by the detection device.

[0067] Adjustment mechanism 34 is used to support and fix the fluorescence detection unit 33, and to compensate for the position deviation of the camera of different types of detection devices through two-dimensional adjustment.

[0068] The power interface unit 35 is used to connect a power supply and provide power to the auxiliary analysis device.

[0069] This auxiliary analysis device allows users to place the paper-based microfluidic chip to be tested in the fluorescence detection unit 33 and capture the fluorescence signal using a camera on a detection device (such as a smartphone). The adjustment mechanism 34 helps users adjust the chip's position and angle to achieve optimal fluorescence signal detection. The power interface unit 35 provides the necessary power supply.

[0070] In one embodiment of the present invention, the fluorescence detection unit 33 includes:

[0071] Base 332, used to connect and secure other components of fluorescence detection unit 33;

[0072] The housing 331 is fixedly connected to the base 332 and has a detection hole on the top and an LED light source 333 on the bottom. The detection hole and the opening on the upper housing 31 are both located on the beam of the LED light source 333 to ensure that the light source can illuminate the detection area of ​​the paper-based microfluidic chip; in one embodiment, the center wavelength of the LED light source 333 is 525nm and the bandwidth is 100nm.

[0073] A chip holder 335 is inserted into one side of the housing 331 and is used to hold a paper-based microfluidic chip. A square groove 3351 is provided on one side of the chip holder 335 to accommodate the paper-based microfluidic chip. A circular hole 3352 is provided on one side of the square groove 3351. This circular hole 3352 coincides with the detection area of ​​the paper-based microfluidic chip and is positioned on the beam of the LED light source 333 to ensure that the light source can illuminate the detection area of ​​the paper-based microfluidic chip.

[0074] With this design, the base 332 and housing 331 of the fluorescence detection unit 33 provide fixation and protection, while the chip holder 335 is used to place the paper-based microfluidic chip and align it with the detection aperture. The light source is located on the bottom side of the housing; illuminated by the LED light source 333, the fluorescent marker emits a fluorescent signal in the detection area of ​​the paper-based microfluidic chip. The positions of the detection aperture and the openings on the upper housing 31 are aligned with the rays of the LED light source 333, ensuring that the fluorescence signal can be captured and detected.

[0075] This fluorescence detection unit 33 is designed to easily accommodate a paper-based microfluidic chip and provides a light source and detection aperture to excite and capture fluorescence signals. In conjunction with other components, this auxiliary analytical device enables convenient detection of organophosphorus pesticide residues.

[0076] As one embodiment of the present invention, the fluorescence detection unit 33 may further include:

[0077] A first bandpass filter module 334 is used to limit the bandwidth of the LED light source 333 and reduce the interference of excitation light on the measured fluorescence signal. This module includes a first filter 3341 and a first fixing bracket 3342. The first filter 3341 is fixedly connected to one side of the first fixing bracket 3342, which is inserted into one side of the housing 331, located between the chip holder 335 and the LED light source 333. The first filter 3341 is positioned on the LED light source 333 to ensure that only excitation light of a specific wavelength can pass through, reducing interference light of other wavelengths. In one embodiment, the first filter 3341 has a center wavelength of 525nm, a bandwidth of 20nm, a size of 10*10mm, and a thickness of 1mm.

[0078] The second bandpass filter module 336 is used to further reduce the influence of the excitation light and filter out interference from other stray light. This module includes a second filter 3361 and a second mounting bracket 3362. The second filter 3361 is fixedly connected to one side of the second mounting bracket 3362, which is inserted into one side of the housing 331, located between the chip holder 335 and the detection aperture. The second filter 3361 is disposed on the LED light source 333 to further filter out the excitation light and other stray light, ensuring that only the fluorescence signal can pass through. In one embodiment, the second filter 3361 is a high-pass filter with a range of 550-1100nm, a size of 10*10mm, and a thickness of 1mm.

[0079] Specifically, through this design, a first bandpass filter module 334 and a second bandpass filter module 336 are incorporated into the fluorescence detection unit 33 to limit and filter out interference from excitation light and other stray light. This helps to improve the detection sensitivity and accuracy of the fluorescence signal.

[0080] In one embodiment of the present invention, the adjustment mechanism 34 includes a horizontal adjustment knob 341, a vertical adjustment knob 342, and a fixing panel 343. The function of the adjustment mechanism 34 is to compensate for the positional deviations of cameras in different types of smartphones. The horizontal adjustment knob 341 compensates for horizontal deviations, and the vertical adjustment knob 342 compensates for vertical deviations. The fixing panel 343 is used to fix the fluorescence detection unit 33, which is fixed by screw holes 3431 on the fixing panel 343.

[0081] Example 1:

[0082] This invention takes the detection of the organophosphorus pesticide dimethoate as an example. Five different concentrations of dimethoate solutions were prepared: 50 nM, 150 nM, 300 nM, 400 nM, and 600 nM. The preparation process is as follows: 20 μL of dimethoate standard solution (1 mg / ml) was added to 9360 μL of ultrapure water to obtain a 10 μM dimethoate solution; 1, 3, 6, 8, and 12 μL of the 10 μM dimethoate solution were then added to ultrapure water to a final volume of 200 μL to obtain 50 nM, 150 nM, 300 nM, 400 nM, and 600 nM dimethoate solutions to be tested.

[0083] Fluorescence detection is performed using the auxiliary analysis device of this invention. The auxiliary analysis device includes a fluorescence detection unit, a smartphone, and a power interface unit. The fluorescence detection unit consists of an upper housing, a lower housing, the fluorescence detection unit itself, an adjustment base for the fluorescence detection unit, and the power interface unit. The upper housing is used to hold the smartphone and has a built-in circular hole to match the camera position for capturing fluorescence images. The power interface unit can be powered by a power bank with a step-down module (3.3V) or directly by a power adapter.

[0084] Image acquisition was performed using a smartphone in professional mode. The aspect ratio was set to 1:1, the pixel count to 2992*2992, the ISO to 500, the exposure time to 1 second, the focal length to 0.5, and the aperture to F1.8. Fluorescent images of the organophosphorus pesticide dimethoate at different concentrations (50nM, 150nM, 300nM, 400nM, and 600nM) were captured.

[0085] In addition, a fluorescence spectroscopy measurement device was used to acquire fluorescence spectra. This device consists of a fiber optic spectrometer, a diode laser with a center wavelength of 532 nm, a long-pass filter (550-1100 nm), a sample holder, and three 600 μm diameter optical fibers. The spectrometer's wavelength range is 360-1200 nm. Fluorescence spectra of five different concentrations of dimethoate pesticide were acquired using this device, and are shown in Image 9.

[0086] from Figure 8 It can be observed that the fluorescence intensity gradually increases with the increase of omethoate concentration. Meanwhile, from... Figure 9 The fluorescence spectrum shows that the fluorescence intensity gradually increases near 565 nm, which is basically consistent with the fluorescence image results of the device of the present invention.

[0087] These steps and results demonstrate that the apparatus and method of the present invention can conveniently perform fluorescence detection of organophosphorus pesticide residues, and the detection results are consistent with the fluorescence spectrum.

[0088] Example 2:

[0089] When using traditional graphene oxide-based fluorescence biosensing methods for detection, adding 150 nM of the organophosphorus pesticide dimethoate to the fluorescent probe yields a fluorescence image as shown below. Figure 10 As shown in .a, the fluorescence of graphene oxide itself is the main feature at this time, which prevents the fluorescence of the dye modified on the aptamer from being displayed.

[0090] When using the paper-based microfluidic chip of this invention, such as Figure 10 As shown in Figure .b, without the addition of the organophosphorus pesticide dimethoate, no fluorescent signal was observed in the detection area, indicating that the graphene oxide did not flow into the detection area and was entirely fixed in the separation area of ​​the paper-based microfluidic chip. Figure 10 As shown in .c, after adding 150 nM of the organophosphorus pesticide dimethoate to the sample titration region of the paper-based microfluidic chip, a fluorescent signal of the dye modified on the aptamer can be obtained in the detection region, such as... Figure 10 As shown in .c.

[0091] The results show that, compared with traditional fluorescence biosensing methods based on graphene oxide, this microfluidic chip separates graphene oxide in a sample separation region while the sample solution to be tested flows to the detection region, which greatly reduces the interference of the fluorescence signal of graphene oxide itself and improves the detection sensitivity and accuracy.

[0092] Example 3:

[0093] When 600 nM dimethoate is added to the sample titration zone of the paper-based microfluidic sheet in this application, and then detected using the auxiliary analysis device of this application, if filter 3361 is removed, a brighter green signal is displayed in the acquired image, such as... Figure 11 As shown in .a, this part mainly excites the light source's own signal, from which the fluorescent dye signal modified on the aptamer cannot be distinguished; when filter 3361 is used, as... Figure 11 As shown in Figure .b, the acquired images mainly contain signals from fluorescent dyes modified on the aptamers, indicating that the use of filters can effectively avoid interference from excitation light and other stray light, and can quickly detect the fluorescent signals of the labeled dyes.

[0094] The results show that the device of the present invention uses a combined filter module, which can effectively avoid interference from excitation light and other stray light.

[0095] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A paper-based microfluidic chip for detecting organophosphorus pesticide residues, characterized in that, include: The sample titration layer includes a titration zone and a reaction zone, which are connected by a first hydrophilic channel. The reaction zone is immobilized with a fluorescent dye-labeled nucleic acid aptamer adsorbed on the surface of graphene oxide through π-π stacking. The sample detection layer includes a separation zone and a detection zone, which are connected by a second hydrophilic channel. The separation zone is located below the reaction zone, and the pore size of the sample detection layer is smaller than the particle size of graphene particles. The base plate is used to fix the sample titration layer and the sample detection layer into a whole.

2. The paper-based microfluidic chip for detecting organophosphorus pesticide residues according to claim 1, characterized in that, The sample titration layer was prepared using absorbent filter paper.

3. The paper-based microfluidic chip for detecting organophosphorus pesticide residues according to claim 1, characterized in that, The sample detection layer uses a nitrocellulose membrane.

4. A paper-based microfluidic chip for detecting organophosphorus pesticide residues according to claim 1, characterized in that, The sample titration layer has a first hydrophobic region, forming a structural pattern of a titration region, a first hydrophilic channel, and a reaction region; the sample detection layer has a second hydrophobic region, forming a structural pattern of a separation region, a second hydrophilic channel, and a detection region.

5. A method for preparing a paper-based microfluidic chip for detecting organophosphorus pesticide residues according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Mix the nucleic acid aptamer solution with the graphene oxide solution and incubate with shaking at room temperature to obtain a "closed" fluorescent probe; S2. Using a laser printer, print the drawn structural pattern onto the filter paper of the sample titration layer and sample detection layer; S3. Place the printed filter paper in an oven for heat curing. S4. Add and dry the fluorescent probe in the reaction zone, repeating the process multiple times to complete the fabrication of the paper-based microfluidic chip.

6. An auxiliary analytical device for detecting organophosphorus pesticide residues using a paper-based microfluidic chip, based on the paper-based microfluidic chip for detecting organophosphorus pesticide residues according to any one of claims 1-4, characterized in that, include: The upper housing is used to house the detection device with a camera and has an opening that matches the position of the camera; The lower housing, together with the upper housing, forms the outer shell of the device; A fluorescence detection unit is disposed within the lower housing. The fluorescence detection unit can accommodate the paper-based microfluidic chip to be detected and provide an excitation light source to excite the fluorescent markers in the paper-based microfluidic chip, and can be captured by the camera of the detection device. The power interface unit is used to connect to a power source and supply power to the auxiliary analysis device.

7. The auxiliary analytical device for detecting organophosphorus pesticide residues using a paper-based microfluidic chip according to claim 6, characterized in that, It also includes an adjustment mechanism for supporting and fixing the fluorescence detection unit, which compensates for the positional deviation of cameras in different types of detection devices through two-dimensional adjustment.

8. The auxiliary analytical device for detecting organophosphorus pesticide residues using a paper-based microfluidic chip according to claim 6, characterized in that, The fluorescence detection unit includes: The base is used to connect and fix the fluorescence detection unit; The outer casing is fixedly connected to the base. A detection hole is provided on the top of the outer casing, and an LED light source is provided on the bottom side of the outer casing. The detection hole and the opening on the upper casing are both located on the LED light source rays. A chip holder is inserted into one side of the housing. A square groove for placing a paper-based microfluidic chip is provided on one side of the chip holder. A round hole is provided on one side of the square groove. The round hole coincides with the detection area of ​​the paper-based microfluidic chip and is located on the LED light source beam.

9. The auxiliary analytical device for detecting organophosphorus pesticide residues using a paper-based microfluidic chip according to claim 8, characterized in that, The fluorescence detection unit further includes a first bandpass filter module for limiting the bandwidth of the LED light source and reducing the interference of excitation light on the measured fluorescence signal. The first bandpass filter module includes a first filter and a first fixing bracket. The first filter is fixedly connected to one side of the first fixing bracket. The first fixing bracket is inserted into one side of the housing and disposed between the chip bracket and the LED light source. The first filter is disposed on the LED light source beam.

10. The auxiliary analytical device for detecting organophosphorus pesticide residues using a paper-based microfluidic chip according to claim 8, characterized in that, The fluorescence detection unit also includes a second bandpass filter module, which is used to further reduce the influence of excitation light and filter out interference from other stray light. The second bandpass filter module includes a second filter and a second fixing bracket. The second filter is fixedly connected to one side of the second fixing bracket. The second fixing bracket is inserted into one side of the housing and is disposed between the chip holder and the detection hole. The second filter is disposed on the LED light source beam.

Images