Pesticide residue multi-joint detection test paper based on wax line barrier separation technology

Abstract

The invention relates to pesticide residue multi-joint detection test paper based on a wax line barrier separation technology. The pesticide residue multi-joint detection test paper comprises a bottom plate as well as a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are arranged on the bottom plate, the conjugate pad is partially lapped at one end of the nitrocellulose membrane; the sample pad is partially overlapped at one end, far away from the nitrocellulose membrane, of the combination pad; the water absorption pad is partially lapped at the other end of the nitrocellulose membrane; a plurality of wax line barriers extending along the chromatography direction, and a detection line and a quality control line which are vertical to the chromatography direction are immersed in the nitrocellulose membrane; the wax line barrier is immersed into the surface, close to the bottom plate, of the nitrocellulose membrane, and the wax line barrier at least extends to pass through the detection line. The device has the characteristics of being ingenious in structural design, suitable for batch production and preparation, high in detection accuracy, capable of detecting various pesticide residues at the same time and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pesticide detection, and in particular to a pesticide residue multi-association detection test paper based on wax line barrier separation technology. BACKGROUND

[0002] The widespread use of pesticides in agricultural production has significantly improved crop yields and effectively alleviated the problem of food supply caused by global population growth. However, the large-scale use of pesticides has also led to environmental and food pollution. It is estimated that about 90% of pesticide residues enter the human body through the food chain, which can potentially harm health. Although pesticide residues in crops only cause acute poisoning in a few cases, chronic poisoning caused by long-term low-dose exposure is more common, and this cumulative harm gradually damages human health. Therefore, in order to protect public health and environmental safety, it is necessary to strengthen the systematic detection and monitoring of multiple pesticide residues in the entire food supply chain system from crop planting to sales and consumption.

[0003] Currently, pesticide residue detection can be performed using various methods. Instrumental analysis techniques, such as QuEChERS combined with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), gas chromatography-microelectronic capture detector (GC-μECD), and gas chromatography-tandem mass spectrometry (GC-MS / MS), have the advantages of high accuracy and high sensitivity, but they rely on expensive equipment and professional operators, which is costly and difficult to apply to large-scale screening in the field. Therefore, developing a rapid and efficient pesticide residue detection method has become a research hotspot. Immunoassay is widely used in medical, biological, and harmful substance identification fields, mainly including enzyme-linked immunosorbent assay (ELISA) and traditional colloidal gold immunochromatography technology. However, these methods can usually only detect a single target, and there are problems of insufficient efficiency and high cost when facing large-scale on-site screening of multiple pesticide residues.

[0004] Therefore, there is an urgent need to develop a sensitive, simple, rapid, and simultaneous detection of multiple pesticide residues immunochromatography solution. SUMMARY

[0005] The present application aims to solve the problems of the prior art and provide a pesticide residue multi-association detection test paper based on wax line barrier separation technology.

[0006] In a first aspect, the present application provides a pesticide residue multi-association detection test paper based on wax line barrier separation technology, comprising a bottom plate and a sample pad, a conjugate pad, a nitrocellulose membrane, and a water absorption pad arranged on the bottom plate. The conjugate pad is partially overlapped with one end of the nitrocellulose membrane; the sample pad is partially overlapped with the end of the conjugate pad away from the nitrocellulose membrane; and the water absorption pad is partially overlapped with the other end of the nitrocellulose membrane. The nitrocellulose membrane is immersed with a plurality of wax line barriers extending in the chromatographic direction and detection lines and quality control lines arranged perpendicularly to the chromatographic direction; the wax line barriers are immersed to the surface of the nitrocellulose membrane close to the base plate, and the wax line barriers extend at least through the detection lines.

[0007] Further specifically, the wax line barriers extend from the joint of the nitrocellulose membrane and the binding pad to the joint of the nitrocellulose membrane and the water absorption pad.

[0008] Further specifically, the detection lines are arranged on the side of the nitrocellulose membrane close to the water absorption pad, the detection lines are parallel to the quality control lines, and the quality control lines are arranged on the side of the detection lines close to the water absorption pad.

[0009] Further specifically, the distance between two adjacent wax line barriers is 2 mm.

[0010] Further specifically, the joint length of the binding pad and the nitrocellulose membrane is 2-5 mm.

[0011] In a second aspect, the application further provides a pesticide residue detection kit comprising the test paper.

[0012] Further specifically, the kit comprises a kit lower shell provided with a sample adding window and a kit upper shell matched with the kit lower shell and provided with an observation window; the test paper is arranged between the kit lower shell and the kit upper shell, the sample pad is partially exposed on the sample adding window, and the detection lines and the quality control lines are exposed in the sample observation window.

[0013] Further specifically, the kit comprises a shell provided with an exposure hole, the test paper is arranged in the shell, and the sample pad passes through the exposure hole and is partially exposed outside the shell.

[0014] In a third aspect, the application provides a preparation method for preparing the test paper, comprising the following steps: S1, mixing and reacting gold nanoparticles and pesticide antibodies to obtain gold nanoparticle-labeled pesticide residue antibodies; S2, fixing the gold nanoparticle-labeled pesticide residue antibodies on the binding pad; S3, wax printing a plurality of wax line barriers on the NC membrane, and printing detection lines and quality control lines in adjacent wax line barriers; S4, assembling the sample pad, the binding pad, the nitrocellulose membrane, and the water absorption pad according to the structure of the test paper.

[0015] Further specifically, in S3, the wax printing specifically comprises: spraying wax printing ink in the form of droplets on the surface of the NC membrane in the chromatographic direction, heating the NC membrane to melt and penetrate the wax printing ink into the NC membrane, and cooling to obtain the NC membrane with a plurality of wax line barriers.

[0016] Further specifically, in S3, the adjacent detection lines are printed with coated different kinds of pesticide antigens.

[0017] Further specifically, the conjugate pad is modified by a conjugate pad treatment liquid, and the sample pad is modified by a sample pad treatment liquid.

[0018] Advantages of the present application: By setting the wax line barrier extending along the chromatographic direction in the NC membrane, the physical separation of the independent detection channels is realized by using the hydrophobic properties of wax, effectively avoiding sample penetration and signal interference between multiple channels, significantly improving the specificity and accuracy of multi-association detection; the wax line barrier is prepared by wax printing + heating penetration process, which is simple to operate, low in cost, can realize large-scale production, and solves the problem of complex traditional physical cutting process and easy damage to the membrane structure; the detection lines on both sides of the wax line barrier can be coated with different kinds of pesticide antigens, combined with gold nano-labeled specific antibodies, and can simultaneously detect multiple pesticide residues, which is high in detection efficiency, simple in operation, and suitable for on-site rapid screening; the sample pad and the conjugate pad are treated by special treatment liquid, combined with BSA blocking technology, which reduces non-specific adsorption and further improves the reliability of the detection results. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is the schematic diagram of the pesticide residue detection test paper designed by the present application.

[0020] Figure 2 is the optimal pH test result of the fipronil detection test paper strip of the present application.

[0021] Figure 3 is the optimal amount of conjugate antibody test result of the fipronil detection test paper strip of the present application.

[0022] Figure 4 is the optimal amount of coated antigen test result of the fipronil detection test paper strip of the present application.

[0023] In the figure: 1, bottom plate; 2, sample pad; 3, conjugate pad; 4, nitrocellulose membrane; 5, water absorption pad; 6, wax line barrier; 7, detection line; 8, quality control line. DETAILED DESCRIPTION

[0024] The technical solutions of the present application will be described clearly and completely below in combination with the drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0025] In the description of the present application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first", "second", "third" are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0026] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "connecting", "connecting" should be broadly understood, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication inside two elements. For those skilled in the art, the specific meaning of the above-mentioned terms in the present application can be understood according to the specific circumstances. In addition, the technical features involved in different embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0027] The multi-residue detection test paper designed in the present application, as shown in Figure 1 The bottom plate 1, the sample pad 2, the binding pad 3, the nitrocellulose membrane 4 (NC membrane) and the water absorption pad 5 are arranged on the bottom plate 1. The binding pad 3 is partially overlapped with one end of the nitrocellulose membrane 4, the sample pad 2 is partially overlapped with the end of the binding pad 3 away from the nitrocellulose membrane 4, and the water absorption pad 5 is partially overlapped with the other end of the nitrocellulose membrane 4. When liquid is absorbed from the sample pad 2, it can be sequentially chromatographed through the binding pad 3, the nitrocellulose membrane 4 and the water absorption pad 5.

[0028] In order to realize the joint detection of multiple pesticides in the test paper, the present application sets several wax line barriers 6 extending in the chromatography direction in the NC membrane, which realizes the physical separation between the detection channels by using the hydrophobic property of wax, effectively avoids the sample penetration and signal interference between multiple channels, and significantly improves the specificity and accuracy of multi-joint detection. In order to ensure the separation effect, the wax line barrier should be fully penetrated and immersed into the NC membrane to the base surface.

[0029] Preparation of multi-channel nitrocellulose membrane of the present application: using a printer to draw several parallel wax lines on the surface of the NC membrane in the chromatography direction in the form of droplets with a diameter of 50-60 μm, the wax droplets are instantaneously cooled and solidified on the surface. Place the NC membrane on the digital hot plate to heat, melt the wax and penetrate into the NC membrane, and form several wax line barriers extending in the chromatography direction after cooling. Preferably, the distance between adjacent wax line barriers is 2 mm.

[0030] The application provides a method for preparing the gold nanoparticle solution, comprising the following steps: Preparation of gold nanoparticle solution: a certain amount of gold tetrachloride (HAuCl4) and deionized water are configured into a gold tetrachloride solution, the solution is stirred and heated to boiling; sodium citrate solution is added to the boiling gold tetrachloride solution, and the solution is continuously stirred and heated until the color of the solution becomes stable dark red (color change of the solution: colorless transparent→black→light purple→dark red), heating is stopped, and the solution is cooled to room temperature and stored in a refrigerator.

[0031] Preparation of gold nanoparticle labeled pesticide residue antibody: the gold nanoparticle solution is adjusted to pH 9, and a preselected pesticide antibody is added, and the solution is shaken at room temperature for a period of time, then BSA is added to block the unmodified part of the surface of the gold nanoparticles (AuNPs), and the solution is continuously shaken at room temperature for a period of time, and then centrifuged at low temperature, and the supernatant is removed to obtain the gold nanoparticle labeled pesticide residue antibody. The types of pesticides that can be used in the application include: insecticides (organophosphorus pesticides such as dichlorvos, chlorpyrifos, phoxim, etc.; carbamates such as methomyl, carbofuran, etc.; pyrethroids such as lambda-cyhalothrin, deltamethrin, etc.; neonicotinoids such as imidacloprid, thiamethoxam, etc.), fungicides (such as mancozeb, chlorothalonil, copper preparations, tebuconazole, difenoconazole, azoxystrobin, pyraclostrobin, carbendazim, etc.), herbicides (such as benzosulfuron, glyphosate, paraquat, etc.), acaricides (such as pyridaben, spirodiclofen, ajenyin, etc.), rodenticides (such as zinc phosphide, bromadiolone, bromethalin, etc.), and the like.

[0032] The core function of the binding pad 3 is to carry the gold nano-labeled pesticide residue antibody. In order to avoid the inactivation of the gold nano-labeled pesticide residue antibody in the binding pad 3 during long-term storage, a binding pad treatment liquid is designed, which contains 1% BSA, 5% sucrose and 0.5% Tween20, and the rest is PBS solution. The binding pad 3 is soaked in the binding pad treatment liquid for 30 minutes. The gold nano-labeled pesticide residue antibody is sprayed on the dried binding pad 3, and the treated binding pad 3 is obtained after drying. The 1% BSA (bovine serum albumin) in the treatment liquid can occupy the hydrophobic adsorption sites of the binding pad 3 in advance, avoiding the subsequent fixation of the gold nano-labeled antibody due to non-specific adsorption, which can cause structural denaturation or loss of activity. At the same time, BSA can form a protective film on the surface of the antibody, reducing the degradation of the antibody during transportation and storage, and prolonging the shelf life of the test paper. The 5% sucrose in the treatment liquid can act as a stabilizer and excipient. During the drying process, a porous matrix structure is formed, which can wrap the gold nano-labeled antibody, avoiding the aggregation and precipitation of the antibody in the dry environment (aggregation can lead to a decrease in antibody activity and a decrease in detection signal). After the subsequent sample droplet is added, the sucrose quickly dissolves and can assist the uniform release of the antibody, ensuring the binding efficiency of the antibody and the pesticide residue in the sample during the chromatography process.

[0033] The untreated binding pad 3 is prone to denaturation and aggregation, resulting in weak detection signal and poor repeatability. After soaking and drying in the treatment liquid, the antibody structure in the binding pad 3 remains good, the activity remains for a long time, and the binding efficiency of the antibody and the pesticide residue in the sample is high.

[0034] The core function of the sample pad 2 is to receive and filter the sample liquid (such as vegetable extract, water sample), and guide the sample liquid to penetrate uniformly and quickly into the binding pad 3. A sample pad treatment liquid is also designed, which contains 0.5% BSA, 2% NaCl and 0.5% Tween20, and the rest is PBS solution. Specifically, the 2% NaCl in the treatment liquid can adjust the ionic strength of the sample pad 2, optimize the environment for immune reaction (appropriate ionic strength can promote the specific binding of antigen and antibody), and at the same time, it can slightly destroy the agglomeration of colloidal particles that may exist in the sample, avoiding the blockage of the pores of the sample pad 2, and ensuring the chromatography speed of the sample liquid.

[0035] Treat the nitrocellulose membrane: print several wax line barriers on the NC membrane, which extend along the chromatographic direction. Use a microfluidic point sampler to draw different kinds of pesticide antigen-coated detection lines in the independent channels formed by adjacent wax line barriers, and arrange them alternately. The quality control line is arranged in parallel on the side of the detection line close to the water absorption pad. After coating, place the NC membrane in a 37°C oven for complete drying.

[0036] Test paper assembly: adhere and combine the bottom plate 1 and the treated sample pad 2, the combination pad 3, the nitrocellulose membrane 4, and the water absorption pad 5 according to the structure shown in the figure. Figure 1

[0037] The application also tests the optimal detection conditions of the fipronil detection test paper. As shown in the figure, the optimal pH for fipronil test paper detection is 9. As shown in the figure, the optimal antibody concentration for fipronil in the test paper is 15-20 μg / ml. As shown in the figure, the optimal coating antigen concentration for fipronil in the test paper is 0.2 μg / cm. Figure 2 Figure 3 Figure 4

[0038] Example 1: Preparation of a double detection test paper that can simultaneously detect chlorpyrifos and fipronil.

[0039] Preparation of gold nanoparticle solution: weigh 0.0927 g of tetrachloroauric acid and add 188.5 ml of deionized water to prepare a tetrachloroauric acid solution. Stir at a speed of 1000 rpm and heat to boiling; weigh 0.03423 g of anhydrous sodium citrate, dissolve in 30 ml of deionized water, and measure 11.5 ml and add to the boiling tetrachloroauric acid solution. Continue to stir and heat until the solution color is stable and dark red. Cool to room temperature and store in a 4°C refrigerator for standby use.

[0040] Preparation of gold nanoparticle labeled pesticide antibody: (1) Preparation of chlorpyrifos labeled antibody: adjust the pH of the gold nanoparticle solution to 9 with 0.2M K2CO3, add 30 μg of chlorpyrifos monoclonal antibody, and shake at room temperature for 5 minutes; add 30 μL of BS A to block the unmodified part of the gold nanoparticle (AuNPs) surface, shake at room temperature for 5 minutes; centrifuge at 10000 rpm for 10 minutes at 4°C, discard the supernatant, resuspend the antibody-gold nanoparticle complex with 600 μL of 50 mM Tris-HCl (pH=8.0), and store at 4°C for standby use; ​​​​(2) Preparation of fipronil labeled antibody: adjust the pH of the gold nanoparticle solution to 9 with 0.2M K2CO3, add 20 μg fipronil monoclonal antibody, and shake at room temperature for 5 minutes; add 30 μL of BSA to block the unmodified part of the gold nanoparticle (AuNPs) surface, and shake at room temperature for 5 minutes; centrifuge at 10000 rpm for 10 minutes at 4°C, discard the supernatant, resuspend the antibody-gold nanoparticle complex with 600 μL of 50 mM Tris-HCl (pH = 8.0), and store at 4°C for later use.

[0041] Treatment of the binding pad 3 and the sample pad 2: soak the binding pad 3 and the sample pad 2 in the corresponding treatment solution for 30 minutes, respectively, and air dry overnight for later use.

[0042] Labeling of the binding pad 3: mix the chlorpyrifos antibody-gold nanoparticle complex and the fipronil antibody-gold nanoparticle complex at a ratio of 1:2, evenly coat the treated binding pad 3 with the mixture using a pipette, and dry in a 37°C oven for 2 hours.

[0043] Preparation of a double-channel nitrocellulose membrane: (1) Preparation of a wax line barrier: use a printer to draw a wax droplet line on the surface of the NC membrane in the chromatographic direction with droplets of 50-60 μm in diameter, and the wax droplets are instantaneously cooled and solidified; place the NC membrane on a digital hot plate to heat, melt the wax, and penetrate into the NC membrane, and form a completely immersed wax line barrier after cooling; (2) Printing of detection lines and quality control lines: use a microfluidic spotter to draw chlorpyrifos antigen-coated detection lines and fipronil antigen-coated detection lines on both sides of the wax line barrier; the concentration of chlorpyrifos antigen is 0.125 μg / mL, and the concentration of fipronil antigen is 0.5 μg / mL; parallelly arrange the quality control lines on the side of the detection lines close to the absorbent pad; finally, dry the NC membrane completely in a 37°C oven.

[0044] Assembly of the test strip: adhere the treated sample pad 2, binding pad 3, nitrocellulose membrane 4, and absorbent pad 5 of the base plate 1 according to the structure shown in Figure 1 Cut the assembled base plate into a 4 mm wide colloidal gold test strip, and seal it in a moisture-proof bag containing a desiccant for storage.

[0045] The embodiments of the present application are described in detail above, but the present application is not limited to the described embodiments. For those skilled in the art, various changes, modifications, replacements, and variations of the embodiments can be made without departing from the principles and spirits of the present application, and still fall within the protection scope of the present application.

Claims

1. A multi-residue test paper based on wax line barrier separation technology, characterized in that, The test strip comprises a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad arranged on the base plate; The conjugate pad is partially overlapped with one end of the nitrocellulose membrane; the sample pad is partially overlapped with the other end of the nitrocellulose membrane; and the absorbent pad is partially overlapped with the other end of the nitrocellulose membrane; The nitrocellulose membrane is immersed with a plurality of wax barriers extending in the chromatographic direction and detection lines and quality control lines arranged perpendicularly to the chromatographic direction; the wax barriers are immersed to the surface of the nitrocellulose membrane close to the base plate, and the wax barriers extend at least through the detection lines.

2. The test paper according to claim 1, characterized in that, The wax barriers extend from the overlapping part of the nitrocellulose membrane and the conjugate pad to the overlapping part of the nitrocellulose membrane and the absorbent pad.

3. The test paper according to claim 1, characterized by The detection lines are parallel to the quality control lines, and the quality control lines are located on the side of the detection lines close to the absorbent pad.

4. The test paper according to claim 1, characterized by The distance between two adjacent wax barriers is 2mm.

5. The test paper according to claim 1, characterized by The detection lines on both sides of the wax barriers are coated with different types of pesticide antigens.

6. A method for producing the test paper according to any one of claims 1 to 5, characterized by, The method comprises the following steps: S1, mixing and reacting gold nanoparticles with pesticide antibodies to obtain gold nanoparticle-labeled pesticide antibodies; S2, fixing the gold nanoparticle-labeled pesticide antibodies on the conjugate pad; S3, wax printing a plurality of wax barriers on the nitrocellulose membrane, and printing detection lines and quality control lines in adjacent wax barriers; S4, assembling the sample pad, the conjugate pad, the nitrocellulose membrane and the absorbent pad according to the structure of the test strip of any one of claims 1-4.

7. The preparation method according to claim 6, characterized in that, In S1, the gold nanoparticles are prepared by reduction reaction of citric acid and tetrachloroauric acid.

8. The preparation method according to claim 6, characterized in that, In S3, the wax printing is specifically as follows: the wax printing ink is sprayed on the surface of the NC membrane in the form of droplets in the chromatographic direction, the NC membrane is heated to make the wax printing ink melt and penetrate into the NC membrane, and after cooling, the NC membrane with a plurality of wax barriers is obtained.

9. The preparation method according to claim 6, characterized in that, In S3, different types of pesticide antigens are printed on adjacent detection lines.

10. The method of claim 6, wherein, The conjugate pad and the sample pad are both modified.

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