Paper-based multicolor colorimetric method for detecting acetylcholin esterase activity or organophosphorus pesticide

Abstract

The invention discloses a paper-based multicolor colorimetric method for detecting acetylcholin esterase activity or organophosphorus pesticide, and the method comprises the following steps: depositing a nanogold bipyramid on a nylon film, and constructing a paper-based analysis device; the method comprises the following steps: adding a reaction solution containing Au < + > into a paper-based analysis device, and inducing the biconical appearance conversion of the nanogold through a disproportionation reaction of Au < + >, so that the biconical nanogold generates color change and position change of a longitudinal localized surface plasmon resonance peak lambda LLSPR; therefore, visual semi-quantitative detection and quantitative detection of acetylcholin esterase activity or organophosphorus pesticide by the ultraviolet spectrophotometry are realized. Compared with an existing method, the Au < + >-induced nanogold biconical appearance conversion reaction can effectively avoid interference of a reducing agent in an actual sample. Meanwhile, the constructed paper-based analysis method shows better nanogold bipyramid storage stability, reaction reproducibility and storability of detection results, and also has the advantages of high etching efficiency and high detection sensitivity.

Description

Technical Field

[0001] The invention relates to the technical field of biosensors, and in particular to a paper-based multicolor colorimetric method for detecting acetylcholinesterase activity or organophosphorus pesticides. Background Art

[0002] Acetylcholinesterase (AChE, EC 3.1.1.7) is a serine hydrolase that is mainly expressed in the central and peripheral nervous systems, neuromuscular junctions, and erythrocytes. As a key enzyme in the nervous system, the main function of AChE is to terminate nerve impulses by rapidly hydrolyzing the neurotransmitter acetylcholine to produce choline and acetate. In addition, AChE is also involved in physiological processes such as tumorigenesis, amyloid fibril formation, cell apoptosis, inflammation regulation, cell growth, and stem cell differentiation. Abnormal expression of AChE in the human body is associated with human neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, and amyotrophic lateral sclerosis. To treat these diseases, some AChE inhibitors (such as donepezil, rivastigmine, galantamine, and huperzine A) are used as drugs to increase the concentration of ACh in the brain. On the other hand, other AChE inhibitors such as organophosphorus pesticides (OPs) can cause neurotoxicity and may be fatal due to excessive inhibition of AChE activity. Therefore, establishing an analytical method for detecting acetylcholinesterase activity is of great significance for the study of the cholinergic system and the monitoring of organophosphorus pesticide poisoning and environmental pollution.

[0003] At present, many analytical methods have been developed for the detection of acetylcholinesterase activity and organophosphorus pesticides, including colorimetry, fluorescence, electrochemical and chemiluminescence. Compared with other analytical methods, colorimetry has the advantages of being easy to use, low cost, not requiring expensive instruments and being applicable to on-site detection. For example, Ellman's method uses the color reaction between thiocholine, the product of acetylcholine hydrolysis of thioacetylcholine, and 5,5-disulfide-2-nitrobenzoic acid to achieve visual detection of acetylcholinesterase activity and organophosphorus pesticides. However, the accuracy of this method is affected by false positive effects and interference from hemoglobin in the blood. Moreover, the use of organic reagents with low molar absorption coefficients and the detection method of monochromatic intensity changes limits the sensitivity and accuracy of its visual detection. Since the human eye is more sensitive to multicolor changes than monochromatic intensity changes, the development of sensitive multicolor visual detection methods is of great significance for the detection of acetylcholinesterase activity and organophosphorus pesticides on site.

[0004] Gold nanoparticles (AuNBPs) are long strip-shaped gold nanoparticles that have both transverse localized surface plasmon resonance (LSPR) and longitudinal localized surface plasmon resonance (LLSPR). AuNBPs with different aspect ratios have different LLSPR peak positions (λ LLSPR), causing them to show different colors in the visible light region. At the same time, AuNBPs also have significant local electric field enhancement effect, narrow LLSPR peak and high refractive index sensitivity. These excellent properties make AuNBPs an ideal material for constructing highly sensitive multicolor colorimetric sensors. At present, a variety of multicolor colorimetric sensors based on AuNBPs etching or growth methods have been developed. However, most of these sensors are interfered by reducing agents such as ascorbic acid (AA) and prototype nicotinamide adenine dinucleotide (NADH) in actual samples. In addition, in these sensors, the etching / growth of AuNBPs is mostly carried out in liquid, and AuNBPs are inevitably diluted by etching solution / growth solution, which affects the accuracy and sensitivity of visual detection. At the same time, the instability of AuNBPs in solution requires a cumbersome preparation process of AuNBPs concentrate, and there is still the disadvantage that the AuNBPs concentrate and the reacted AuNBPs solution (i.e., the detection result) are not easy to preserve. Therefore, it is necessary to develop a portable immobilized device that is easy to operate and can stabilize the morphology of AuNBPs and to establish a sensitive and efficient AuNBPs etching method to achieve rapid on-site screening and detection of targets including acetylcholinesterase activity and organophosphate pesticides. Summary of the invention

[0005] In order to solve the above-mentioned deficiencies in the prior art, the present invention uses the newly discovered Au + A paper-based multicolorimetric method for detecting acetylcholinesterase activity or organophosphorus pesticides was established based on the disproportionation reaction-induced bipyramidal morphology transformation reaction of nanogold.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: a paper-based multicolor colorimetric method for detecting acetylcholinesterase activity or organophosphorus pesticides is provided, comprising the following steps: depositing nano-gold bipyramids on a nylon membrane to construct a paper-based analytical device; depositing Au + The reaction solution was added to the paper-based analytical device and the Au + The disproportionation reaction induces the transformation of the nano-gold bipyramid morphology, which causes the nano-gold bipyramid to produce color changes and longitudinal localized surface plasmon resonance peak λ LLSPR The position changes can realize visual semi-quantitative detection and UV spectrophotometric quantitative detection of acetylcholinesterase activity or organophosphorus pesticides.

[0007] The beneficial effects of the present invention are as follows: the present invention is newly discovered Au + Based on the disproportionation reaction-induced bipyramidal morphology transformation reaction of nano-gold, a novel Au nanoparticle was established to regulate the activity of acetylcholinesterase. + The detection system of the disproportionation reaction-induced bipyramidal morphology transformation reaction of gold nanoparticles. + Disproportionation reaction occurs to generate Au 0 and Au 3+, Au 0 will be deposited on the side of the nanogold bipyramid, while Au 3+ The Au nanoparticles are specifically etched with bipyramidal tips. + The disproportionation reaction can change the morphology of AuNBPs, resulting in changes in the color of AuNBPs and the λ LLSPR In addition, by inhibiting Au + The disproportionation reaction occurs, resulting in Au + The disproportionation-induced morphological change of AuNBPs was inhibited. + The disproportionation reaction induces the transformation of the nano-gold bipyramid morphology and the color change and λ of the nano-gold bipyramid caused by the reaction LLSPR Changes to achieve visual semi-quantitative detection and UV spectrophotometric quantitative detection of acetylcholinesterase activity or organophosphorus pesticides.

[0008] In the method of the present invention, Au is regulated by acetylcholinesterase activity. + The reaction of disproportionation-induced changes in the bipyramidal morphology of gold nanoparticles. Acetylcholinesterase can affect Au in many ways. + dismutation reaction, such as the enzymatic hydrolysis product obtained by the enzymatic hydrolysis reaction of acetylcholinesterase to affect Au + Au can also be affected by other methods involving acetylcholinesterase. + That is to say, as long as acetylcholinesterase can participate and Au + The disproportionation-induced changes in the bipyramidal morphology of nano-gold are affected, all of which are within the scope of protection of the present invention.

[0009] On the basis of the above technical solution, the present invention can also be improved as follows:

[0010] Furthermore, when Au + The aspect ratio of the gold nanoparticles can be increased by inducing a disproportionation reaction. LLSPR The color of the nanogold bipyramids gradually shifted to blue, and changed from brown-red to red-purple, indicating that the sample to be tested contained acetylcholinesterase.

[0011] The beneficial effect of adopting the above further technical solution is that as the activity of acetylcholinesterase increases, the Au + As the concentration increases, the morphology of the AuNBPs gradually changes from a bipyramid to an ellipsoid and an irregular sphere, causing the color of the AuNBPs to change from brownish red to purple-red (brownish red, brown, grass green, green, blue-green, blue, purple, and red-purple), accompanied by the λ LLSPRBlue shift. This change can be used to determine whether the sample contains acetylcholinesterase.

[0012] Furthermore, when a certain amount of acetylcholinesterase was added to the reaction solution in advance, the λ LLSPR The color of the nanogold bipyramids changes from red-purple to brown-red, indicating that the sample contains organophosphorus pesticides.

[0013] The beneficial effect of adopting the above further technical solution is that when the sample contains OPs, OPs can inhibit the activity of AChE in the reaction solution, resulting in the Au that can undergo disproportionation reaction. + As the concentration of OPs increased, the morphology of the AuNBPs gradually changed from irregular spheres to ellipsoids and bipyramids, causing the color of the AuNBPs to change from purple to brownish red (red-purple, purple, blue, blue-green, green, grass-green, brown and brownish red), accompanied by the λ LLSPR The blue shift gradually disappears. This change can be used to determine whether the sample contains organophosphorus pesticides.

[0014] In summary, the increase in AChE activity caused the color of AuNBPs to change from brown-red to red-purple, and the λ LLSPR The color of AuNBPs gradually shifted to blue, and the increase of OPs pesticide concentration caused the color of AuNBPs to change from reddish purple to brownish red. LLSPR Gradually red shift. This principle and result can be used to detect whether the sample contains acetylcholinesterase or organophosphorus pesticides.

[0015] Furthermore, when the activity of acetylcholinesterase was detected, the + The reaction solution includes: choline oxidase, acetylcholine chloride, tri(2-carboxyethyl)phosphine (TCEP), hexadecyltrimethylammonium bromide (CTAB), HCl and Au + Solution;

[0016] The detection process is:

[0017] Nanogold bipyramids were deposited on nylon membranes to construct paper-based analytical devices;

[0018] The sample to be tested is mixed with a buffer solution containing choline oxidase, acetylcholine chloride and TCEP to obtain an enzymatic solution;

[0019] The enzymatic solution was incubated and then the Au solution containing CTAB and HCl was added. + solution, and mixing to obtain a reaction solution;

[0020] After the reaction solution is incubated, it is added to a paper-based analysis device and reacted at 35-50°C for 2-10 minutes. The color of the nano-gold bipyramid deposition film and / or the extinction spectrum of the nano-gold bipyramid deposition film in the range of 400-1000nm are observed, and the nano-gold bipyramid λ is compared with the standard paper-based color card and / or the extinction spectrum is compared with the standard paper-based color card. LLSPR The displacement was substituted into the standard working curve to determine the acetylcholinesterase activity.

[0021] The beneficial effect of adopting the above further technical scheme is: adding a sample or standard containing acetylcholinesterase to a reaction solution containing acetylcholine chloride, choline oxidase and TCEP, acetylcholine chloride is first hydrolyzed by acetylcholinesterase and choline oxidase to generate H2O2; H2O2 can consume TCEP, that is, H2O2 oxidizes TCEP to TCEPO; then, adding Au containing CTAB and HCl to the enzymatic hydrolysis solution + Solution, TCEPO cannot chelate Au + , which leads to Au + Finally, the mixed solution, i.e., the reaction solution, is added to a paper-based analytical device to complete the AuNBPs reaction on the nylon membrane. + Therefore, the increase of AChE activity enables AuNBPs to undergo disproportionation reaction. + As the concentration increases, the λ of AuNBPs on the nylon membrane after reaction LLSPR Gradually blue-shifted, and the color of AuNBPs changed from brown-red to red-purple.

[0022] Furthermore, the incubation conditions of the enzymatic solution are: incubation at 35-40°C for 25-35 min; the incubation conditions of the reaction solution are: incubation at 45-55°C for 3-7 min; + The choline oxidase activity in the reaction solution is 5-60 U / L, the concentration of acetylcholine chloride is 50-400 μM, the concentration of TCEP solution is 14-22 μM, the concentration of CTAB is 20-60 mM, the concentration of HCl is 20-60 mM, and the concentration of Au + The concentration is 10-20 μM. The preferred incubation conditions for the enzymatic solution are: incubation at 37°C for 30 min; the incubation conditions for the reaction solution are: incubation at 50°C for 5 min; + The choline oxidase activity in the reaction solution was 40 U / L, the concentration of acetylcholine chloride was 100 μM, the concentration of TCEP solution was 20 μM, the concentration of CTAB was 40 mM, the concentration of HCl was 20 mM, and the concentration of Au + The concentration is 15 μM.

[0023] The beneficial effect of adopting the above further technical solution is: by optimizing the concentration of each component in the above reaction solution, the efficiency of the reaction can be improved and its detection sensitivity can be improved.

[0024] Furthermore, when detecting organophosphorus pesticides, Au + The reaction solution includes: acetylcholinesterase, choline oxidase, acetylcholine chloride, TCEP, CTAB, HCl and Au + Solution;

[0025] The detection process is:

[0026] Nanogold bipyramids were deposited on nylon membranes to construct paper-based analytical devices;

[0027] Mixing the sample to be tested with a buffer solution containing acetylcholinesterase to obtain a mixed solution;

[0028] The mixed solution is incubated, and then a buffer solution containing choline oxidase, acetylcholine chloride and TCEP is added and mixed to obtain an enzymatic solution;

[0029] The enzymatic solution was incubated and Au containing CTAB and HCl was added. + solution, and mixing to obtain a reaction solution;

[0030] After the reaction solution is incubated, it is added to a paper-based analysis device and reacted at 35-50°C for 2-10 minutes. The color of the nano-gold bipyramid deposition film and / or the extinction spectrum of the nano-gold bipyramid deposition film in the range of 400-1000nm are observed, and the nano-gold bipyramid λ is compared with the standard paper-based color card and / or the extinction spectrum is compared with the standard paper-based color card. LLSPR The displacement was substituted into the standard working curve to determine the concentration of organophosphorus pesticides.

[0031] The beneficial effect of adopting the above further technical scheme is: adding a sample or standard containing OPs to a reaction solution containing acetylcholine chloride, acetylcholinesterase, choline oxidase and TCEP, OPs can inhibit the activity of AChE, thereby retaining the substrate acetylcholine chloride and TCEP, and TCEP can chelate Au + , leading to Au + The disproportionation reaction cannot be carried out. Finally, the mixed solution, i.e., the reaction solution, is added to the paper-based analysis device to complete the Au on the nylon membrane containing AuNBPs. + Therefore, the increase in OPs concentration inhibits the activity of AChE in the reaction solution, resulting in the AuNBPs that can undergo disproportionation reaction. + As the concentration decreased, the color of AuNBPs on the nylon membrane gradually changed from reddish purple to brownish red, and the λ LLSPR Gradually red shift.

[0032] Furthermore, when detecting organophosphorus pesticides, the incubation conditions of the mixed solution are: incubation at 35-40°C for 15-25 minutes; the incubation conditions of the enzymatic solution are: incubation at 35-40°C for 25-35 minutes; the incubation conditions of the reaction solution are: incubation at 45-55°C for 3-7 minutes; + The activity of acetylcholinesterase in the reaction solution is 1-3U / L, the activity of choline oxidase is 5-60U / L, the concentration of acetylcholine chloride is 50-400μM, the concentration of TCEP solution is 14-22μM, the concentration of CTAB is 20-60mM, the concentration of HCl is 20-60mM, and the concentration of Au + The concentration is 10-20 μM. Preferably, the incubation conditions of the mixed solution are: incubation at 37°C for 20 min; the incubation conditions of the enzymatic solution are: incubation at 37°C for 30 min; the incubation conditions of the reaction solution are: incubation at 50°C for 5 min; + In the reaction system, the activity of acetylcholinesterase was 2U / L, the activity of choline oxidase was 40U / L, the concentration of acetylcholine chloride was 100μM, the concentration of TCEP solution was 20μM, the concentration of CTAB was 40mM, the concentration of HCl was 20mM, and the concentration of Au + The concentration is 15 μM.

[0033] The beneficial effect of adopting the above further technical solution is: by optimizing the concentration of each component in the above reaction solution, the efficiency of the reaction can be improved and its detection sensitivity can be improved.

[0034] Furthermore, when detecting the enzyme of acetylcholinesterase or detecting organophosphorus pesticides, the reaction solution is added to the paper-based analysis device and reacted for 6 minutes at 50° C. Under this reaction condition, the reaction efficiency and detection sensitivity can be improved.

[0035] The present invention has the following beneficial effects:

[0036] 1. The present invention provides a paper-based multicolor colorimetric method for detecting acetylcholinesterase activity and organophosphorus pesticides, and its detection principle involves Au + It consists of three parts: the reaction of AuNBPs morphology changes mediated by the dual-enzyme hydrolysis strategy and PAD (paper-based analytical device).

[0037] Au + The reactions that mediate the morphological changes of AuNBPs are as follows Figure 3 As shown in A. When Au + A disproportionation reaction occurs on the surface of AuNBPs to generate Au 0 and Au 3+ After that, Au 0 will be deposited on the sides of AuNBPs, resulting in an increase in width, while Au3+ The AuNBPs tip was specifically etched, resulting in a decrease in length. + The net result of the disproportionation reaction can be seen as the transfer of gold atoms at the tip of AuNBPs to the side, so the reaction is called Au + Mediate the morphological transformation of AuNBPs. + With the increase of concentration, the morphology of the nanogold bipyramids gradually changed from bipyramids to ellipsoids and irregular spheres, accompanied by eight bright color changes, specifically from brown-red to brown, grass green, green, blue-green, blue, purple and red-purple.

[0038] Dual enzyme digestion strategy Figure 3 As shown in B: When the sample contains acetylcholinesterase (path A), acetylcholine chloride is first hydrolyzed by acetylcholinesterase and choline oxidase to generate H2O2, which then oxidizes TCEP to generate the oxidized product TCEPO. Because TCEPO cannot chelate Au + , so Au + When acetylcholinesterase and organophosphorus pesticides are present at the same time (path B), organophosphorus pesticides can effectively inhibit the activity of acetylcholinesterase, thereby retaining the substrates acetylcholine chloride and TCEP. TCEP can chelate Au + , leading to Au + Unable to undergo disproportionation reaction.

[0039] The paper-based analytical device (PAD) contains a nylon membrane deposited with gold nanoparticles (see Figure 3 C) When the reaction solution in the dual enzyme enzymatic hydrolysis strategy is added to the paper-based analytical device, the Au in the solution that is not chelated by TCEP + It will react with the gold nanopipette on the nylon membrane + The reaction that mediates the change of the bipyramidal morphology of the nano-gold is due to the acetylcholinesterase activity and the free Au + The concentration of organophosphorus pesticides is proportional to the free Au + The concentration of organophosphorus pesticides is inversely proportional to the color of the gold nanoparticles on the nylon membrane. Therefore, the increase in acetylcholinesterase activity causes the color of the gold nanoparticles on the nylon membrane to gradually change from brown-red to brown, grass green, green, blue-green, blue, purple and red-purple, while the increase in the concentration of organophosphorus pesticides causes the color of the gold nanoparticles on the nylon membrane to gradually change from red-purple back to purple, blue-purple, blue, blue-green, green, grass green and brown. In summary, this study developed a novel Au nanoparticle-based + PMS (paper-based multicolor sensor) mediated by the change of nanogold bipyramidal morphology can be used to detect acetylcholinesterase activity or organophosphorus pesticide concentration.

[0040] 2. Compared with the existing colorimetric method for detecting acetylcholinesterase activity and organophosphorus pesticides, the colorimetric method established by the present invention has the advantages of simple operation, high throughput, high sensitivity and multi-color signals. The designed paper-based analysis device can detect eight samples at the same time. The nanogold bipyramids enriched on the nylon membrane after deposition will not be diluted by the reaction solution, ensuring the sensitivity of human eye detection and instrument detection. The method presents 7-8 color changes according to the changes in acetylcholinesterase activity and organophosphorus pesticide concentration, which is helpful for semi-quantitative detection with naked eyes.

[0041] Compared with the existing colorimetric method based on etching reaction to change the bipyramidal morphology of gold nanoparticles, Au + Inducing the transformation reaction of the nano-gold bipyramid morphology can effectively avoid the interference of the reducing agent in the actual sample. At the same time, the preparation of the nano-gold cone deposition film is simple, and the λ LLSPR It has good reproducibility and can be stored for a long time, solving the problems of cumbersome preparation of nanogold bipyramid concentrate in liquid, poor reproducibility between batches, and inability to store for a long time. In addition, compared with the colorimetric method of nanogold bipyramid morphology reaction in liquid, the detection results of the reaction on the constructed paper-based analysis device can be stably stored and show excellent reaction reproducibility, high etching efficiency and high detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Flow chart of nano-gold bipyramid deposition; (A) the process of printing wax on nylon membrane; (B) the assembly of deposition device: top view of each component of the deposition device, the size of the drilled acrylic plate, and the top view and side view of the device; (C) top view of the nano-gold bipyramid deposition film.

[0043] Figure 2 The figures are a schematic diagram and a physical picture of a paper-based analysis device; among them, (A) is a schematic diagram of the principle of the paper-based analysis device, and (B) is a physical picture of the paper-based analysis device.

[0044] Figure 3 Based on Au + Schematic diagram of PMS-mediated transformation of gold nanoparticles into bipyramidal morphologies for determination of acetylcholinesterase activity and organophosphorus pesticides.

[0045] Figure 4 The results of AChE detection using a paper-based analytical device; (A) the color of AuNBPs on nylon membrane at different AChE activities, (B) the extinction spectrum of AuNBPs, (C) the Δλ of AuNBPs LLSPR Exponential relationship with AChE activity.

[0046] Figure 5The results of DDVP detection using a paper-based analytical device; (A) the color of AuNBPs on the nylon membrane at different DDVP concentrations, (B) the extinction spectrum of AuNBPs, (C) the Δλ of AuNBPs LLSPR Exponential relationship with DDVP concentration. DETAILED DESCRIPTION

[0047] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0048] Example 1: Preparation of gold seed solution

[0049] After soaking the glass bottle in aqua regia overnight, wash it with deionized water and dry it. Then, add CTAB (64mM, 8mL), citric acid solution (50mM, 1mL) and HAuCl4 solution (2.5mM, 1mL) to the glass bottle in sequence. Then, add sodium borohydride solution (25mM, 0.25mL) to the glass bottle under magnetic stirring (1200rpm). After stirring at room temperature for 2 minutes, transfer the glass bottle to a water bath and incubate at 80℃ for 90 minutes to complete the preparation of the gold seed solution.

[0050] Example 2: Preparation of Nano-gold Bipyramid Growth Solution

[0051] 90 mM CTAB, 0.2 mM HAuCl4, 20 mM HCl, 0.1 mM AgNO3, 2 mM L-ascorbic acid (AA) and 0.2 mL of gold seed solution were mixed and incubated in a 40 °C water bath for 4 min to obtain the nanogold bipyramid growth solution.

[0052] The above-mentioned nano-gold bipyramid growth solution was mixed with TCEP solution (0.2 mM, 10 mL) for the preparation of nano-gold bipyramid deposition film.

[0053] Example 3: Preparation of Nano-gold Biconical Deposition Film

[0054] The preparation of the nano-gold bipyramid deposition film includes three steps: preparation of the wax Indonesian dragon film, assembly of the deposition device, and deposition of the nano-gold bipyramids on the nylon membrane. The construction flow chart is shown in Figure 1 , the specific process is as follows:

[0055] ① Punch two small holes (3 mm in diameter) on a square nylon membrane (40 mm × 40 mm), and then use a stainless steel mold to print the melted wax on the nylon membrane to form four wax circles with a diameter of about 12 mm to obtain a wax Indonesian dragon membrane.

[0056] ② From bottom to top, stack the untreated acrylic plate, absorbent cotton layer, wax Indonesian film and perforated acrylic plate in sequence, and fix them with two clips to complete the assembly of the deposition device. In the deposition device, the perforated acrylic plate has two small holes (3mm in diameter) and four large holes (7mm in diameter), and a silicone rubber sealing ring (7mm in inner diameter, 2mm in ring width) is glued under each large hole. Two positioning rods are inserted into the two corresponding small holes on the perforated acrylic plate and the wax Indonesian film, respectively, to align the two. Therefore, the silicone sealing ring can be located in the corresponding wax ring on the nylon membrane to ensure that the nanogold bipyramids can be deposited in the wax ring.

[0057] ③ The synthesized nano-gold bipyramid growth solution was dripped into the large hole of the device. The AuNBPs growth solution can pass through the membrane and be absorbed by the absorbent cotton below. The nano-gold bipyramids are deposited on the membrane to form a dark red circle with a diameter of about 7 mm. Finally, the prepared nano-gold bipyramid deposition membrane was ultrasonically cleaned with deionized water, dried and stored in a refrigerator at 4°C.

[0058] Example 4: Construction of a paper-based analytical device

[0059] The paper-based analytical device consists of a custom-made metal heater, two flow guide components and an oscillating tank. The metal heater has two slots, each of which has four large holes with a diameter of 8 mm and two small holes with a diameter of 3 mm. The flow guide component consists of an acrylic base plate, a water-absorbing cotton layer, a perforated acrylic plate and a nano-gold biconical deposition film. The acrylic base plate is fixed with two long positioning rods, which can pass through the corresponding small holes on the perforated acrylic plate, the nano-gold biconical deposition film and the metal heater in turn to align them. In addition to the two small holes (3 mm in diameter) for positioning, the treated acrylic plate also has four large holes (9 mm in diameter). A silicone rubber sealing ring (9 mm in inner diameter, 2 mm in ring width) is glued above each large hole and filled with cotton. This design ensures that the reaction solution can pass through each nano-gold biconical deposition area on the membrane independently and unidirectionally. After inserting the two assembled flow guide components into the slots of the heater, the heater is installed on the oscillating tank, and finally three wing nuts are tightened to fix the entire device. During the reaction, the heater on the upper layer of the device ensured a constant temperature (50°C) during the reaction, while the oscillator on the lower layer provided an oscillation rate (750rpm) to ensure the uniformity of the reaction. In addition, the constructed device can detect up to eight samples at the same time.

[0060] The schematic diagram and physical diagram of the paper-based analysis device are shown in Figure 2 .

[0061] Example 5: Au on nylon membrane + Mediating the morphological transformation of AuNBPs

[0062] Au was deposited on the nylon membrane with AuNBPs. + To mediate the morphological transformation of AuNBPs, it is necessary to consider the effects of different experimental factors on the reaction efficiency and sensitivity. The reaction efficiency was evaluated by studying the HCl concentration, CTAB concentration, reaction temperature and reaction time in the paper-based analytical device, and the presence of different reducing agents in the reaction system. + The concentration was used to evaluate the sensitivity of the reaction, as follows:

[0063] 1. Set different HCl concentrations and CTAB concentrations. The HCl concentrations were set to 5mM, 10mM, 20mM, 40mM and 60mM, and the CTAB concentrations were set to 5mM, 10mM, 20mM, 40mM and 60mM.

[0064] The Au + Solution (Au + After incubation at 50°C for 5 min, Au + The solution was added to the paper-based analytical device prepared in Example 4. After the reaction was completed, the color of AuNBPs on the nylon membrane was observed and the extinction spectrum within the range of 400-1000 nm was measured.

[0065] The experimental results show that with the increase of HCl concentration and CTAB concentration, the Δλ of AuNBPs LLSPR The signal increased and reached a signal plateau when the HCl concentration was 20 mM and the CTAB concentration was 40 mM, so 20 mM HCl and 40 mM CTAB were selected as the optimal conditions.

[0066] 2. The deposition volume of AuNBPs (100μL, 125μL, 150μL, 175μL) was optimized. The deposition volume of AuNBPs determines the amount of AuNBPs deposited on the nylon membrane and thus determines the intensity of the AuNBPs color. The experimental results show that with the increase of the AuNBPs deposition volume, the color of the AuNBPs on the nylon membrane gradually deepens, but at the same Au + Color and Δλ of AuNBPs with different deposition volumes as a function of concentration LLSPR The results were basically consistent, indicating that the reaction on the nylon membrane was efficient and uniform. Considering the feasibility of naked eye detection and economic cost, 175 μL was selected as the optimal deposition volume.

[0067] 3. The reaction temperatures in the paper-based analytical device were set to 35°C, 40°C, 45°C and 50°C, and the reaction times were 2 min, 4 min, 6 min, 8 min and 10 min. The effects of reaction temperature and reaction time on the reaction efficiency were studied.

[0068] The experimental results show that as the temperature increases from 35℃ to 50℃, the λ LLSPR The wavelength shift increases, and considering that the wax on the nylon membrane tends to melt above 50°C, 50°C is selected as the optimal reaction temperature.

[0069] As the time increases from 2 min to 6 min, the Δλ of AuNBPs LLSPR increased significantly, while Δλ LLSPR This may be because the water absorption capacity of the water-absorbing cotton layer under the AuNBPs deposition film is close to saturation, which hinders the Au + The optimal reaction time is 6 min.

[0070] 4. Detection of different Au under the above optimal conditions + The sensitivity of the reaction was evaluated by adjusting the concentration (0, 0.3μM, 0.5μM, 1.3μM, 1.8μM, 2.5μM, 4.0μM, 10μM). + As the concentration increased from 0 to 10 μM, the AuNBPs on the nylon membrane showed eight bright color changes (brown-red, brown, grass-green, green, cyan, blue, purple, and red-purple), accompanied by the λ LLSPR Gradually blue shift. In the range of 0.2 to 4.0 μM Au + Within the concentration range, the Δλ of AuNBPs LLSPR With Au + The concentration of Au shows a good exponential relationship. + Within the concentration range, the Δλ of AuNBPs LLSPR Also with Au + The concentration showed a good exponential relationship. In contrast, the detection sensitivity of the reaction on the nylon membrane was about one order of magnitude lower than that in aqueous solution, which was due to the enrichment of AuNBPs on the nylon membrane and the high efficiency of the reaction on the nylon membrane.

[0071] In the existing detection technology, the gold nanopipian is stored in liquid, but the gold nanopipian cannot be stored in liquid for a long time. Similarly, the detection results obtained by this detection technology are also based on the color and λ of the AuNBPs in the liquid after the reaction. LLSPRThe concentration change of the target object is reflected by the change in the concentration of AuNBPs. Due to the instability of AuNBPs in liquid, the test results are difficult to preserve. Therefore, it is often necessary to use an ultraviolet spectrophotometer capable of rapid spectral scanning to promptly scan the extinction spectrum of the test results in order to achieve high-throughput and accurate detection. This requires expensive instruments and the detection time is limited. The present application deposits the nanogold bipyramids on a nylon membrane, so that the nanogold bipyramids can be stored for a long time and have good detection sensitivity. At the same time, the test results can also be preserved for a long time. This can achieve visual semi-quantitative detection on site, and then bring the sample back to the laboratory for quantitative detection by ultraviolet spectrophotometry.

[0072] 5. The present invention also studies the effects of different reducing agents on Au + The influence of disproportionation on the morphological transformation reaction process of AuNBPs is as follows:

[0073] The reaction was carried out in the presence of different reducing agents, including TCEP, AA, NADH, and HQ (hydroquinone). Among them, only TCEP chelated Au. + The results showed that the addition of TCEP (3 μM) resulted in the LLSPR Only a small shift occurred, while the results of the experiment with the addition of other reducing agents (30 μM) were similar to those without the addition of reducing agents, both of which produced obvious λ LLSPR These results not only confirm that the reaction is not interfered by the reducing agent, but also confirm that the reaction is carried out through Au + The disproportionation changes the morphology of AuNBPs, and TCEP has the function of specifically regulating this reaction.

[0074] Example 6: Detection of acetylcholinesterase activity using a paper-based analytical device

[0075] When the paper-based analysis device in Example 4 is used to detect acetylcholinesterase activity, the specific process is as follows:

[0076] (1) PB buffer (900 μL) containing different concentrations of acetylcholinesterase (0-1.0 U / L), 40 U / L cholinesterase (CHO), 100 μM acetylcholine chloride and 26.7 μM TCEP was prepared and incubated at 37°C for 30 min.

[0077] (2) Mix 60 μM HAuCl4, 180 μM NADH, 160 mM CTAB and 80 mM HCl to prepare Au + Solution.

[0078] (3) 300 μL of Au +The solution was added to the PB buffer obtained in step (1), and reacted at 50° C. for 5 minutes to obtain a reaction solution.

[0079] (4) Take 1.2 mL of the reaction solution and add it to the paper-based analysis device for Au + Mediate the AuNBPs morphology transformation reaction. After 6 minutes, add 100 μL of TCEP solution (500 μM) to stop the reaction. Finally, take out the nylon membrane after the reaction, record the color of the nanogold bipyramidal region, and measure the extinction spectrum in the range of 400-1000 nm.

[0080] The results show that as the AChE activity increases from 0 to 1.0 U / L, the AuNBPs on the nylon membrane show eight bright color changes (from brown-red to brown, grass green, green, blue-green, blue, purple and red-purple). LLSPR Also blue shifted, and Δλ LLSPR It has a good exponential relationship with AChE activity in the range of 0.03-1.0U / L. The naked eye detection limit of this method is 0.05U / L, and the spectrophotometer detection limit is 0.026U / L. Compared with other methods, the paper analysis method provided by the present invention not only has multiple color changes, but also has better sensitivity than other colorimetric methods and some fluorescence methods.

[0081] In the above detection process, the present invention also performs the following optimization process:

[0082] 1. Optimize the three experimental conditions: TCEP concentration, ACh concentration and CHO activity.

[0083] The TCEP concentrations were designed as: 14 μM, 16 μM, 18 μM, 20 μM, and 22 μM;

[0084] The ACh concentrations were designed as: 50 μM, 100 μM, 200 μM, 300 μM, and 400 μM;

[0085] The CHO activity is designed to be: 5U / L, 10U / L, 20U / L, 40U / L, 60U / L.

[0086] When the concentrations of TCEP and ACh were 20 μM and 100 μM, respectively, the Δλ of AuNBPs LLSPR When CHO activity increases to 40U / L, Δλ LLSPR Therefore, the optimal experimental conditions for TCEP concentration, ACh concentration and CHO activity were 20 μM, 100 μM and 40 U / L, respectively.

[0087] 2. In the early stage, the present invention has confirmed that Au +The reaction process of AuNBPs morphological transformation mediated by disproportionation is not affected by AA, NADH and HQ. In order to evaluate the ability of PMS to resist the interference of reducing substances when detecting AChE activity, the present invention still selected three common reducing agents (AA, NADH and HQ, all at a concentration of 200 μM) as interfering substances. It can be seen from the results that when using PMS to detect AChE activity (0.2 U / L AChE), the Δλ obtained by the sample containing reducing substances is LLSPR The Δλ obtained for the control sample without reducing substances is LLSPR This indicates that the PMS detection of AChE activity is not affected by these reducing agents. Since reducing substances such as AA and NADH are often present in actual samples, the above characteristics will greatly improve the application ability of this detection method in detecting actual samples.

[0088] In addition, in order to evaluate the anti-interference ability and specificity of PMS in detecting AChE activity, five common ions with high content in serum (Na + , K + Mg 2+ , Ca 2+ and Zn 2+ , the concentration was 100 μM) and three common protein enzymes (alkaline phosphatase ALP, ascorbate oxidase AO and glucose oxidase GOx, the concentration was 2 U / L) as interferents for testing.

[0089] The results show that the AuNBPs color and λ of the sample containing only interferences and no AChE LLSPR The experimental results are similar to those of the background sample. Similarly, the color and λ of AuNBPs in the sample containing both interferents and AChE (0.2 U / L) LLSPR The experimental results are similar to those of the control sample containing the same concentration of AChE. The experimental results show that all interferents do not affect the Au on the nylon membrane. + The AuNBPs morphology transformation reaction and AChE activity were mediated, which also showed that this method could specifically detect AChE activity.

[0090] Example 7: Detection of organophosphorus pesticides using a paper-based analytical device

[0091] When the paper-based analysis device in Example 4 is used to detect organophosphorus pesticides, the specific process is as follows:

[0092] (1) 81 μL of PB buffer containing different concentrations of dichlorvos (0-2 mg / L, specifically 0, 0.03 mg / L, 0.05 mg / L, 0.10 mg / L, 0.30 mg / L, 0.50 mg / L, 1.0 mg / L, 2.0 mg / L) was mixed with 9 μL of PB buffer containing acetylcholinesterase (200 U / L) and incubated at 37°C for 20 min.

[0093] (2) Add 810 μL of PB buffer containing 44.4 U / L choline oxidase, 111.1 μM acetylcholine chloride and 29.6 μM TCEP to the mixture obtained in step (1), and continue incubating at 37°C for 30 minutes.

[0094] (3) Mix 60 μM HAuCl4, 180 μM NADH, 160 mM CTAB and 80 mM HCl to prepare Au + Solution.

[0095] (4) 300 μL of Au + The solution was added to the PB buffer solution obtained in step (2), and reacted at 50° C. for 5 minutes to obtain a reaction solution.

[0096] (5) Take 1.2 mL of the reaction solution and add it to the paper-based analysis device for Au + Mediate the AuNBP morphology transformation reaction. After 6 minutes, add 100 μL of TCEP solution (500 μM) to stop the reaction. Finally, take out the nylon membrane after the reaction, record the color of the nanogold bipyramidal region, and measure the extinction spectrum in the range of 400-1000 nm.

[0097] The results show that as the DDVP concentration increases from 0 to 2.0 mg / L, the AuNBPs on the nylon membrane show seven bright color changes (from purple to blue-purple, blue, blue-green, green, grass green and brown). LLSPR A red shift is also produced, and Δλ LLSPR It has a good exponential relationship with DDVP concentration in the range of 0.03-2.0 mg / L. The naked eye detection limit of this method is 0.03 mg / L, and the detection limit of the spectrophotometer is calculated to be 0.028 mg / L. Compared with the reported methods for detecting OPs, this PMS has multiple color changes and higher sensitivity in detecting OPs.

[0098] In addition, in order to evaluate the anti-interference ability and specificity of PMS in detecting OPs, the present invention investigated four cations (Na + , K + Mg 2+ and Ca 2+, the concentration was 100 μM) and two proteins (ascorbate oxidase AO and bovine serum albumin BSA, the concentrations were 2 U / L and 100 mg / L respectively) as interferences on the detection of DDVP by PMS. The signal responses of PMS to the detection of seven OPs (dichlorvos DDVP, methamidophos MAP, trichlorfon DEP, paraoxon PXN, phoxim PHO, omethoate OMT and parathion PAR, the concentrations were 2 mg / L) and six non-OPs (bifenthrin BF, cypermethrin FNP, barnyardgrass QNC, boscalid BSC, 2,4-dichlorophenoxyacetic acid 2,4-D and carboxin CBX, the concentrations were 2 mg / L) were also investigated.

[0099] The results show that the color and λ of AuNBPs in samples containing the interfering substance DDVP LLSPR The experimental results of the control sample containing only DDVP are similar, while the color and λ of AuNBPs of the sample containing only interfering substances without DDVP are different. LLSPR The experimental results are similar to those of the background samples. The experimental results show that all the above interferents do not affect the PMS detection of OPs. Compared with the background samples, the seven OPs can cause significant color changes of AuNBPs and obvious λ LLSPR , while the other six non-OPs maintained the AuNBPs color and λ similar to the background sample. LLSPR The above experimental results show that PMS has excellent anti-interference ability and specificity in the rapid detection and screening of OPs.

[0100] According to the above analysis and research, the paper-based analysis device and detection reagent provided by the present invention can be used as a paper-based multicolor sensor (PMS) to detect acetylcholinesterase activity and organophosphorus pesticides in actual samples, as follows:

[0101] Experimental Example 1:

[0102] The constructed paper-based analytical device was used to detect the activity of acetylcholinesterase in red blood cell samples. The specific process is as follows:

[0103] (1) The whole blood sample was centrifuged at 3500 rpm for 10 minutes to obtain red blood cells, and then the red blood cells were washed twice with 10 times the sample volume of PBS buffer. Then different concentrations of acetylcholinesterase were added to the red blood cell sample, and then diluted 50,000 times with PB buffer (the different concentrations of acetylcholinesterase were added here to calculate the recovery rate to show that the test results were accurate).

[0104] (2) 870 μL of the red blood cell sample obtained above was mixed with 30 μL of PB buffer containing 120 U / L choline oxidase, 3 mM acetylcholine chloride and 800 μM TCEP, and incubated at 37° C. for 30 minutes.

[0105] (3) Mix 60 μM HAuCl4, 180 μM NADH, 160 mM CTAB and 80 mM HCl to prepare Au + Solution.

[0106] (4) 300 μL of Au + The solution was added to the PB buffer obtained in step (2), and reacted at 50° C. for 5 minutes to obtain a reaction solution.

[0107] (5) Take 1.2 mL of the reaction solution and add it to the paper-based analysis device for Au + Mediate the AuNBPs morphology transformation reaction. After 6 minutes, add 100 μL of TCEP solution (500 μM) to stop the reaction. Finally, take out the nylon membrane after the reaction, record the color of the nanogold bipyramidal region, and measure the extinction spectrum in the range of 400-1000 nm.

[0108] The obtained color was compared with the constructed standard paper-based color card. At the same time, the extinction spectrum of the actual sample in the range of 400-1000nm was determined using a microplate reader, and the displacement of the longitudinal localized surface plasmon resonance peak of the nanogold bipyramid was substituted into the standard working curve (which can be obtained according to the steps in Example 6). The acetylcholinesterase activity in the red blood cell sample was obtained to be 5.6U / mL, the recovery rate of the spiked sample was 96.7% to 96.9%, and the relative standard deviation of 5 measurements was less than 4% (see Table 1 for details).

[0109] Table 1 Detection results of acetylcholinesterase activity in red blood cell samples

[0110]

[0111] Experimental Example 2:

[0112] The constructed paper-based analytical device was used to detect the activity of organophosphorus pesticides in river water, rice and vegetable samples. The specific process is as follows:

[0113] (1) The water sample was treated as follows: the water sample from the Minjiang River in Fuzhou was filtered through a 0.22 μm nylon membrane and then diluted 10 times with PB buffer containing 0.1% methanol for later use.

[0114] (2) The rice was treated as follows: the rice was ground and mixed with dichloromethane at a ratio of 0.5 g / mL and vortexed for 15 minutes. The sample was then centrifuged at 6000 rpm for 5 minutes and filtered through a 0.22 μm filter membrane. 0.5 mL of the extract was rotary evaporated at 2000 rpm and 30° C. for 20 minutes. The residue was dissolved in 0.5 mL of PB buffer containing 0.1% methanol for later use.

[0115] (3) The vegetable Shanghai Qing was treated as follows: the Shanghai Qing was chopped and mixed with acetonitrile at a ratio of 0.5 g / mL and vortexed for 15 minutes. Then, the sample was centrifuged at 6000 rpm for 5 minutes. 20 mL of the supernatant was mixed with 2 g of MgSO4 and 1 g of N-propylethylenediamine, vortexed again for 1 minute, and filtered with a 0.22 μm filter membrane. Then, 0.5 mL of the extract was rotary evaporated at 2000 rpm and 30°C for 30 minutes. The residue was dissolved in 0.5 mL of PB buffer containing 0.1% methanol for later use.

[0116] (4) Take 81 μL of the treated water sample, rice sample and Shanghai green sample and mix them with 9 μL of PB buffer containing acetylcholinesterase (200 U / L) and incubate at 37°C for 20 minutes. Then, add 810 μL of PB buffer containing 44.4 U / L choline oxidase, 111.1 μM acetylcholine chloride and 29.6 μM TCEP to the mixture and incubate at 37°C for 30 minutes. Then, add 300 μL of Au + The solution was reacted at 50°C for 5 minutes, and finally 1.2 mL of the reaction solution was added to the paper-based analysis device for Au + Mediate the AuNBPs morphology transformation reaction. After 6 minutes, add 100 μL of TCEP solution (500 μM) to stop the reaction. Finally, take out the nylon membrane after the reaction, record the color of the nanogold bipyramidal region, and measure the extinction spectrum in the range of 400-1000 nm.

[0117] The above Au + The solution was prepared by mixing 60 μM HAuCl4, 180 μM NADH, 160 mM CTAB and 80 mM HCl. + Solution.

[0118] The obtained color was compared with the constructed standard paper-based color card. At the same time, the extinction spectrum of the actual sample in the range of 400-1000nm was determined using a microplate reader. The displacement of the longitudinal localized surface plasmon resonance peak of the nanogold bipyramid was substituted into the standard working curve (which can be obtained according to the steps in Example 7). The recovery rate of the dichlorvos concentration in the river water, rice and bok choy samples was 85.4-93.9%, and the relative standard deviation of the 5 measurements was less than 5% (see Table 2 for details).

[0119] Table 2 Detection results of dichlorvos in river water, rice and Shanghai green samples

[0120]

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A paper-based multicolor colorimetric method for detecting acetylcholinesterase activity or organophosphorus pesticides, characterized in that: The following steps are involved: The gold nanopipette was deposited on the nylon membrane to construct a paper-based analytical device; + The reaction solution was added to the paper-based analytical device and the Au + The disproportionation reaction induces the transformation of the nano-gold bipyramid morphology, which causes the nano-gold bipyramid to produce color changes and longitudinal localized surface plasmon resonance peak λ LLSPR The position changes can realize visual semi-quantitative detection and UV spectrophotometric quantitative detection of acetylcholinesterase activity or organophosphorus pesticides.

2. The paper-based multicolor colorimetric method for detecting acetylcholinesterase activity or organophosphorus pesticides according to claim 1, characterized in that: When Au + The aspect ratio of the gold nanoparticles can be increased by inducing a disproportionation reaction. LLSPR The color of the gold nanopipette changes from brown-red to red-purple, indicating that the sample contains acetylcholinesterase.

3. The paper-based multicolor colorimetric method for detecting acetylcholinesterase activity or organophosphorus pesticides according to claim 1, characterized in that: When Au + The disproportionation reaction cannot occur, and the morphological change of the nano-gold bipyramid is inhibited, which eventually leads to the λ LLSPR The color of the nanogold bipyramids changes from red-purple to brown-red, indicating that the sample to be tested contains organophosphorus pesticides.

4. The paper-based multicolor colorimetric method for detecting acetylcholinesterase activity or organophosphorus pesticides according to claim 1 or 2, characterized in that: When detecting acetylcholinesterase activity, the + The reaction solution includes: choline oxidase, acetylcholine chloride, TCEP, CTAB, HCl and Au + Solution; The detection process is: Nanogold bipyramids were deposited on nylon membranes to construct paper-based analytical devices; The sample to be tested is mixed with a buffer solution containing choline oxidase, acetylcholine chloride and TCEP to obtain an enzymatic solution; The enzymatic solution was incubated and then the Au solution containing CTAB and HCl was added. + solution, and mixing to obtain a reaction solution; After the reaction solution is incubated, it is added to a paper-based analysis device and reacted at 35-50°C for 2-10 minutes. The color of the nano-gold bipyramid deposition film and / or the extinction spectrum of the nano-gold bipyramid deposition film in the range of 400-1000nm are observed, and the nano-gold bipyramid λ is compared with the standard paper-based color card and / or the extinction spectrum is compared with the standard paper-based color card. LLSPR The displacement was substituted into the standard working curve to determine the acetylcholinesterase activity.

5. The paper-based multicolor colorimetric method for detecting acetylcholinesterase activity or organophosphorus pesticides according to claim 4, characterized in that: The incubation conditions of the enzymatic solution are: incubate at 35-40℃ for 25-35min; the incubation conditions of the reaction solution are: incubate at 45-55℃ for 3-7min; + The choline oxidase activity in the reaction solution is 5-60 U / L, the concentration of acetylcholine chloride is 50-400 μM, the concentration of TCEP solution is 14-22 μM, the concentration of CTAB is 20-60 mM, the concentration of HCl is 20-60 mM, and the concentration of Au + The concentration is 10-20 μM.

6. The paper-based multicolor colorimetric method for detecting acetylcholinesterase activity or organophosphorus pesticides according to claim 5, characterized in that: The incubation conditions of the enzymatic solution were: incubation at 37°C for 30 min; the incubation conditions of the reaction solution were: incubation at 50°C for 5 min; + The choline oxidase activity in the reaction solution was 40 U / L, the concentration of acetylcholine chloride was 100 μM, the concentration of TCEP solution was 20 μM, the concentration of CTAB was 40 mM, the concentration of HCl was 20 mM, and the concentration of Au + The concentration is 15 μM.

7. The paper-based multicolor colorimetric method for detecting acetylcholinesterase activity or organophosphorus pesticides according to claim 1 or 2, characterized in that: When detecting organophosphorus pesticides, Au + The reaction solution includes: acetylcholinesterase, choline oxidase, acetylcholine chloride, TCEP, CTAB, HCl and Au + Solution; The detection process is: Nanogold bipyramids were deposited on nylon membranes to construct paper-based analytical devices; Mixing the sample to be tested with a buffer solution containing acetylcholinesterase to obtain a mixed solution; The mixed solution is incubated, and then a buffer solution containing choline oxidase, acetylcholine chloride and TCEP is added and mixed to obtain an enzymatic solution; The enzymatic solution was incubated and Au containing CTAB and HCl was added. + solution, and mixing to obtain a reaction solution; After the reaction solution is incubated, it is added to a paper-based analysis device and reacted at 35-50°C for 2-10 minutes. The color of the nano-gold bipyramid deposition film and / or the extinction spectrum of the nano-gold bipyramid deposition film in the range of 400-1000nm are observed, and the nano-gold bipyramid λ is compared with the standard paper-based color card and / or the extinction spectrum is compared with the standard paper-based color card. LLSPR The displacement was substituted into the standard working curve to determine the concentration of organophosphorus pesticides.

8. The paper-based multicolor colorimetric method for detecting acetylcholinesterase activity or organophosphorus pesticides according to claim 7, characterized in that: The incubation conditions of the mixed solution are: incubate at 35-40℃ for 15-25min; the incubation conditions of the enzymatic solution are: incubate at 35-40℃ for 25-35min; the incubation conditions of the reaction solution are: incubate at 45-55℃ for 3-7min; + The activity of acetylcholinesterase in the reaction solution is 1-3U / L, the activity of choline oxidase is 5-60U / L, the concentration of acetylcholine chloride is 50-400μM, the concentration of TCEP solution is 14-22μM, the concentration of CTAB is 20-60mM, the concentration of HCl is 20-60mM, and the concentration of Au + The concentration is 10-20 μM.

9. The paper-based multicolor colorimetric method for detecting acetylcholinesterase activity or organophosphorus pesticides according to claim 8, characterized in that: The incubation conditions of the mixed solution were: incubation at 37°C for 20 min; the incubation conditions of the enzymatic solution were: incubation at 37°C for 30 min; the incubation conditions of the reaction solution were: incubation at 50°C for 5 min; + In the reaction system, the activity of acetylcholinesterase was 2U / L, the activity of choline oxidase was 40U / L, the concentration of acetylcholine chloride was 100μM, the concentration of TCEP solution was 20μM, the concentration of CTAB was 40mM, the concentration of HCl was 20mM, and the concentration of Au + The concentration is 15 μM.

10. The paper-based multicolor colorimetric method for detecting acetylcholinesterase activity or organophosphorus pesticides according to any one of claims 4 to 9, characterized in that: The reaction solution was added to a paper-based analytical device and reacted at 50°C for 6 minutes.

Images