JP2007068531A - Detection method for agrochemicals, detection kit for agrochemicals, and detection strip for agrochemicals - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detection method for agrochemicals which can quickly, simply, safely and inexpensively detect the residue of an agrochemical inhibiting neurotransmitter systems; to provide a detection kit agrochemicals, and to provide a detection strip for agrochemicals. <P>SOLUTION: The mechanism of acetylcholine esterase activity inhibition with an agrochemical is utilized. This detection method for detecting agrochemicals in a specimen comprises mixing the specimen with the acetylcholine esterase, adding the mixture solution to acetylthiocholine to react the mixture solution with the acetylthiocholine, and then detecting the produced thiocholine. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a pesticide detection method, a pesticide detection kit, and a pesticide detection strip for detecting pesticides remaining in a sample such as food.

  In recent decades, agriculture has used a large amount of agricultural chemicals to provide a stable supply of food as the global population increases. However, due to their use, residual chemical substances are found in drinking water and food, which has a harmful effect on humans.

  Among them, some pesticides, especially organophosphorus pesticides and carbamate pesticides, have been shown to cause acute toxicity, especially in small children. Late neuropathy is well known as a long-term effect of pesticide pesticides such as organophosphorus. This is called organophosphate-induced delayed (delayed) neurotoxin. Acute poisoning occurs by inhibiting acetylcholinesterase, an enzyme involved in neurotransmission, and delayed neuropathy is characterized by weakness and ataxia of the extremities that appear 1-2 weeks after acute organophosphate poisoning and subsequent paralysis. is there. Pathologically there is degeneration of long axons in the spinal cord and peripheral nerves. Degeneration of specific parts of the brain has also been revealed.

  Under such circumstances, the development of technologies that enable detection of residual pesticides has become an urgent task for the stable supply of safe and secure food and drinking water, prevention of pollution damage, and countermeasures. Pesticide detection methods and detection devices have been proposed. Currently, methods for detecting and measuring pesticide residues in agricultural crops are widely performed by instrumental analysis such as Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC), and Mass Spectrometer (MS). These methods have the advantages of high sensitivity, high accuracy, and the ability to analyze many pesticides at once if the same method is used to extract pesticides from crops.

  In addition, the development of a measurement method called bioassay (biological sample method), which is an application of immunoassay and applies this immunological technique to the analysis of pesticide residues, is progressing. According to this, the target substance (here, pesticide) can be identified using the reaction between the antigen and the antibody, and the amount of the substance can be measured from the degree of the reaction. Until now, antibodies with large molecular weights such as proteins could be produced with relative ease, but it was difficult to produce antibodies with substances containing metal elements, such as pesticides, depending on the type of low molecular weight substances. However, in recent years, the pavement for the production of antibodies against such substances has made it possible to analyze pesticides by immunoassay. When analyzed by this immunoassay, expensive analytical instruments such as instrumental analysis are not required, and only a relatively inexpensive instrument and a commercially available immunoassay analysis kit need be prepared. It is possible to perform analysis of 50 points or more.

  Another analytical method is the nitrobenzyl pyridine method. 4- (4-Nitrobenzyl) pyridine has been used as a color reagent for alkylating agents such as methyl iodide and is later used as a color reagent for organophosphorus pesticides in thin-layer chromatographs. This is how it came to be. There is also a sensor using nitrifying bacteria, and this method is a detection method using the property that nitrifying bacteria are inhibited even at extremely low concentrations with respect to various chemical substances.

For example, Non-Patent Document 1 below discloses a method for detecting residual agricultural chemicals by GC and HPLC.
J. et al. Sherma, G .; Zweig, Anal. Chem. 55 (1983), p. 57R

  However, the detection of pesticides by instrumental analysis such as GC, HPLC, MS, etc. mainly involves analysis for each pesticide, and it takes time for the inspection. Depending on the target pesticide, even a skilled researcher can analyze about 5 to 10 points. It is not rare that it takes several days. In addition, since a dangerous organic solvent is used, air conditioning equipment is necessary, the analyzer itself is expensive, and the maintenance cost and labor cost of the apparatus are high, so that a very large cost is required. In addition, there are problems that the operation is complicated, the number of samples that can be analyzed with one analytical instrument is small, and a high-purity organic solvent is used in a large amount at the time of pesticide extraction, and therefore dangerous work is involved.

  In addition, in pesticide analysis by immunoassay, one kit is required for one pesticide, and not all pesticides registered in Japan have immunoassay analysis kits. In addition, these analysis kits have a short period of use, may be difficult to analyze with different crops, have cross-reactivity, and this method is also a method for confirming safety during the production process. I'm not.

  In addition, the nitrobenzyl pyridine method has drawbacks such as requiring an apparatus that can be heated at 100 ° C. and requiring ether to visually determine, such as propopas that do not develop color in a trace amount. . The method using nitrifying bacteria has a drawback that the apparatus is large and expensive.

  As described above, the currently used methods have many problems and have not been used at each site. From the viewpoint of food safety, simulta- neously screening residues for a large number of pesticides at the same time, and quickly, easily, safely and inexpensively detect pesticide residues that are most harmful to the human body and inhibit the neurotransmission system. There is a strong demand for the development of detection methods.

  Therefore, the present invention has been proposed in view of such conventional circumstances, and a pesticide detection method, a pesticide detection kit, which can detect the residue of a pesticide that inhibits the neurotransmission system quickly, simply, safely and inexpensively, And it aims at providing an agrochemical detection strip.

  The inventors have been studying over a long period of time to achieve the above-mentioned object. As a result, in order to detect the residue of agricultural chemicals that inhibit the neurotransmission system quickly, simply, safely and inexpensively, the knowledge that it is effective to use the mechanism of acetylcholinesterase or cholinesterase activity inhibition by agricultural chemicals is effective. I came to get.

  Here, for the sake of understanding, the mechanism of the neurotransmission system will be described. FIG. 1 is an explanatory diagram for explaining the mechanism of the nerve transmission system.

In mammals including humans, individual muscle fibers (muscle cells) are dominated by a single motor nerve fiber. The part where the motor nerve terminal end and the muscle cell are in contact with each other is called a neuromuscular junction or end plate, and has a disk shape with a diameter of about 20 μm. The nerve endings end ^{Ca 2+} channels that selectively transmits ^{Ca 2+} is present densely. When the action potential reaches the nerve terminal end, the Ca ²⁺ channel opens due to depolarization (increase in permeability to Ca ²⁺ ), and Ca ²⁺ flows into the terminal end due to the concentration gradient, and acetylcholine is released. The details of the release mechanism of this transmitter are unclear, but its release depends on the Ca ²⁺ concentration in the nerve terminal end. The released acetylcholine stirs the nerve-muscle gap of about 50 nm, binds to the acetylcholine receptor present on the surface of the muscle cell membrane, and the acetylcholine receptor channel opens. This receptor channel is permeable to cations (Na ⁺ , K ⁺ , Ca ²⁺ ), so that the neuromuscular junction of myocytes exhibits depolarization. This depolarization is called end plate potential. When a muscle cell is depolarized, the permeability to Na ⁺ is increased and an action potential is generated, as in the case of nerve fibers, and this excitation is conducted to the entire length of the muscle cell and the muscle contracts. Under normal conditions, muscle cells should remain depolarized as long as acetylcholine released from motor nerve endings persists. However, the end plate potential peaks at about 1 msec and then decreases. This is because acetylcholine released from the nerve terminal is rapidly decomposed into choline and acetic acid by acetylcholinesterase present at the neuromuscular junction. The degradation product choline is taken up by the motor nerve terminal end, participates in the synthesis of acetylcholine again, and is released as a transmitter.

  Next, the mechanism of neurotransmitter inhibition by pesticides will be described. Since the release of acetylcholine and the response of its receptors are essential for normal nerve-muscle transmission, these abnormalities naturally cause impaired muscle activity. Pesticides that inhibit the neurotransmission system, such as organophosphorus and carbamate pesticides, irreversibly bind to acetylcholinesterase (AChE), which is an enzyme that degrades acetylcholine, which is a neurotransmitter. Therefore, these pesticides inhibit the action of AChE, which degrades acetylcholine, at the nerve synapse, resulting in excess acetylcholine. As a result, normal nerve transmission is not performed, causing various poisonings. This is the same when cholinesterase is used as an enzyme.

  The present invention has been completed on the basis of this neurotransmitter alienation mechanism, and utilizes the fact that acetylcholinesterase or cholinesterase is inhibited by nervous system inhibitory pesticides such as organophosphorus and carbamate pesticides and loses enzyme activity. Thus, pesticides are detected. FIG. 2 is an explanatory diagram for explaining the detection principle of the present invention. Acetylcholinesterase reacts with acetylthiocholine to produce thiocholine. On the other hand, a nervous system inhibiting pesticide binds acetylcholinesterase irreversibly and inhibits its action. Therefore, the sample and acetylcholinesterase are mixed, and the mixed solution is added to acetylthiocholine to detect the produced thiocholine. Depending on the amount of pesticide present in the sample, the activity of acetylcholinesterase is inhibited and the amount of thiocholine produced changes. By detecting the thiocholine produced, the pesticide in the sample is indirectly detected. By measuring the amount of thiocholine, it is possible to quantitatively detect the pesticide in the sample. The same applies to the case where cholinesterase is used as an enzyme.

  Various means for detecting thiocholine are conceivable, and examples thereof include a color former or a luminescent agent that reacts with thiocholine to develop color or emit light. As the color former, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) is suitable. DTNB forms a yellow pigment when it reacts with thiocholine. The amount of thiocholine produced varies depending on the concentration of the pesticide in the sample, and a yellow pigment is formed depending on the amount of thiocholine. The amount of thiocholine can be measured according to the degree of color development. If there is an agricultural chemical in the sample, the agricultural chemical irreversibly forms a complex with acetylcholinesterase or cholinesterase, and the reaction does not proceed and the color does not develop. On the contrary, when there is no pesticide (or the amount is small), the color develops without being inhibited.

  The present invention has been completed based on the above detection principle. That is, the pesticide detection method according to the present invention is characterized in that acetylcholinesterase or cholinesterase and a sample are mixed, the mixed solution is added to acetylthiocholine and reacted to detect the produced thiocholine.

  When acetylcholinesterase or cholinesterase is mixed with a sample, the acetylcholinesterase or cholinesterase activity is irreversibly bound to the pesticide that inhibits neurotransmitters such as organophosphorus and carbamate pesticides in the sample. Is inhibited. When the mixed solution is added to acetylthiocholine, acetylcholine and non-bonded acetylcholinesterase or cholinesterase and acetylthiocholine react with each other to produce thiocholine. That is, the amount of thiocholine produced varies depending on the concentration of the pesticide in the sample. By detecting the produced thiocholine, it becomes possible to indirectly detect the pesticide in the sample. According to the present invention, it is not necessary to analyze each type of pesticide separately, and it is possible to widely detect pesticides that inhibit neurotransmission systems such as organophosphorus pesticides and carbamate pesticides. Any of the steps can be performed easily and quickly, and since no dangerous elements are included, the safety is high. A large-scale device or an expensive solvent is not necessary, and detection can be performed at low cost.

  The acetylthiocholine is preferably fixed on a substrate. According to this invention, since acetylthiocholine is fixed to the substrate, it is easy to handle. In this fixed state, liquid acetylthiocholine may be dropped onto the substrate and dried and fixed, or liquid, gel, or polymer acetylthiocholine may be stored and fixed in a storage space provided on the substrate. It may be what you do.

  For the detection of the thiocholine, it is preferable to use a color former or a luminescent agent that reacts with thiocholine to develop color or emit light. According to the present invention, visual confirmation is possible by using a color former or a light-emitting agent that reacts with thiocholine to develop color or emit light.

  The color former or luminescent agent is preferably mixed with acetylthiocholine and fixed to the substrate. According to the present invention, the thiocholine production step and the coloring or coloring can be performed by a single operation of adding a mixed solution of a sample and acetylcholinesterase or cholinesterase to the color former or luminescent agent and acetylthiocholine fixed on the substrate. The light emission process is performed continuously.

  It is preferable to evaporate water after adding the mixed solution to the acetylthiocholine. Here, the evaporation of moisture may be slight evaporation or complete evaporation (drying). According to the present invention, when the sample and acetylcholinesterase or a mixed solution of cholinesterase are added to acetylthiocholine to be easily dried and the moisture is evaporated, the enzymatic reaction is quickly stopped and the pesticide is quickly removed. Can be detected.

  In mixing the acetylcholinesterase or cholinesterase with the sample, it is preferable to adjust the amount of acetylcholinesterase or cholinesterase mixed with the sample. According to this invention, the detection sensitivity of a pesticide (the threshold value of the detectable pesticide concentration) can be adjusted by adjusting the amount of acetylcholinesterase or cholinesterase mixed with the sample.

  The agrochemical detection kit of the present invention preferably comprises acetylthiocholine immobilized on a substrate, acetylcholinesterase or cholinesterase housed in a container, and detection means capable of detecting thiocholine.

  When the sample and acetylcholinesterase or cholinesterase contained in the container are mixed, the pesticide in the sample and acetylcholinesterase or cholinesterase are irreversibly bound, and the activity of acetylcholinesterase or cholinesterase is inhibited depending on the concentration of the pesticide in the sample. The When the mixed solution is dropped onto a substrate on which acetylthiocholine is fixed, thiocholine is produced by an enzymatic reaction of acetylcholinesterase that is not bound to pesticide or cholinesterase and acetylthiocholine. Agrochemicals can be indirectly detected by detecting the produced thiocholine by a detection means.

  The detection means is preferably a color former or a luminescent agent that develops color or emits light by reacting with thiocholine. Visual confirmation is possible by using a color former or a light-emitting agent.

  The color former or luminescent agent is preferably mixed with acetylthiocholine and fixed to the substrate. According to this invention, since the color former or the luminescent agent is mixed with acetylthiocholine and fixed to the substrate, handling is easy. A single operation of adding a mixture of a sample and acetylcholinesterase or cholinesterase to the color former or luminescent agent fixed on the substrate and acetylthiocholine, the thiocholine production process and the color development or luminescence process are continued. Done.

  The fixation is preferably dry fixation. According to this invention, since the added liquid mixture is easy to dry and the enzymatic reaction is stopped by drying, the agricultural chemical can be detected quickly.

  It is preferable that the amount of acetylcholinesterase or cholinesterase stored in the container can be adjusted. According to the present invention, when the sample and acetylcholinesterase or cholinesterase are mixed, the amount of acetylcholinesterase or cholinesterase added can be adjusted, and the detection sensitivity (the threshold value of the detectable pesticide concentration) can be adjusted.

  The agrochemical detection strip of the present invention comprises acetylcholinesterase or a region holding cholinesterase and a region where acetylthiocholine is immobilized.

  According to this invention, when the sample is impregnated in the region holding acetylcholinesterase or cholinesterase, the sample and acetylcholinesterase or cholinesterase are mixed, and the sample inhibits neurotransmission systems such as organophosphorus and carbamate pesticides. When an agrochemical is included, acetylcholinesterase (enzyme) or cholinesterase binds irreversibly, and the mixed solution reaches the region where acetylthiocholine is immobilized while the binding proceeds. In the region where acetylthiocholine is immobilized, acetylcholine and non-bonded acetylcholinesterase or cholinesterase and acetylthiocholine react with each other to produce thiocholine. That is, the amount of thiocholine produced in the region where acetylthiocholine is immobilized changes according to the concentration of the pesticide in the sample, and the amount of thiocholine is detected to detect the pesticide in the sample. Is possible. Simply impregnating acetylcholinesterase or a region holding cholinesterase with a reagent, the binding of acetylcholinesterase or cholinesterase and pesticide and the enzymatic reaction of acetylcholinesterase or cholinesterase and acetylthiocholine are carried out on one strip. Moreover, it can be detected quickly, and since it does not contain dangerous elements, it is highly safe. A large-scale device or an effective solvent is not necessary, and can be detected at low cost.

  In the agrochemical detection strip of the present invention, it is preferable that a coloring agent or a luminescent agent that develops color or emits light by reacting with thiocholine is further fixed in the region where the acetylthiocholine is immobilized. According to the present invention, the thiocholine produced in the region where acetylthiocholine is immobilized reacts with the color former or the luminescent agent to develop color or emit light, so that visual confirmation on the strip is possible.

  According to the agrochemical detection method, the agrochemical detection kit, and the agrochemical detection strip of the present invention, thiocholine produced by an enzymatic reaction between acetylthiocholine and acetylcholinesterase or cholinesterase is detected using the inhibition of the activity of acetylcholinesterase by the agrochemical. Because it can detect pesticides contained in the sample, complicated and dangerous work such as analysis and large-scale facilities are unnecessary, and it can be detected quickly, easily, safely, and inexpensively. Is possible.

  If a color former or a luminescent agent is used as the thiocholine detection means, visual confirmation is possible. By adding the mixed solution to the acetylthiocholine and then drying it, the enzyme reaction can be stopped, the color development state and the luminescence state can be confirmed, and the agrochemical can be detected quickly.

  When mixing the acetylcholinesterase or cholinesterase with the sample, the detection sensitivity of the pesticide (the threshold of the detectable pesticide concentration) can be adjusted by adjusting the amount of acetylcholinesterase mixed with the sample. In the agricultural chemical inspection, it is often inspected whether the agricultural chemical concentration in the sample is above or below a threshold value. By adjusting the detection sensitivity of a pesticide (the threshold of detectable pesticide concentration), it can be determined whether the concentration of pesticide in the sample is less than or less than the threshold based on the presence or absence of color development or luminescence, and the determination becomes easy.

  In addition, the agrochemical detection kit and the agrochemical detection strip of the present invention have a simple configuration that does not require a large-scale member or an expensive solvent, and can be reduced in size and cost. In addition, detection work and determination of results are easy, and dangerous work is unnecessary.

  Because of these advantages, the pesticide detection method, the pesticide detection kit, and the pesticide detection strip of the present invention can be widely used, and at various stages in the market, it inhibits the nerve transmission system that is most concerned about harm to the human body. By detecting pesticides to be used, safety can be secured, pollution damage prevention and countermeasures can be strengthened.

  Moreover, it is more effective when the agrochemical detection method, the agrochemical detection kit, and the agrochemical detection strip of the present invention are used for primary screening. That is, a sample is first subjected to simple, rapid, and inexpensive inspection using the agrochemical detection method, the agrochemical detection kit, and the agrochemical detection strip of the present invention. Perform an inspection. Undetected items are shipped as they are. As a result, it is possible to maintain accuracy while performing food inspection simply, quickly, and inexpensively, and can contribute to efficient pesticide inspection of food and stable supply of safe food. .

  Hereinafter, the agrochemical detection method, the agrochemical detection kit, and the agrochemical detection strip according to the present invention will be described in detail. In this embodiment, the case where acetylcholinesterase is used as an enzyme will be described as an example, but the same applies to the case where cholinesterase is used as an enzyme.

  The pesticides that can be detected by the pesticide detection method, pesticide detection kit, and pesticide detection strip of the present invention are pesticides that inhibit neurotransmission systems such as organophosphorus pesticides and carbamate pesticides. For example, the following are examples of neurotransmitter-inhibiting pesticides contained in rice. Examples of organophosphorus pesticides include Diazinon, Malathion, Parathion, Ethoprophos, Etrimfos, Chlorpyrifos, Chlorfenvinphos, Terbufos, Fentrothion, Fenthion, Phenthos and Butamis. Examples of carbamate pesticides include Aldicarb, Isoprocarb, Esprocarb, Oxamyl, Carbaryl, Thiobencarb, Pyricarbarb, Fenobucarb, Propamocarb, Bendiocarb, Methiocarb and the like.

<Pesticide detection method>
First, the agrochemical detection method of the present invention will be described. In the agrochemical detection method of the present invention, acetylcholinesterase and a sample are mixed, the mixed solution is added to acetylthiocholine to cause an enzyme reaction, and thiocholine produced as a result of the enzyme reaction is detected, thereby indirectly. It detects pesticides in samples.

  Here, the sample refers to various things that may be contaminated with agricultural chemicals, such as food and drinking water. Examples of food include fruits such as apples and mandarin oranges, agricultural products such as vegetables and cereals, processed foods such as frozen foods and frozen foods, and tap water and soft drinks as drinking water. The sample is not limited to these, and the present invention is widely applicable to various samples.

  First, as a pretreatment, the sample is brought into a state that can be inspected according to the present invention. In the case where the sample is of a high water content such as apples and mandarin oranges, the components are extracted in the state of fruit juice and used as a sample. Also, if the sample is poor in water such as rice, extract the components by crushing finely in a mixer, pulverizing the rice in hexane, methanol, etc., then evaporate the solvent and use the buffer. And re-dissolve to prepare an extract, which is used as a sample.

  Next, a pretreated solution sample and acetylcholinesterase are mixed to prepare a mixed solution A, which is allowed to stand for a predetermined time. When the pesticide contains a pesticide that inhibits the neurotransmission system, the pesticide and acetylcholinesterase are irreversibly bound to inhibit the activity of acetylcholinesterase. The amount of acetylcholinesterase that is inhibited varies depending on the concentration of the pesticide contained in the sample.

  Next, a predetermined amount of the mixed solution A of the agricultural chemical and acetylcholinesterase left for a predetermined time is added to acetylthiocholine. Acetylcholinesterase unbound with pesticides reacts with acetylthiocholine to produce thiocholine. Acetylcholinesterase combined with pesticides is inactive and does not cause enzymatic reaction. As a result, the amount of thiocholine produced reflects the pesticide concentration in the sample. When the pesticide concentration is high, the amount of thiocholine decreases, and when the pesticide concentration is low, the amount of thiocholine increases.

  Next, the produced thiocholine is detected. By detecting thiocholine, it becomes possible to detect pesticides in the sample.

For detection of thiocholine, it is preferable to use a color former or a luminescent agent that reacts with thiocholine to develop color or emit light. Visual confirmation is possible by using a color former or a light-emitting agent. As the color former, DTNB (5,5′-dithiobis-2-nitrobenzoic acid) is preferably used. When the reaction solution in which thiocholine was produced and DTNB were mixed, a yellow dye (5′-Mercapoto-2-nitrobenzoic)
acid) and develops a yellow color. As the amount of thiocholine increases, the degree of color development increases, and the amount of thiocholine, that is, the concentration of pesticide can be easily determined by simply checking the degree of color development visually.

  For detection of thiocholine, thiocholine may be detected separately by adding a mixed solution A of an agricultural chemical and acetylcholinesterase to acetylthiocholine and then separately using a color former or a luminescent agent. In addition, detection means such as acetylthiocholine and a color former or a luminescent agent may be mixed in advance, and a mixed solution A of an agricultural chemical and acetylcholinesterase may be added thereto. According to this, the mixed solution is added. In a single operation, thiocholine detection (formation of a yellow pigment in the case of DTNB) is continuously performed from the enzyme reaction.

  In addition, acetylthiocholine or acetylcholine and detection means (DTNB or the like) are preferably dried and fixed on a filter paper or the like. Thereby, when it mixes with the liquid mixture A and an enzyme reaction is carried out, a reaction liquid becomes easy to evaporate. As the reaction solution evaporates, the enzyme reaction stops and the enzyme reaction does not proceed any further, so that the colored state can be maintained. When acetylthiocholine is dried and fixed on a filter paper or the like, the enzymatic reaction stops and the color development state is maintained, so that the examination can be performed promptly.

  Further, in the agricultural chemical inspection, it is often inspected whether the agricultural chemical concentration in the sample is equal to or higher than a threshold value. When preparing the mixed solution A of the sample and acetylcholinesterase, it is possible to adjust the detection sensitivity (the threshold value of the detectable pesticide concentration) by adjusting the amount of acetylcholinesterase mixed with the sample. Depending on the amount of acetylcholinesterase mixed, the amount of acetylcholinesterase whose activity is inhibited by the pesticide in the sample varies. If a certain concentration of pesticide in the sample and the mixed acetylcholinesterase form a complex without excess or deficiency, thiocholine is not generated at the pesticide concentration above the threshold, with the pesticide concentration as the threshold, and at the pesticide concentration below the threshold, the pesticide Depending on the concentration, thiocholine is produced. Only by confirming whether thiocholine has been detected (that is, whether or not color has developed), it is possible to easily determine whether the concentration of the pesticide is greater than or less than a threshold value.

<Agricultural chemical detection kit>
Next, the agrochemical detection kit K of the present invention will be described. FIG. 3A is a plan view of the agrochemical detection kit K, and FIG. 3B is an exploded perspective view of the substrate 1. The agrochemical detection kit K includes acetylthiocholine fixed to the substrate 1, acetylcholinesterase housed in the container 2, and detection means 3 capable of detecting thiocholine.

  The substrate 1 is a chip-shaped substrate including a resin-made container 1a such as a plastic having a recess in the central portion and an absorbent body 1b such as a filter paper disposed in the recess. As shown in FIG. 3B, the structure is such that the absorbent body 1b is fitted in the recess of the container 1a, and the absorbent body 1c is fixed to the container 1a with an O-ring 1c. The absorber 1b can be replaced by removing the O-ring 1c, and can be reused simply by replacing the absorber 1b. Acetylthiocholine is dried and fixed on the absorber 1. Dry fixation means a state in which acetylthiocholine is first dropped onto the absorber 1 and dried to fix the components to the absorber 1. The substrate 1 preferably has a shape in which the reaction liquid in the recess of the container 1a is likely to evaporate. In the present embodiment, the recess has a large opening upward.

  The container 2 is made of a resin such as plastic, and acetylcholinesterase is accommodated in the container 2. The container 2 is provided with a takeout port so that a desired amount of acetylcholinesterase can be taken out and mixed with the sample.

  The detection means 3 may have any function that detects thiocholine and emits a signal in response to thiocholine. Examples thereof include a color former or a luminescent agent that develops color or emits light in response to thiocholine. In this embodiment, DTNB is used which reacts with thiocholine to form a yellow pigment, and the degree of color development changes according to the amount of thiocholine. This detection means (DTNB) 3 is mixed with acetylthiocholine, dried and fixed on the filter paper of the substrate 1, and is integrated with acetylthiocholine.

  This pesticide detection kit K is used as follows. First, as a pretreatment, a sample solution is prepared and used as a sample. This is the same as that described in the agrochemical detection method.

  Next, the sample on the solution and the acetylcholinesterase stored in the container 2 are mixed to prepare a mixed solution A. When the sample contains an agricultural chemical, it binds to acetylcholinesterase to form a complex, and the activity of acetylcholinesterase is inhibited depending on the concentration of the agricultural chemical.

  Next, the mixed solution A is dropped onto the absorbent body 1b on which the acetylthiocholine of the substrate 1 and the detection means 3 are dried and fixed. When acetylcholinesterase unbound with the pesticide is present in the mixed solution A, it reacts with acetylthiocholine immobilized on the absorber 1 to produce thiocholine. The detection means (DTNB) fixed to the absorber 1 reacts with the generated thiocholine to form a yellow pigment and develop a color. Since color develops according to the amount of thiocholine, the amount of thiocholine can be determined by visually confirming the degree of color development. That is, when the pesticide concentration in the sample is high, the degree of color development is weak, and when the pesticide concentration is low, the degree of color development is strong. The pesticide concentration in the sample can be confirmed by visually confirming this colored state.

  In addition, since acetylthiocholine is dried and fixed to the substrate 1, the reaction solution of acetylthiocholine and the mixed solution A is easily evaporated. When the reaction solution evaporates, the enzyme reaction stops, and the enzyme reaction does not proceed any further, so that the colored state can be maintained and the inspection can be performed quickly. This point is the same as the above-described pesticide detection method.

  Further, the detection means (DTNB) 3 may be separately housed in a separate container without being dried and fixed to the substrate 1. FIG. 4 is a plan view of an agrochemical detection kit K of another embodiment. Only acetylcholinesterase is dried and fixed on the substrate 1. After the mixed solution A is dropped onto the substrate 1, the detection means (DTNB) 3 housed individually is dropped onto the absorber 1 b of the substrate 1. As a result, the dropped detection means (DTNB) 3 reacts with thiocholine produced by the reaction between acetylthiocholine dried and fixed on the substrate 1 and the mixed solution A, and the yellow color is in a degree corresponding to the amount of thiocholine. Color develops.

  Moreover, when making the liquid mixture A of a sample and acetylcholinesterase, the point which becomes possible to adjust detection sensitivity by adjusting the mixing amount of the acetylcholinesterase with respect to a sample is the same as the said pesticide detection method. .

  In the present embodiment, acetylthiocholine is dried and fixed to the base 1. For example, a storage space is provided in the base 1, and liquid, gel, polymer, or other acetylthiocholine is stored in the storage space. You may fix by doing.

<Agricultural chemical detection strip>
Next, the agrochemical detection strip S of the present invention will be described. FIG. 5 is a perspective view of the agrochemical detection strip S. FIG. The agrochemical detection strip S has a strip shape and includes an impregnation portion 10 that is a region holding acetylcholinesterase, and a detection unit 11 that is a region where acetylthiocholine and a color former are immobilized. In the present embodiment, a member that fulfills the functions of the respective parts 10 and 11 is attached to a strip-shaped support 12. An impregnation unit 10 is provided on the upstream side of the agrochemical detection strip S, and a detection unit 11 is provided on the downstream side with a distance H from the impregnation unit 10. A sample pad 13 is provided so that the impregnation part 10 is sandwiched between the support 12 and an absorption pad 14 is provided downstream of the detection part 11.

  As the support 12, a membrane used in this type of immunochromatography can be used without limitation, and for example, nitrocellulose or the like can be used. In this embodiment, strip-shaped nitrocellulose is used.

  The impregnation unit 10 holds the acetylcholinesterase in a movable (developable) state. In the present embodiment, glass wool formed into a strip shape is used by impregnating and holding a solution containing acetylcholinesterase. A sample pad (for example, nitrocellulose excellent in water absorption) 13 is disposed on the upper surface of the glass wool, and the reaction time is set so that the sample is easily held in the impregnation portion 10 disposed between the support 12 and the sample pad 13. Secured. Moreover, the impregnation part 10 is made of glass wool having a relatively low adsorption capacity so that movement (development) of acetylcholinesterase or the like in the direction of the detection part 11 is performed smoothly. The member of the impregnation part 10 is not limited to this, and for example, a glass fiber nonwoven fabric, a cellulose cloth, a porous plastic cloth such as polyethylene, polypropylene, or the like can be used.

  The detection unit 11 is fixed with acetylthiocholine and a color former that reacts with thiocholine and develops color. The member of the detection unit 11 only needs to be able to fix acetylthiocholine and the color former, and in the present embodiment, strip-shaped nitrocellulose is used. In addition, nylon or PVDF can be used. Acetylthiocholine or a color former may be directly immobilized on a predetermined position of the support 12. Instead of the color former, a luminescent agent that reacts with thiocholine to emit light may be used.

  A gap H is provided between the impregnation unit 10 and the detection unit 11. Since the reaction proceeds between the acetylcholinesterase held in the impregnation unit 10 and the sample impregnated in the impregnation unit 10 until reaching the detection unit 11, the interval H plays a role of adjusting the reaction time. Therefore, the interval H is preferably a distance corresponding to a desired reaction time.

  The absorption pad 14 is nitrocellulose or the like excellent in water absorption, and promotes movement (development) of the sample solution or the like from the upstream side to the downstream side, or absorbs excess sample solution or the like.

  The pesticide detection strip S is used for an immunochromatographic test in which a sample is absorbed at one end of the support 12 and the sample is developed in the lateral direction by using a capillary phenomenon. First, as a pretreatment, a sample solution is prepared and used as a sample. This is the same as that described in the agrochemical detection method.

  Next, the impregnation part 10 of the pesticide detection strip S is impregnated with a solution sample. When the acetylcholinesterase held in the impregnation unit 10 and the impregnated sample are mixed and the pesticide is contained in the sample, the acetylcholinesterase is combined with the acetylcholinesterase to form a complex, depending on the concentration of the pesticide As a result, the activity of acetylcholinesterase is inhibited.

  The impregnated sample, acetylcholinesterase, and the like are developed on the pesticide detection strip S from upstream to downstream and reach the detection unit 11. The reaction between the pesticide and acetylcholinesterase is proceeding during the development, and the reaction progresses to a moderate level when reaching the detection unit 11 due to the interval H. At this time, the action of spreading from the impregnation unit 10 toward the detection unit 11 works due to the absorption force of the absorption pad 14, and the sample or the like reaches the detection unit 11 more reliably. Further, the extra sample after reaching the absorption pad 14 is absorbed by the absorption pad 14.

  When acetylcholinesterase unbound with pesticide is present in the sample that has reached the detection unit 11, it reacts with acetylthiocholine immobilized on the detection unit 11 to generate thiocholine. Further, the color former fixed to the detection unit 11 reacts with the generated thiocholine to form a dye and develop a color. Since the color former develops a color with an intensity corresponding to the amount of thiocholine, the amount of thiocholine can be determined by visually confirming the degree of color development. That is, when the pesticide concentration in the sample is low, the degree of color development is strong, and when the pesticide concentration is high, the degree of color development is weak. The pesticide concentration in the sample can be confirmed by visually confirming this colored state. What is necessary is just to confirm the grade of light emission, when the light-emitting agent is fix | immobilized.

  Note that only acetylthiocholine may be fixed to the detection unit 11 and measurement may be performed by a separately prepared thiocholine measurement means. However, as in the present embodiment, when the color developing agent or the light emitting agent is fixed to the detection unit 11, when the sample reaches the detection unit 11, the result can be visually confirmed by color development or light emission. And it can be done promptly.

  As described above, the agrochemical detection method, the agrochemical detection kit, and the agrochemical detection strip of the present invention require a short reaction time and work time, and do not require complicated and dangerous work such as analysis, and large and expensive equipment. It is. Since a dedicated analysis or the like is not required for each type of agricultural chemical, neurotransmitter-inhibiting agricultural chemicals can be widely detected only by the agricultural chemical detection method and the agricultural chemical detection kit of the present invention. Therefore, it is possible to detect agrochemicals in a sample quickly, simply, safely and inexpensively. Quantitative detection is possible by using a detection means that changes the signal according to the amount of thiocholine. For example, if a color former such as DTNB or a luminescent agent is used, the detection result can be easily confirmed visually. it can.

  In addition, in the agricultural chemical inspection, it is common to inspect whether a sample has an agricultural chemical concentration equal to or higher than a threshold value. According to the agrochemical detection method and the agrochemical detection kit of the present invention, by adjusting the amount of acetylcholinesterase mixed with the sample, the detection sensitivity (threshold of the detectable agrochemical concentration) can be adjusted, and whether or not it has been detected. It is possible to easily determine whether the pesticide concentration is greater than or less than a threshold value simply by confirming.

  From the above advantages, the agrochemical detection method and the agrochemical detection kit of the present invention can be widely used and can be used in various scenes such as food production, shipment, sale, and purchase. Thereby, a stable supply of safe and secure food can be achieved, and prevention and countermeasures against pollution damage can be strengthened.

<Experiment 1> Production of Pesticide Detection Kit In the following, the pesticide detection kit of the present invention was produced using diazinon, which is an organophosphorus pesticide, as an agrochemical to be detected. Since the regulated value of diazinon is defined as 0.1 ppm, the agrochemical detection kit was manufactured based on the results of the following experiments so that diazinon can be detected when the concentration is 0.1 ppm or more.

  First, we examined acetylcholinesterase, which proceeds rapidly in a 96-well plate, and then used a diazinon oxon body (Fig. 6), an organophosphorus pesticide, so that this reaction system can detect residual pesticides sufficiently. We examined whether to have. And the reaction system which can control color development according to the residual agricultural chemical regulation value was examined. Based on these results, conditions of DTNB, acetylthiocholine, acetylcholinesterase to be immobilized on a chip-like substrate were examined.

(1-1) Sample and apparatus Acetyl cholinesterasefrom is used for enzyme activity measurement.
bovineserum (Wako Pure Chemical Industries), Acetylcholinesterase
eel (SIGMA), Acetylthiocholine chloride (SIGMA), 5,5′-dithiobis-2-nitrobenzoicacid (DTNB, DOJINDO), and Diazinon oxon standard (Wako Pure Chemical Industries) were used. Filter paper 1 (ADVANTEC) was used as a membrane to be mounted in the central recess of the chip-like substrate.

  The pH of the PBS was adjusted with a glass electrode type hydrogen ion concentration indicator SS002 (Horiba Seisakusho). The 96-well plate was a 96-well microplate (NUNK immunomodule), and the absorbance was measured with Multi-spectrometer (Dainippon Pharmaceutical Co., Ltd.). The absorbance at a glass electrode type hydrogen ion concentration indicator SS002 (Horiba Seisakusho) and a chip-shaped substrate was measured with a diode-type photoelectric photometer (Biomedia).

(1-2) Reaction characteristics of acetylcholinesterase Diazinon, an organophosphorus pesticide, is stable under neutral conditions, but slowly hydrolyzed under alkaline conditions and more rapidly under acidic conditions. The sample was prepared with 50 mM PBS (pH 7). All reactions were performed at room temperature (25 ° C.).

(1-2-1) Examination of acetylcholinesterase The type of acetylcholinesterase used was examined. There are two types of subjects to be studied: those derived from bovine serum and those derived from electric eel. For each type, prepare acetylcholinesterase at a concentration of 0.9 U / ml and 0.45 U / ml, and use each type and each concentration of acetylcholinesterase to obtain acetylcholinesterase (40 μl) and 0.43 μmol / ml. Acetylcholine ATCh (40 μl), 10 μmol / ml DTNB (8 μl) and pH 7 PBS (200 μl) were mixed, reacted at room temperature, and the absorbance (λ = 410 nm) was measured over time using a multi-spectrometer. It was measured. FIG. 7 shows the result. As a result of the experiment, bovine serum-derived acetylcholinesterase derived from bovine serum was used as an enzyme because the reaction rate was high.

(1-2-2) Reaction Characteristics in Inhibition of Bovine Serum-Derived Acetylcholinesterase In preparing the pesticide detection kit, diazinon, a kind of organophosphorus pesticide, was used. 0.9 U / ml bovine serum-derived AChE (40 μl) and diazinon oxon body (25 ppm to 0.05 ppt) (40 μl) were mixed and allowed to stand for 2 minutes. After inhibiting the enzyme activity of Ache, 0.43 μmol / ml ATCh (40 μl and 10 μmol / ml) 8 μl of DTNB and 200 μl of 50 mM PBS were mixed and reacted, and the absorbance (λ = 410 nm) after 10 minutes was measured.

  0.9 U / ml bovine serum-derived AChE (40 μl) and diazinon oxon body (5-0.025 ppb) (40 μl) were mixed (2 to 60 minutes) and allowed to stand to inhibit the enzyme activity of AChE, 0.43 μmol / ml The reaction was carried out by mixing 8 μl of 10 μmol / ml DTNB and 160 μl of 50 mM PBS, and measuring the absorbance after 10 minutes (λ = 410 nm). We examined whether the reaction system used this time was sensitive enough to detect residual pesticides in foods.

  After reacting AChE with the diazinone oxon body, which is an organophosphorus pesticide as an inhibitor, to form an enzyme-inhibitor complex, the substrate ATCh and the color former DTNB were added to measure the residual enzyme activity. It was found that 5 ppb diazinon oxon body can be measured in this reaction system (FIG. 8).

  Next, as a result of changing the enzyme-inhibitor complex formation reaction time and measuring the residual enzyme activity, the formation time can be measured up to 0.5 ppb in 10 minutes, 0.25 ppb in 20 minutes, and 0 in 60 minutes. It was found that measurement was possible up to 1 ppb (FIG. 9). From this result, it was found that there is sufficient sensitivity to detect the residual regulation value of diazinon.

(1-2-3) Optimization of reaction conditions Detection was performed using diazinon which is an organophosphorus pesticide. Then, a reaction system (a reaction system that develops color when the diazinon concentration is 0.1 ppm or more and does not develop color when the diazinon concentration is 0.2 ppm or less) according to the residual regulation value (0.1 ppm) of diazinon in food was examined. .

  The amount of ATCh, DTNB and diazinon added was kept constant, and the amount of 2.4 U / ml AChE was changed to examine the color development. Specifically, 2.4 U / ml bovine serum-derived AChE (30, 60, 70 μl) and diazinon solution (1 to 0.001 ppm, 5 μl) were mixed and allowed to stand for 5 minutes to inhibit the enzyme activity of AChE. Thereafter, 0.43 μM ATCh (40 μl), 10 μM DTNB (8 μl) and PBS were mixed and reacted, and the absorbance (l = 410 nm) was measured over time.

  The result is shown in FIG. When the color development is measured by changing the amount of AChE to 30, 60, 70 μl, when 70 μl of 2.4 U / ml AChE is added, the reaction system develops color at a diazinon concentration of 0.1 ppm or less and does not develop color at 0.2 ppm or more. Was able to be produced.

(1-3) Production of agrochemical detection kit (1-3-1) Examination of immobilization amount of DTNB to membrane Based on the result of optimization of the above reaction conditions, optimization of immobilization of DTNB and ATCh to membrane We studied
8, 20, 50 μl of 10 μmol / ml DTNB was dropped on the membrane, and air-dried overnight for immobilization. 2.4 U / ml bovine serum-derived AChE (70 μl) and 0.43 μmol / ml ATCh (30 μl) were mixed and reacted, dropped onto the membrane, and the absorbance was measured over time using a diode-type photoelectric photometer. The amount of DTNB immobilized on the membrane was examined.

  From the above results, 8 μl of 10 μmol / ml DTNB was immobilized on the membrane. However, when DTNB is dropped on the membrane, the solution spreads and is fixed, and thus the color development may be reduced. Therefore, a large amount of 20, 50 μl was immobilized on the membrane and the three color developments were compared to examine the amount of immobilization. Even when the reaction is performed on the membrane and the color development is seen, all three do not change and the background does not change (FIG. 11). Therefore, the amount of 10 μmol / ml DTNB immobilized on the membrane was 8 μl.

(1-3-2) Examination of Optimal AChE Amount on DTNB Immobilized Membrane 8 μl of 10 μmol / ml DTNB was dropped on the membrane, and air-dried overnight for immobilization. 2.4 U / ml bovine serum-derived AChE (70, 80, 100, 120 μl) and diazinon oxon body (1 to 0.001 ppm) 5 μl were mixed and allowed to stand for 5 minutes to inhibit the enzyme activity of AChE. 30 μl of ATCh (43 μmol / ml) was mixed and reacted, 100 μl was dropped onto the membrane, and the absorbance was measured over time. By changing the amount of AChE, a reaction system capable of controlling color development in accordance with the residual regulation value of diazinon in foods was examined.

  Based on the above results, using a chip-like substrate on which 8 μl of 10 μmol / ml DTNB was immobilized, the amount of AChE was changed and reacted on the membrane, and color development was observed. From the result of the above (1-2-3), 70 μl of AChE was dropped, but almost no color was observed at a diazinon concentration of 0.1 ppm. Therefore, when the amount of AChE is 70 μl, it is considered that most of the complex with the diazinon oxon body is formed and no enzyme activity remains. Then, the reaction system which increases the amount of AChE to be reacted and develops color at 0.1 ppm was examined. When the amount of AChE reacted with the diazinon oxon body was increased to 80, 100, and 120 μl, the color was developed at a diazinon concentration of 0.1 ppm at 120 μl, and the color development was suppressed at 0.2 ppm (FIG. 12).

(1-3-3) Examination of ATCh Immobilization Concentration on Membrane 8 μl of 10 μM DTNB and (0.43, 1.0, 2.0, 3.0, 4.3 μmol / ml) ATCh 30 μl dropped on the membrane Then, it was naturally dried overnight and fixed. 100 μl of 2.4 U / ml bovine serum-derived AChE was dropped onto the membrane, and the absorbance was measured over time. The ATCh concentration was changed and the optimum immobilization concentration was examined.

  From the above results, 8 μl of 10 μmol / ml DTNB was immobilized on the membrane. From the above results, 0.43 μmol / ml ATCh (30 μl) was dropped onto the membrane and immobilized. However, when ATCh is dropped on the membrane, the solution spreads and is fixed, and it is considered that the color development becomes thin. In addition, ATCh may be denatured because it is naturally dried at room temperature. Therefore, 30 μl of ATCh having a high concentration (1.0, 2.0, 3.0, 4.3 μmol / ml) was also immobilized on the membrane, and the immobilized concentration was examined by comparing the color development. Even if the concentration of ATCh is 0.43 μmol / ml, the color development is thin, and as the concentration increases, the color development becomes deeper. However, when ATCh is 3.0, 4.0 μmol / ml, the background value is also increased. It is getting higher (FIGS. 13 and 14). Therefore, the reaction was allowed to produce a certain degree of color development, and 2.0 μmol / ml at which the background did not develop color was defined as the ATCh immobilization concentration.

(1-3-4) Preparation of pesticide detection kit (examination of optimal AChE amount)
8 μl of 10 μmol / ml DTNB and 30 μl of 2.0 μmol / ml ATCh were dropped on the membrane, followed by natural drying overnight and immobilization to produce an agrochemical detection kit. Then, using this pesticide detection kit, the construction of a reaction system that can control the color development according to the regulation value of diazinon was examined.

  A reaction system capable of controlling color development in accordance with the residual regulation value of diazinon in food by changing ATCh, DTNB, and diazinon constant and changing the amount of 2.4 U / ml AChE added. Specifically, 2.4 U / ml bovine serum-derived AChE (80, 100, 120 μl) and diazinon solution (1-0.001 ppm) 5 μl were mixed and allowed to stand for 5 minutes to inhibit the enzyme activity of AChE. Thereafter, 100 μl (85 μl was dropped when the amount of AChE was 80 μl) was dropped on the membrane, and the absorbance was measured over time.

  FIG. 15 shows the result. As the amount of AChE was increased to 80, 100, and 120 μl, color development at a diazinon concentration of 0.1 ppm also increased. The difference in color development from 0.2 ppm or less when AChE was 120 μl was well understood.

  From the above, after reacting bovine serum-derived AChE with the diazinone oxon body as an organophosphorus pesticide which is an inhibitor to form an enzyme-inhibitor complex, ATCh and DTNB were added to measure the residual enzyme activity. It was found that the reaction system conducted this time can measure up to 0.1 ppb, and that it has sufficient sensitivity to detect the residual regulation value of diazinon.

  A reaction system that can control the color development according to the residual regulation value of diazinon was investigated. 2.4 U / ml AChE (70 μl) and diazinon oxon body (5 μl) were mixed and allowed to stand for 5 minutes to inhibit the enzyme activity of AChE. Then, 0.43 μmol / ml ACh (30 μl) and 10 μmol / m DTNB (8 μl) were added. By mixing and reacting with 160 μl of PBS, color development could be controlled in accordance with the residual regulation value.

  Based on the above findings, the conditions of DTNB and ATCh to be immobilized on the membrane were examined. As a result, 10 μml / ml DTNB was immobilized at 8 μl and 2.0 μmol / ml ATCh at 30 μl and dried overnight at room temperature. Used as a pesticide detection kit. Using the pesticide detection kit prepared here, a reaction system capable of controlling color development in accordance with the residual regulation value of diazinon was examined. By mixing 120 μl of 2.4 U / ml AChE with 5 μl of diazinon oxon body and allowing to stand for 5 minutes and inhibiting the enzyme activity of AChE, the color development could be controlled in accordance with the residual regulation value.

  In this example, color development was evaluated using inhibition of diazinon, which is an organophosphorus pesticide, using a pesticide detection kit in which reactants were immobilized on a membrane. As a result, it was possible to control the color development according to the regulation value of residual agricultural chemicals. Therefore, the pesticide detection was performed from the actual food sample using the pesticide detection kit produced here.

<Experiment 2> Examination of pesticide detection from food Based on the above findings, we investigated the effect of ingredients in food on AChE, added diazinon oxon body to actual food and detected it with pesticide detection kit, or residual A reaction system capable of controlling color development according to the regulation value was examined.

(2-1) Sample and apparatus As a food sample, 100% of Made Maid apple juice concentrated reduced juice (Nippon Coca-Cola) and 100% of Minute Maid orange juice concentrated reduced fruit juice (Nippon Coca Cola) were used. In order to extract diazinon from food, hexane (Wako Pure Chemical Industries), sodium chloride (Wako Pure Chemical Industries), and acetone (Wako Pure Chemical Industries) were used. In order to adjust the pH of the food sample, a glass electrode type hydrogen ion concentration meter SS974 (Horiba) was used. Otherwise, the same reagents and equipment used in Chapter 2 were used.

(2-2) Detection of pesticides in foods Using the pesticide detection kit produced in Experiment 1 above, pesticides in foods were detected.

(2-2-1) Detection of pesticides in apple juice (2-2-1-1) Examination of acetylcholinesterase inhibition by apple juice The inhibition of AChE by components in apple juice was examined using the produced pesticide detection kit. . First, the pH of apple juice was measured and adjusted to 7. 2.4 U / ml of AChE (120 μl) and apple juice (5 μl) were mixed and allowed to stand for 5 minutes, and 100 μl of this mixed solution was dropped onto the membrane, and the absorbance was measured over time.

  The pesticide detection method of this invention was implemented using the produced agrochemical detection kit, and apple juice was directly measured. The pH of the apple juice itself was 3.75. This was adjusted to pH 7 and used for measurement. Residual enzyme activity was almost 0% at both pH 3.75 and pH 7 (FIG. 16). From this, it is thought that there exists a component which inhibits AChE in the component of apple juice.

(2-2-1-2) Examination of inhibition of acetylcholinesterase by organic solvent extraction solution and detection of diazinon in apple juice To 100 μl of apple juice adjusted to pH 7, add 100 μl of hexane and 100 μl of 5% aqueous sodium chloride solution and shake for about 1 minute. Then, the aqueous layer and the organic layer were separated, and 50 μl of hexane was further added to the aqueous layer and shaken for 1 minute. All organic layers were collected, and a solution in which 100 μl of 50 mM PBS (pH 7) was added to a place where all hexane was blown with nitrogen gas was used as an apple juice extract solution. It was examined whether this solution inhibits AChE. Further, the recovery rate was determined by measuring by GC-MS how much diazinon was extracted by hexane. A diazinon oxon body was extracted by the above-described extraction method in addition to apple juice so that the diazinon concentration was 0.01 to 1 ppm. When measured by GC-MS, the diazinon oxon body was dissolved in hexane and used for the measurement.

  Apple juice adjusted to pH 7 was extracted with hexane, and it was examined whether the extracted solution inhibited AChE. When AChE (120 to 170 μl) and 5 μl of the extraction solution were mixed, reacted, and dropped onto a chip-shaped substrate, the residual enzyme activity was almost 100% and no inhibition was observed (FIGS. 17, 18, and 19). Therefore, since diazinon is hydrophobic, it is extracted with an organic solvent, and since the component that inhibits AChE in apple juice is not included in the extraction solution, the extracted solution can be used for the measurement of residual pesticides. It is done. Moreover, the recovery rate was calculated | required using GC-MS how much diazinon was extracted with hexane. First, a calibration curve with respect to the peak area and retention time in the diazinon oxon body was obtained (FIG. 20). Next, the solution extracted from apple juice was measured to determine the peak area, and the recovery rate of the diazinon oxon body was determined from the calibration curve obtained earlier, which was 85% (FIG. 21).

  Next, it added to apple juice so that a diazinon density | concentration might be set to 0.01-1 ppm, and the diazinon oxon body was extracted with the extraction method just before, and was used for residual pesticide detection. 2.4 U / ml AChE (120-170 μl) and apple juice extract solution (diazinone concentration 1-0.001 ppm) 5 μl were mixed and allowed to stand for 5 minutes to inhibit the enzyme activity of AChE, and then 100 μl was dropped on the membrane. The absorbance was measured over time. We examined a reaction system that can control the color development according to the residual regulation value of diazinon in apples.

  A reaction system that uses a solution of apple juice extracted with diazinon (0.01-1ppm) extracted with hexane to detect residual pesticides and changes the amount of AChE to control the color development according to the regulation value of diazinon in apples. It was investigated. As the amount of AChE reacted with the diazinon oxon body was increased to 120, 140, 150, and 160 μl, color development at a diazinon concentration of 0.1 ppm also increased. When AChE was 160 μl, the difference in color development between the diazinon concentrations of 0.1 ppm and 0.2 ppm or more was well understood. When the amount of AChE is 170 μl, coloring occurs even when the diazinon concentration is 0.2 ppm, and the amount of enzyme is too large (FIGS. 17, 18, and 19). Therefore, in the case of apples, it is considered optimal when the amount of AChE is 160 μl.

(2-2-2) Pesticide detection in orange juice (2-2-2-1) Investigation of acetylcholinesterase inhibition by orange juice and detection of diazinon in orange juice Components in orange juice using the prepared pesticide detection kit The inhibition of AChE was investigated. The pH of the orange juice was measured and the pH was adjusted to 7. 120 μl of 2.4 U / ml AChE and 5 μl of orange juice were mixed and allowed to stand for 5 minutes. 100 μl of this mixed solution was dropped on the membrane, and the absorbance was measured over time.

Next, it added to orange juice so that a diazinon density | concentration might be set to 0.01-1 ppm, and it detected as it was, without extracting. 2.4 U / ml AChE (120-160 μl) and orange juice (diazinone concentration 1-0.001 ppm) 5 μl were mixed and allowed to stand for 5 minutes to inhibit the enzyme activity of AChE, and then 100 μl was dropped on the membrane. Absorbance was measured over time. A reaction system was studied in which the amount of AChE was changed, and the color development could be controlled in accordance with the residual regulation value in orange of diazinon.

  The agrochemical detection method of the present invention was carried out using the agrochemical detection kit of the above example, and orange juice was directly measured. The pH of the orange juice itself was 3.94. This was adjusted to pH 7 and used for measurement. Residual enzyme activity exceeded 100% for both pH 3.94 and 7 (FIGS. 22, 23, and 24). From this, it was found that in the case of orange juice, residual pesticides can be measured directly without extraction.

  Therefore, an orange juice added with diazinon (0.01 to 1 ppm) was used for residual pesticide detection, and a reaction system capable of controlling the color development according to the residual regulation value of diazinon in orange by changing the amount of AChE was examined. When the amount of AChE reacted with the diazinon oxon body was increased to 120, 130, 140, 150, 160 μl, the color development at a diazinon concentration of 0.1 ppm also increased. When AChE was 160 μl, the difference in coloration between the diazinon concentrations of 0.1 ppm and 0.2 ppm or more was clearly seen (FIGS. 22, 23, and 24). Therefore, in the case of orange, it is considered optimal when the amount of AChE is 160 μl. Further, the amount of AChE was 120 μl at the time of preparing the agrochemical detection kit in the above example, but the optimum amount this time is 160 μl, and the amount of AChE is increased as compared with the case of measuring by adding diazinon to PBS. This is due to factors such as the fact that the components in orange juice affect ATCh, which is a substrate, and are not easily decomposed into thiocholine, and that DTNB, which is a thiocholine and color former, is less likely to react with components in orange juice. It is thought that the amount of AChE has increased.

  From the above results, by carrying out the pesticide detection method of the present invention using the pesticide detection kit of the present invention, organophosphorus pesticides can be detected from foods, and some foods can be measured as real samples without being extracted. It was shown to be a simple primary screening tool. For carbamate pesticides, almost the same results were obtained, confirming that they were effective.

<Experiment 3> Production of Pesticide Detection Strip In producing a pesticide detection strip, the reaction time and the pesticide concentration were varied, and the extent to which acetylcholinesterase was inhibited by each concentration of pesticide was examined. In the experiment, diazinone, which is an organophosphorus pesticide, acetylcholinesterase (AChE) (WAKO, 2.4 U / ml), and DTNB (5,5′-dithiobis-2) which changes to blue according to the concentration of thiocholine as a color former. -Nitrobenzoic acid (DOJINDO), acetylthiocholine chloride (Sigma) was used.

  First, 40 μl of diazinon oxon body was mixed with 40 μl of AChE derived from 0.9 U / ml bovine serum while gradually changing from 0 ppb to 5 ppb. Each was allowed to stand for 2 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, and 60 minutes to change the reaction time, thereby inhibiting the enzyme activity of AChE. Subsequently, 0.43 μmol / ml ATCh (40 μl), 10 mmol / ml DTNB (8 μl) and 50 mM PBS (160 μl) were mixed and reacted, and the absorbance after 10 minutes (λ = 410 nm) was measured. The result is shown in FIG.

  From the results of the above experiments, as the reaction time progresses, the binding between the diazinon oxon body and AChE is promoted, the enzyme activity is inhibited, and the absorbance is low. From this, it can be seen that even a low concentration pesticide can be measured by taking a sufficient reaction time. In the case of a qualitative inspection such as the presence or absence of residual pesticides, it is possible to increase the reliability by taking a sufficient reaction time. Therefore, when aiming at a qualitative inspection, it is desirable to take a sufficient distance between the impregnation part of the agrochemical detection strip and the detection part.

  Further, when the reaction time is short (2 to 10 minutes), the absorbance of the diazinon oxon body having a relatively high concentration (5 ppb to 0.25 ppb) varies, and the reaction time is long (20 to 60 minutes). ), The absorbance of the diazinon oxon body having a relatively low concentration (0.1 ppb to 0.0025 ppb) varies, and it can be seen that the concentration that can be judged depends on the reaction time. From the above, it is possible to detect a pesticide at a desired concentration by adjusting the reaction time between the diazinon oxon body and acetylcholinesterase. Therefore, for the purpose of quantitative inspection, it is desirable to adjust the distance between the impregnation part of the agrochemical detection strip and the detection part according to the concentration of the agricultural chemical to be detected.

  Based on the results of the above experiment, a pesticide detection strip was prepared. As the support, strip-shaped nitrocellulose having a width of 4 mm and a length of 55 mm was used. The impregnation part was held by dropping 100 μl of 2.4 U / ml AChE onto glass wool having a width of 4 mm and a length of 8 mm, and attached to one end of the support. A sample pad having a width of 4 mm and a length of 16 mm was disposed on the upper surface of the glass wool, and the glass wool was sandwiched between the sample pad and the support. The detection unit is dried and fixed with acetylthiocholine and a color developing agent that reacts with thiocholine and develops a blue color on strip-shaped nitrocellulose with a width of 4 mm and a length of 5 mm, and is attached at an interval of 18 mm from the impregnation unit. It was. The reaction time of about 2 minutes was ensured by an interval of 18 mm provided between the impregnation part and the detection part. Furthermore, an absorption pad was attached to the other end side of the support so as to partially overlap the detection unit.

  The pesticide detection method was carried out using the produced pesticide detection strip. First, 400 μl of PBS (phosphate buffer solution) with 5 ppm diazinon oxon body was prepared as a sample, and impregnated into the impregnated portion of the produced agrochemical detection strip B and developed. For comparison, 400 μl of sample PBS not containing pesticide was impregnated into the impregnated portion of the produced pesticide detection strip A and developed. FIG. 26 shows a state after 7 minutes from the start of development. The detection part of the pesticide detection strip A impregnated with a material containing no pesticide began to turn blue. The pesticide detection strip B impregnated with materials containing pesticides showed no color change. It can be seen that a diazinon oxon body having a concentration of 5 ppm can be detected in the pesticide detection strip B of this example.

  The agrochemical detection strip of the present invention is not limited to the above-described embodiment, and various kinds of Pesticide detection strips can be obtained by adjusting the amount of AChE fixed to the impregnation part and the inhibition time (that is, the distance from the impregnation part to the detection part). It is possible to detect the concentration of pesticides. Low concentrations of pesticides can also be detected. For example, by detecting low concentrations of residual pesticides in food, it is possible to improve food safety with a simple test.

Explanatory drawing explaining the mechanism of neurotransmission system Explanatory drawing explaining the agrochemical detection principle of this invention (A) is a top view of the agrochemical detection kit of this Embodiment, (b) is a disassembled perspective view of a base | substrate. Plan view of a pesticide detection kit according to another embodiment Perspective view of the pesticide detection strip of the present embodiment Structural formula of diazinon oxon, an organophosphorus pesticide A graph of the results of two acetylcholinesterase studies Graphical representation of residual activity of AChE when using various diazinon concentrations A graph of residual enzyme activity when the inhibition reaction time of AChE and diazinon is changed. A graph of the results of optimization of reaction conditions The figure which shows the examination result of the amount of DTNB immobilization The figure which shows the examination result of AChE in the chip-shaped base | substrate which fix | immobilized DTNB A graph of ATCh immobilization results Photo showing the result of ATCh immobilization amount The figure which shows the examination result of the optimum amount of AChE The figure which shows the examination result of acetylcholinesterase inhibition by apple juice Figure showing the detection result of diazinon in apple extract (effect of apple extract on AChE) Diagram showing the detection result of diazinon in apple extract (graph of color change of chip-like substrate over time in detection of diazinon) Diagram showing the detection result of diazinon in apple extract (photo showing the color development of chip-shaped substrate in diazinon detection) Calibration curve of diazinon oxon body Graph showing the peak area of diazinon extraction solution The figure which shows the detection result of diazinon in orange (Effect of apple extract on AChE) Diagram showing the detection result of diazinon in orange (graph of color change of chip-like substrate over time in detection of diazinon) Diagram showing the detection result of diazinon in orange (photo showing the color development of the chip-like substrate in the detection of diazinon) Graph showing the results of examining the degree of inhibition of acetylcholinesterase by various concentrations of pesticides depending on the reaction time Photo showing the results of pesticide detection using the pesticide detection strip of this example

Explanation of symbols

K pesticide detection kit 1 base 1a container 1b absorber 1c o-ring 2 container 3 detection means S pesticide detection strip 10 impregnation part 11 detection part 12 support 13 sample pad 14 absorption pad H provided between the impregnation part and the detection part interval

Claims (13)

  A method for detecting pesticides, comprising mixing acetylcholinesterase or cholinesterase and a sample, adding the mixture to acetylthiocholine, causing the mixture to react, and detecting the produced thiocholine.   The method for detecting an agrochemical according to claim 1, wherein the acetylthiocholine is fixed to a substrate.   The pesticide detection method according to claim 1 or 2, wherein the thiocholine is detected using a color former that develops color by reacting with thiocholine or a luminescent agent that emits light.   4. The method for detecting a pesticide according to claim 3, wherein the color former or the luminescent agent is mixed with acetylthiocholine and fixed to the substrate.   The method for detecting a pesticide according to any one of claims 1 to 4, wherein the mixed solution is evaporated after being added to the acetylthiocholine.   The method for detecting an agrochemical according to any one of claims 1 to 5, wherein, when the acetylcholinesterase or cholinesterase and the sample are mixed, an amount of acetylthiocholine mixed with the sample is adjusted.   A pesticide detection kit comprising: acetylthiocholine fixed to a substrate; acetylcholinesterase or cholinesterase housed in a container; and a detection means capable of detecting thiocholine.   The agrochemical detection kit according to claim 7, wherein the detection means is a color former or a luminescent agent that develops color or emits light by reacting with thiocholine.   9. The agrochemical detection kit according to claim 8, wherein the color former or the luminescent agent is mixed with acetylthiocholine and fixed to a substrate.   The agrochemical detection kit according to any one of claims 7 to 9, wherein the fixation is dry fixation.   The agrochemical detection kit according to any one of claims 7 to 10, wherein the amount of acetylcholinesterase or cholinesterase stored in the container can be adjusted.   An agrochemical detection strip comprising acetylcholinesterase or a region holding cholinesterase and a region where acetylthiocholine is immobilized. 13. The agrochemical detection strip according to claim 12, wherein a color former or a luminescent agent that develops color or emits light by reacting with thiocholine is immobilized in the region where the acetylthiocholine is immobilized.

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