An electrochemical detection chip, detection kit and detection system based on enzyme inhibition method

Abstract

The invention discloses an electrochemical detection chip based on enzyme inhibition method, comprising: an electrode chip (1), at least having a working electrode (11) and a counter electrode (12), the working electrode (11) having at least an enzyme for electrochemical detection fixed thereon; a shell mechanism (2), wrapped on the electrode chip (1) and provided with an electrode lead outlet, and provided with a sample addition hole (221) at a position corresponding to the working electrode (11); a carrier sheet (4) prefabricated with a solid substrate is provided in the area outside the sample addition hole, and an auxiliary mechanism is provided to drive the carrier sheet (4) to extend into the sample addition hole after the enzyme reacts with a liquid sample containing a target analyte in the sample addition hole for a certain period of time. When performing detection, the sample to be detected is first dripped through the sample addition hole for a certain period of time, and then the carrier sheet prefabricated with the substrate is extended into the sample addition hole by the auxiliary mechanism, which greatly enhances the convenience and stability of operation and is conducive to rapid on-site detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of electrochemical detection chips, and in particular relates to an electrochemical detection chip based on an enzyme inhibition method, and a detection kit and a detection system comprising the chip. Background Art

[0002] The principle of analyzing the target by enzyme inhibition is as follows: the target to be measured inhibits the enzyme, affecting the reaction between the enzyme and the corresponding substrate, so that the corresponding signal output by the change of the substrate is also affected. The output signal is inversely proportional to the concentration of the target to be measured, thereby inferring the existence of the target to be measured or the amount of the target to be measured. This method is widely used in the detection of organophosphorus or carbamate pesticides, and can also be used for the determination of benzoic acid, a preservative in food (see Li Qingbo's "Sensor, Inhibition Kinetics and Application Based on Inorganic / Biological Organic Nanohybrid Membrane Electrode" Master's Thesis of Yangzhou University) or the determination of L-cysteine ​​(see Analytica Chimica Acta 399 (1999) 287–293). As long as the enzyme inhibition system and the corresponding enzyme and substrate reaction system are developed, it is possible to open up new application scenarios. In these scenarios, a two-step reaction is necessary, in which the enzyme inhibition reaction is the first reaction, and then the remaining enzyme reacts with the substrate to output a signal.

[0003] Taking the detection of organophosphorus or carbamate pesticides as an example, the principle of enzyme inhibition method is derived from the insecticidal mechanism of the aforementioned pesticides: organophosphorus and carbamate pesticides can inhibit the activity of acetylcholinesterase in the insect nervous system, thereby causing the accumulation of acetylcholine, a neurotransmitter, affecting the normal nerve conduction of insects, causing them to be poisoned and die. The inhibition rate of organophosphorus and carbamate pesticides on enzymes is positively correlated with their concentration. Based on the principle of enzyme inhibition method, the extract from the fruit and vegetable sample is contacted with enzymes such as acetylcholinesterase and wheat bran esterase. If there is no pesticide residue or the amount is very small in the extract, the activity of the enzyme is not inhibited and the corresponding substrate can be hydrolyzed; on the contrary, if the pesticide residue is relatively high, the activity of the enzyme is inhibited and the amount of substrate hydrolyzed is significantly reduced. By detecting the amount of substrate hydrolysis, an observable signal can be output to qualitatively determine the presence of pesticide residues, or by calculating the inhibition rate, the amount of pesticide residues in the sample extract can be quantitatively calculated.

[0004] Based on the above principle, enzyme inhibition test paper method (rapid color card) and enzyme inhibition spectrophotometry have been used in practice to detect organophosphorus pesticides and carbamate pesticides. However, the former is only a qualitative test, the latter instrument is large in size, and the quantitative results are easily interfered by pigments in fruits and vegetables.

[0005] In recent years, electrochemical detection of pesticide residues based on the principle of enzyme inhibition has attracted attention. The electrochemical method outputs signals quickly and is expected to become an effective means of on-site detection. Similarly, electrochemical detection based on the principle of enzyme inhibition requires at least two steps of reaction: first, the liquid containing pesticide residues needs to be contacted with the enzyme fixed on the electrode, so that the pesticide and enzyme undergo a first reaction for a certain period of time to inhibit the enzyme, and then an appropriate amount of substrate is used to undergo a second reaction with the remaining enzyme. The substrate is hydrolyzed by the enzyme to produce electroactive substances, based on which an electrical signal for detection can be output. The higher the amount of pesticide, the more enzyme inhibition, the less the amount of substrate hydrolyzed, and the weaker the output electrical signal.

[0006] Chinese patent publication number CN104198559B discloses an electrochemical biosensor for organophosphorus pesticide detection. In the embodiment, it is clear that a screen-printed electrode with acetylcholinesterase fixed thereon is placed in a certain concentration of organophosphorus pesticide solution for inhibition for 15 minutes, and then placed in a certain concentration of substrate-acetylcholine chloride solution, and the steady-state current is measured to calculate the content of organophosphorus. In this method, the enzyme electrode needs to be placed in different solutions twice. Therefore, during on-site detection, in addition to preparing sample extracts from fruits and vegetables, it is also necessary to prepare substrate solutions in advance, and the two solutions are in different containers. The enzyme-fixed electrode contacts the two solutions in turn to achieve the test goal.

[0007] Chinese patent publication number CN101587092A discloses a portable electrochemical biosensor for rapid detection of organophosphorus pesticide residues. The embodiment discloses another method of contacting a liquid with an enzyme electrode: a reaction pool is fixed on a screen-printed electrode fixed with acetylcholinesterase, 10 μL of control solution or sample extract is first added to the reaction pool, left for more than 15 minutes, and then 2 μL of substrate solution is dripped into the reaction pool, and the response current signal is read after 2 minutes. In this method. Although there is only one reaction pool on an enzyme electrode, which accommodates two samples in batches, the two samples still need to be prepared separately and dripped in sequence.

[0008] In the above schemes, the corresponding solution needs to be added dropwise twice, which is inconvenient in operation; another problem of the above method is that the substrate in solution is unstable and easily inactivated, and the substrate solution must be freshly prepared before use, otherwise it will lead to error in the results, which undoubtedly forms a significant restriction on the on-site performance of such methods. Therefore, there is still a need in the art to develop a simple, portable, rapid, accurate and quantitative detection device. Summary of the invention

[0009] The present invention is made to solve the above-mentioned shortcomings, and provides a novel portable electrochemical detection chip with an external substrate carrier sheet based on the enzyme inhibition method to overcome the existing problems in the art. The chip prefabricates the substrate that undergoes the second reaction with the enzyme in a carrier sheet in a solid form, and the prefabricated substrate-containing carrier sheet is first separated from the sample well, and then immersed in the solution of the sample well when necessary to release the substrate for reaction. This technical solution can save the amount of substrate, enhance the convenience of carrying and using the chip, and ensure the accuracy of the detection results and the timeliness of the chip.

[0010] For the purpose of illustrating the present invention, the two-step reaction for electrochemical detection of target analytes based on enzyme inhibition is defined as follows:

[0011] First reaction: The reaction between the target analyte and the enzyme. If the target analyte is contained in the sample extract, the first reaction will inhibit the enzyme.

[0012] Second reaction: The reaction between the substrate and the enzyme. Under the catalysis of the enzyme, the substrate is changed, and the changed product has electrochemical activity, and the corresponding electrical signal can be collected. This reaction must start after the first reaction has proceeded to a certain extent, so it is called the second reaction here.

[0013] The electrochemical detection method based on enzyme inhibition is not limited to any application scenario, and can be used as long as an enzyme inhibition system and a corresponding enzyme-substrate reaction system are developed. For example, the reaction system is the substrate-enzyme system of organophosphorus or carbamate pesticides mentioned in the background technology section, the substrate-enzyme system of benzoic acid or L-cysteine, etc.

[0014] In some embodiments, when detecting organophosphorus pesticides or carbamate pesticides, available enzyme systems include, but are not limited to, acetylcholinesterase system, butyrylcholinesterase system, tyrosinase system, class B plant esterase system, and the like.

[0015] When the enzyme fixed on the working electrode is acetylcholinesterase, the substrate can be selected from thioacetylcholine chloride and thioacetylcholine iodide; when the enzyme fixed on the working electrode is butyrylcholinesterase, the substrate can be selected from thiobutyrylcholine chloride and thiobutyrylcholine iodide; when the enzyme fixed on the working electrode is a class B plant esterase, such as wheat bran esterase, the substrate is -naphthyl acetate; when the enzyme fixed on the working electrode is tyrosinase, the substrate can be selected from catechol or 1,2-naphthoquinone-4-sulfonic acid.

[0016] In a preferred embodiment, the target analyte is an organophosphorus or carbamate pesticide; the substrate is thioacetylcholine chloride; and the corresponding enzyme immobilized on the working electrode is acetylcholinesterase.

[0017] In some embodiments, in order to improve the reaction sensitivity, an electron mediator material is also fixed on the working electrode. The electron mediator can reduce the oxidation potential of the product produced by the enzyme-catalyzed substrate hydrolysis, improve the anti-interference ability of the electrode and the sensitivity of detection. At the same time, some electron mediators oxidize very quickly on the electrode surface, which can greatly shorten the detection time. For example, Prussian blue or potassium ferrocyanide.

[0018] In a preferred embodiment, the target analyte is an organophosphorus or carbamate pesticide; the substrate is thioacetylcholine chloride; the corresponding enzyme fixed on the working electrode is acetylcholinesterase; and the electron mediator material is Prussian blue. Prussian blue can greatly reduce the oxidation potential of thiocholine chloride produced by the hydrolysis of thioacetylcholine chloride catalyzed by acetylcholinesterase, and has a good peak shape and a large peak current, effectively improving the sensitivity of detection.

[0019] In a first aspect, the present invention provides an electrochemical detection chip based on enzyme inhibition method, the structure of which is designed based on the above principle, and the specific structure is as follows, including:

[0020] An electrode chip (1) comprises at least a working electrode (11) and a counter electrode (12); at least an enzyme for electrochemical detection is fixed on the working electrode (11);

[0021] A shell mechanism (2) is wrapped around the electrode chip (1) and is provided with an electrode lead outlet, and a sample loading hole (221) is provided on the upper surface at a position corresponding to the working electrode (11) for adding a liquid sample containing a target analyte; a carrier sheet (3) prefabricated with a solid substrate is provided in the area outside the sample loading hole, and an auxiliary mechanism is provided for driving the carrier sheet (3) to extend into the sample loading hole after the enzyme reacts with the liquid sample containing the target analyte in the sample loading hole for a certain period of time.

[0022] In the present invention, a reference electrode (13) is also provided on the electrode chip (1), and the three electrodes are made on a substrate by a screen printing process to form an electrode chip, and the leads of the three electrodes are assembled at the front end of the chip.

[0023] The carrier sheet (3) and the sample loading hole (221) are first separated, and when the first reaction between the enzyme and the liquid sample in the sample loading hole is completed to a certain extent, the carrier sheet is immersed in the solution of the sample loading hole to carry out the second reaction. At this time, the solid substrate prefabricated at the carrier sheet (4) can be dissolved and used to carry out the second reaction with the enzyme to output an electrical signal.

[0024] In a simple implementation scheme provided by the present invention, the carrier sheet (4) prefabricated with the solid substrate is packaged separately, and when necessary, the packaging bag is opened and the carrier sheet is placed in the sample addition hole. In this case, the auxiliary mechanism is the hand of the sample tester, which may be slightly inconvenient in operation, but it can still solve the relevant technical problems.

[0025] In a preferred embodiment provided by the present invention, an outer cover (3) connected to the outer shell mechanism (2) is provided, and a carrier sheet (4) prefabricated with a solid substrate is fixed at a corresponding position of the outer cover (3). When the outer cover (3) is attached to the outer shell, the carrier sheet (4) extends into the sample loading hole (221).

[0026] Preferably, the housing structure (2) comprises a lower cover (21) and an upper cover (22) which are detachably connected. The lower cover (21) encapsulates the electrode chip (1) and is provided with an electrode lead outlet; the upper cover (22) is provided with a sample addition hole (221).

[0027] The auxiliary mechanism is an outer cover (3) mounted on the upper cover (22) in a folded form, on which a separately packaged carrier sheet (4) is arranged. The position of the carrier sheet (4) corresponds to the sample loading hole. When the outer cover (3) is folded and attached to the upper cover (22), the carrier sheet (4) is placed in the sample loading hole (221) and coincides with the position of the sample loading hole (221).

[0028] More preferably, the carrier sheet (4) is fixed on the outer cover (3) in a protruding form, and the outer surface is sealed by a clean film. When the carrier sheet (4) needs to be attached to the outer shell (2), the clean film is torn off to keep the substrate area clean before the reaction.

[0029] Further preferably, in order to achieve the connection between the upper and lower covers, the lower cover (21) and the upper cover (22) are installed by means of a plurality of buckles and slots. Specifically, a plurality of slots are evenly arranged on the periphery of the lower cover (21), and a plurality of buckles are correspondingly arranged on the bottom end of the upper cover (22).

[0030] In order to achieve a tight fit between the upper cover (22) and the outer cover (23), the upper cover (22) and the outer cover (23) are respectively provided with mutually matching buckles, so that the chip can be kept clean before initial use. When the buckles are opened, the sample addition hole (221) is exposed and can be used to receive liquid samples.

[0031] In another preferred embodiment provided by the present invention, the structure of the housing mechanism (2) remains unchanged, and comprises a lower cover (21) and an upper cover (22), and the upper cover (22) is provided with a sample addition hole (221).

[0032] The auxiliary mechanism (5) is slidably mounted on the upper cover (22), and one end close to the sample loading hole (221) is connected to the carrier sheet (4) via a flexible connecting belt (6). The carrier sheet (3) is laid flat on the upper cover (22) and is located between the auxiliary mechanism (5) and the sample loading hole (221).

[0033] Preferably, the auxiliary mechanism (5) comprises a slide groove (51) arranged on the housing and a slide plate (52) installed in the slide groove, wherein the upper surface of the slide plate (52) is provided with a toggle convex strip (521), and the lower surface is provided with a slider (522). When the first reaction is completed, the slide plate (52) is pushed to move in the slide groove, and the carrier sheet (4) is pushed forward and folded down into the sample loading hole (221). The flexible connecting belt (6) is preferably a foldable loose-leaf.

[0034] In this solution, the outer surface of the carrier sheet (4) is still sealed by the clean film. When the carrier sheet (4) needs to be moved to the sample loading hole, the clean film is torn off to keep the substrate area clean before the reaction.

[0035] In a variation of the preferred embodiment, a cavity may be processed in the upper cover of the housing, and the carrier sheet (4) may be placed in the cavity and pushed into the sample loading hole by the sliding block.

[0036] The substrate prefabrication method of the present invention is as follows: the substrate is prefabricated in a carrier sheet by spraying or injecting a substrate solution, and then used after drying. The carrier sheet material includes polyester cellulose film, glass cellulose film, etc., preferably polyester cellulose film.

[0037] There are many methods for fixing the enzyme on the working electrode, including but not limited to coating, embedding, deposition, etc., and the enzyme can be fixed by a known method as long as it does not affect the reaction activity of the enzyme.

[0038] When the above chip is used for detection, when the target analyte extract is injected into the sample loading hole of the chip, the enzyme on the working electrode first combines with the target analyte in the extract to perform a first reaction. After a certain period of time, a carrier sheet prefabricated with a substrate is attached or pushed to the sample loading hole. The carrier sheet is immersed in the liquid in the sample loading hole, the substrate is dissolved, and a second reaction occurs with the enzyme. The detection result can be obtained by collecting the electrical signal.

[0039] The second aspect of the present invention also provides an electrochemical detection kit based on enzyme inhibition method, comprising an electrochemical detection chip and a buffer solution, an extraction solution or a standard substance required for detection and analysis. The electrochemical detection chip is as described above.

[0040] The third aspect of the present invention provides an electrochemical detection system based on enzyme inhibition method, comprising: the above-mentioned electrochemical detection kit and an electrochemical detection device connected thereto.

[0041] The beneficial effects of the present invention are as follows:

[0042] Firstly, the present invention provides a carrier sheet prefabricated with a substrate, and arranges the carrier sheet on the area outside the sample loading hole, thereby meeting the two-step reaction requirement of electrochemical detection of the target analyte based on the enzyme inhibition method, and overcoming the trouble of carrying and adding additional substrate solution, reducing the use of substrate, extending the timeliness of the prefabricated substrate, and enhancing the consistency and stability of the detection. The detection method can better meet the needs of convenient and rapid detection.

[0043] Secondly, when the device of the present invention is detecting, the sample to be detected is first added through the sample addition hole for the first reaction. After the first reaction is carried out for a certain period of time, the carrier sheet prefabricated with the substrate is extended into the sample addition hole through the auxiliary mechanism, which greatly enhances the convenience and stability of the operation and is conducive to rapid on-site detection. The test shows that the detection limit of the electrochemical chip of the present invention for detecting dichlorvos and furadan samples is lower than the national standard, showing a higher detection sensitivity; for mixed pesticides, a fairly sensitive detection effect can also be obtained.

[0044] Third, the electrochemical detection chip of the present invention is small, convenient and safe to carry, and easy to connect with electrochemical detection equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The structure of the electrochemical detection chip of the present invention is shown in FIG. Figure 1 , wherein (a) is a schematic diagram of the overall structure, and (b) is a schematic diagram of the structure of the electrode chip;

[0046] Figure 2 Another structural schematic diagram of the upper cover and auxiliary mechanism of the electrochemical detection chip of the present invention, wherein (a) is a schematic diagram of the separate structure of the upper cover, the auxiliary mechanism and the carrier sheet, (b) is a schematic diagram of the initial assembly structure of the upper cover, the auxiliary mechanism and the carrier sheet, and (c) is a schematic diagram of the state when the carrier sheet is extended into the sample loading hole;

[0047] Figure 3 The standard curve for the electrochemical detection of dichlorvos;

[0048] Figure 4 This is the standard curve for the electrochemical detection of carbofuran;

[0049] Figure 5 The standard curve for the electrochemical detection of organophosphorus mixed standard (16 kinds) pesticide standards;

[0050] Figure 6 The standard curve for the electrochemical detection of carbamate mixed pesticide standards;

[0051] Reference numerals

[0052] 1 electrode chip; 11 working electrode; 12 counter electrode; 13 reference electrode; 21 lower cover; 22 upper cover; 221 sample loading hole; 32 buckle buckle groove; 3 outer cover; 31 buckle; 4 carrier sheet prefabricated with substrate; 5 auxiliary mechanism; 51 slide groove; 52 slide plate; 521 toggle convex strip; 522 slide block; 6 foldable loose-leaf DETAILED DESCRIPTION

[0053] The implementation of the present invention is described in detail below in conjunction with the drawings and embodiments of the present invention. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0054] Example 1: Electrochemical detection chip structure

[0055] Figure 1 The figure shows a schematic diagram of one structure of the electrochemical detection chip of the present invention. Figure 1 As shown, the detection chip includes an electrode chip 1 and a shell structure 2 that wraps the electrode chip, and one end of the shell is connected to an outer cover 3.

[0056] The electrode chip 1 includes a working electrode 11, a counter electrode 12 and a reference electrode 13 located on both sides of the working electrode 11, which are made on a substrate by screen printing to form an electrode chip. The leads of the three electrodes are assembled at the front end of the chip, and the leads extend from the front end opening of the shell. The working electrode 11 is fixed with an enzyme for electrochemical detection.

[0057] The housing mechanism 2 includes a detachable lower cover 21 and an upper cover 22. The lower cover 21 encapsulates the electrode chip 1 and is provided with an electrode lead outlet; the upper cover 22 is provided with a sample addition hole 221 corresponding to the working electrode 11 for adding a liquid sample containing a target analyte.

[0058] The outer cover 3 is foldably mounted on the upper cover 22, and a carrier sheet 4 with a prefabricated substrate is fixed in a protruding form at the corresponding position. When the outer cover 3 is fitted with the housing, the carrier sheet 4 extends into the sample loading hole 221. In order to achieve a close fit between the two, a groove 32 for snap-fit ​​is also provided near the front end of the upper cover 22, and correspondingly, a snap 31 is provided at the corresponding position of the outer cover 3. When the two are fastened, the outer cover 3 and the upper cover 22 are closely fitted, so that the chip can be kept clean before initial use. When the snap is opened, the sample loading hole 221 is exposed and can be used to receive liquid samples.

[0059] In this embodiment, the carrier sheet is preferably a polyester cellulose membrane purchased from Shanghai Jieyi Biotechnology Co., Ltd., model MA0280, with a thickness of 0.33 mm. A suitable size is placed flat, 5 μL of 100 mM thioacetylcholine chloride PBS buffer is dropped on it, and after natural drying, it is fixed on the corresponding position of the outer cover 3. The carrier sheet can also be a glass cellulose membrane, such as GL0145. After testing, the substrate in the polyester cellulose membrane dissolves faster.

[0060] When using the above chip for detection, first take out the chip, flip the outer cover upward to unlock the buckle, and expose the sample loading hole. When the target analyte extract is injected into the sample loading hole of the chip, the enzyme on the working electrode first combines with the target analyte in the extract to perform a first reaction. After a certain period of time, re-fasten the buckle, and the carrier sheet prefabricated with the substrate is again attached to the sample loading hole and immersed in the liquid in the sample loading hole. The substrate dissolves and undergoes a second reaction with the enzyme. The detection result can be obtained by collecting the electrical signal.

[0061] Another structural form of the electrochemical detection chip of the present invention abandons the structural design of the outer cover and arranges the auxiliary mechanism on the upper cover. Figure 2 A schematic structural diagram of another form of an upper cover, an auxiliary mechanism and a carrier sheet of the electrochemical detection chip of the present invention.

[0062] like Figure 2 As shown, the upper cover 22 is provided with a sample addition hole 221 corresponding to the working electrode 11 for adding a liquid sample containing a target analyte.

[0063] The auxiliary mechanism 5 is slidably mounted on the upper cover 22, and one end close to the sample loading hole 221 is connected to the carrier sheet 4 through a foldable hinge 6. The carrier sheet 3 is laid flat on the upper cover 22, and is located between the auxiliary mechanism 5 and the sample loading hole 221.

[0064] The auxiliary mechanism 5 includes a slide groove 51 provided on the housing and a slide plate 52 installed in the slide groove. The upper surface of the slide plate 52 is provided with a cam 521 for easy force application, and the lower surface is provided with a slider 522. When the first reaction is completed to a certain extent, the slide plate 52 is pushed to move in the slide groove, and the carrier sheet 4 is pushed forward and folded down into the sample loading hole 221 to be completely immersed in the solution of the sample loading hole, so as to facilitate better dissolution of the substrate.

[0065] Example 2: Fabrication of screen-printed electrode chip

[0066] The working electrode and counter electrode of the screen-printed electrode are carbon electrodes, and the reference electrode is an Ag / AgCl electrode, which can be made using conventional screen-printing technology. For example, the following production steps can be used:

[0067] (1) Customize a screen with a suitable aperture, clean it, pour an appropriate amount of silver paste into the screen frame, place the PET substrate on the positioned machine, put down the screen frame, and control the distance between the printing plate and the substrate to be between 5-8mm. Adjust the height of the ink return knife to be level with the screen, and adjust the height of the scraper to keep the angle between the scraper and the screen at 15-20°. Set the printing parameters on the machine, place the PET substrate, and use the ink return knife to smooth the silver paste for printing conductors and contacts, and print from the screen holes in the hollow graphic part of the screen onto the PET substrate. After the scraper scrapes, the screen rebounds and separates from the PET substrate. Lift the screen frame and remove the PET substrate from the machine;

[0068] (2) After the silver paste is dry, replace the stencil and overprint a layer of carbon paste on the working electrode and counter electrode areas.

[0069] (3) After the carbon paste is dry, replace the screen and overprint the silver chloride paste in the reference electrode area;

[0070] (4) Replace the screen, add insulating ink to the screen frame, align the screen, print a layer of insulating ink on the PET substrate, bake at 120°C for 15 minutes, and complete the printing of the entire electrode.

[0071] Example 3 Immobilization of Prussian Blue and Acetylcholinesterase on the Working Electrode

[0072] In order to further enhance the electrical signal, a layer of electron mediator material - Prussian blue or potassium ferrocyanide can be fixed on the working electrode before fixing acetylcholinesterase. Prussian blue can greatly reduce the oxidation potential of thiocholine chloride produced by the hydrolysis of thioacetylcholine chloride catalyzed by acetylcholinesterase, and has a good peak shape and a large peak current, which can effectively improve the sensitivity of detection.

[0073] Prussian blue is deposited on the surface of the screen-printed working electrode by electrodeposition. The specific method is as follows: the electrodeposition solution is dripped onto the surface of the working electrode, and Prussian blue is electrodeposited through the time-current curve IT. After deposition, the electrodeposition solution is shaken off to form a Prussian blue film on the surface of the working electrode, and then further activated by cyclic voltammetry in a mixed solution of HCL and KCL. The activated electrode is gently rinsed once with pure water and baked in an oven at 100°C for 1h. Among them, the formula of the electrodeposition solution is (1.5mL system): 150μL of 20mM FeCl3.6H2O, 150μL of 20mM K3[Fe(CN)6], 750μL of 0.1M HCl, 250μL of 0.3M KCl, and 200μL of pure water. The configuration of the activation solution is: 0.1MKCl solution and 10mM HCl solution are mixed in equal proportions.

[0074] The method for immobilizing acetylcholinesterase is as follows:

[0075] 0.2% Nafion117, 0.1 mg / mL carboxylated multi-walled carbon nanotubes, and 8 mg / mL acetylcholinesterase were mixed in a volume ratio of 1:1:3, and 5 μL of the mixed solution was dripped onto the surface of the working electrode and dried at 25°C and a humidity range of 55%-65%. The electrode chip with the enzyme fixed was stored at 4°C.

[0076] Nafion117 was purchased from Sigma; acetylcholinesterase was purchased from Sigma or prepared by conventional methods; multi-walled carbon nanotubes were purchased from Shenzhen Nanoport Co., Ltd. Carboxylated multi-walled carbon nanotubes were made by ourselves. The production method is as follows: multi-walled carbon nanotubes were soaked in concentrated hydrochloric acid solution for 2 days, filtered, washed several times with 0.1M NaOH, and then washed several times with ultrapure water. After drying, 1g of multi-walled carbon nanotubes was dissolved in 90mL of concentrated sulfuric acid, ultrasonically dispersed, and then 30mL of concentrated nitric acid was added. Oil bath at 60-80 degrees for 10h. Centrifuged at 4000rpm for 10min-20min, and the supernatant was discarded. A mixture of concentrated sulfuric acid and hydrogen peroxide (concentrated sulfuric acid: hydrogen peroxide = 4:1) was added to the precipitate, and reacted at 70℃ for 30min. Centrifuged at 4000rpm for 10min, the supernatant was discarded, and then washed several times with ultrapure water and dried. On the one hand, the use of carboxylated multi-walled carbon nanotubes can effectively load enzymes and increase the amount of enzyme immobilization, thereby improving the catalytic performance of the enzyme. On the other hand, it provides three-dimensional space for enzyme molecules, which can better maintain their biological functions and help enhance conductive properties.

[0077] Example 4 Detection of pesticide standards

[0078] Take the above Figure 1 The detection chip shown is placed flat, and the electrode lead contacts of the screen-printed electrodes are connected to the corresponding contacts of the electrochemical detection device. The electrochemical detection device uses the Chenhua electrochemical workstation (model CHI660E). Open the outer cover to expose the sample well, add 50μL of blank solution (PBS buffer solution) to the sample well, connect the electrochemical detection device, and 600s after adding the sample, buckle the outer cover to make the carrier sheet with the substrate pre-set contact with the sample solution, and immediately perform the time-current curve test, test potential: 0.1V, test time: 30s, and record the current value I0 corresponding to the 30th second. Among them, the PBS buffer solution is a pH 7.4 phosphate buffer solution.

[0079] Prepare PBS buffer solutions of pesticide standards of different concentrations and add them to the above Figure 1 50 μL of pesticide standard solution of different concentrations was dripped into the sample wells of the detection chip shown in the figure, and the electrochemical detection device was connected. 600 seconds after the sample was added, the outer cover was buckled to make the carrier sheet with the substrate pre-set in contact with the sample solution, and the time-current curve test was immediately performed. The test potential was 0.1V, the test time was 30 seconds, and the current value I corresponding to the 30th second was recorded.i .

[0080] Pesticide inhibition rate I R =(I0-I i ) / I0.

[0081] The following standards were tested in this experiment: dichlorvos, carbofuran, mixed standard (16 kinds) of organophosphorus pesticides, and mixed standard of carbamate pesticides. All of them were purchased from Henan Wanjia Standard Material Research and Development Center Co., Ltd.

[0082] When detecting dichlorvos, the concentrations are: 0.1ng / mL, 0.3ng / mL, 1ng / mL, 3ng / mL, 10ng / mL, 30ng / mL, and the corresponding current signals are measured to calculate I R , the standard curve is as follows Figure 3 As shown, the fitting equation is:

[0083]

[0084] It can detect dichlorvos at a concentration of 0.1ng / mL. If the detection limit is calculated based on a 50% inhibition rate, the detection limit is 1.12ng / mL. Compared with the method listed in the national standard "Rapid detection of organophosphorus and carbamate pesticide residues in vegetables" (GB / T5009.199-2003), converted to a weight ratio, it is 0.0056mg / kg, which is only about 1 / 18 of the detection limit (0.1mg / kg) of dichlorvos by enzyme inhibition rate (spectrophotometry) in the above national standard, and the detection sensitivity is higher.

[0085] When testing furadan, the concentrations are: 0.1ng / mL, 0.3ng / mL, 1ng / mL, 3ng / mL, 10ng / mL, 30ng / mL, 100ng / mL, and the corresponding current signals are measured to calculate I R , the standard curve is as follows Figure 4 As shown, the fitting equation is:

[0086]

[0087] Furadan concentration of 0.1ng / mL can be detected. If the detection limit is calculated based on 50% inhibition rate, the detection limit is 1.45ng / mL. Compared with the method listed in the national standard "Rapid detection of organophosphorus and carbamate pesticide residues in vegetables" (GB / T5009.199-2003), the weight ratio is 0.0072mg / kg. It is 1 / 7 of the detection limit (0.05mg / kg) of the enzyme inhibition rate (spectrophotometry) of the above national standard for furadan, and the detection sensitivity is higher.

[0088] When detecting organophosphorus mixed standard (16 kinds) pesticide standards, the concentrations are: 0.3ng / mL, 1ng / mL, 3ng / mL, 10ng / mL, and the corresponding current signals are measured to calculate I R , the standard curve is as follows Figure 5 As shown, the fitting equation is:

[0089]

[0090] It can detect 16 kinds of organophosphorus mixed standard pesticides at a concentration of 0.3ng / mL. If the detection limit is calculated based on a 50% inhibition rate, the detection limit is 1.116ng / mL. According to the method listed in the national standard "Rapid detection of organophosphorus and carbamate pesticide residues in vegetables" (GB / T 5009.199-2003), the weight ratio is 0.0056mg / kg.

[0091] When testing carbamate mixed pesticide standards, the concentrations are: 0.1ng / mL, 0.3ng / mL, 1ng / mL, 3ng / mL, 10ng / mL, 30ng / mL, 100ng / mL, and the corresponding current signals are measured to calculate I R , the standard curve is as follows Figure 6 As shown, the fitting equation is:

[0092]

[0093] It can detect carbamate mixed standards at a concentration of 0.1ng / mL. If the detection limit is calculated based on a 50% inhibition rate, the detection limit is 6.74ng / mL. According to the method listed in the national standard "Rapid detection of organophosphorus and carbamate pesticide residues in vegetables" (GB / T 5009.199-2003), the weight ratio is 0.0337mg / kg.

[0094] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. An electrochemical detection chip based on enzyme inhibition method, characterized in that: include: An electrode chip (1) comprises at least a working electrode (11) and a counter electrode (12), wherein at least an enzyme for electrochemical detection is fixed on the working electrode (11); A shell mechanism (2) is wrapped around the electrode chip (1) and is provided with an electrode lead outlet, and a sample loading hole (221) is provided at a position on the upper surface corresponding to the working electrode (11); a carrier sheet (4) prefabricated with a solid substrate is provided in the area outside the sample loading hole, and an auxiliary mechanism is provided to drive the carrier sheet (4) to extend into the sample loading hole after the enzyme reacts with the liquid sample containing the target analyte in the sample loading hole for a certain period of time, The housing mechanism (2) comprises a lower cover (21) and an upper cover (22); the upper cover (22) is provided with the sample loading hole (221); the auxiliary mechanism (5) is mounted on the upper cover (22); one end close to the sample loading hole (221) is connected to the carrier sheet (4) via a flexible connecting belt (6); the carrier sheet (4) is laid flat on the upper cover (22) and is located between the auxiliary mechanism (5) and the sample loading hole (221); The auxiliary mechanism (5) comprises a slide groove (51) arranged on the housing and a slide plate (52) installed in the slide groove. The slide plate (52) is provided with a moving convex strip (521) on the upper surface and a sliding block (522) on the lower surface. The flexible connecting belt (6) is a foldable loose-leaf.

2. The electrochemical detection chip based on enzyme inhibition method according to claim 1, characterized in that: in, The electrode chip (1) is also provided with a reference electrode (13). The three electrodes are manufactured on a substrate by a screen printing process to form an electrode chip, and the leads of the three electrodes are assembled at the front end of the chip.

3. The electrochemical detection chip based on enzyme inhibition method according to claim 1, characterized in that: in, The carrier sheet (4) is individually packaged by a clean film.

4. An electrochemical detection kit based on enzyme inhibition method, characterized in that: It includes an electrochemical detection chip and a buffer solution, an extraction solution or a standard substance required for detection and analysis. Wherein, the electrochemical detection chip is the electrochemical detection chip based on enzyme inhibition method according to any one of claims 1 to 3.

5. An electrochemical detection system based on enzyme inhibition method, characterized in that: include: An electrochemical detection kit and an electrochemical detection device connected thereto, wherein the electrochemical detection kit is as described in claim 4.

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