An electrochemical detection device, detection system and detection method based on enzyme inhibition method

Abstract

The application provides an electrochemical detection device based on enzyme inhibition method, a detection system, a detection method and a software product. The electrochemical detection device is connected with an electrochemical detection chip, receives a detection signal of the electrochemical detection chip, and processes the signal. The electrochemical detection device comprises an electrochemical detection chip interface, a chip plug-in detection module, a sample adding detection module, an electrochemical detection module and a microprocessor. The microprocessor receives the detection signal of the above module; after receiving the sample adding signal of the sample adding detection module, the microprocessor outputs the current signal collected by the electrochemical detection module according to a timing instruction until the set time interval is reached. The detection device of the application is matched with a one-time sample adding type electrochemical detection chip. The detection device starts timing after sample adding to ensure that the two-step reaction time matches the detection structure of the electrochemical detection chip. The electrochemical signal is automatically collected after the timing is ended. No manual intervention is needed between the two-step reactions, which can ensure the consistency of the detection and is beneficial to on-site detection.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical detection and analysis technology, specifically relating to an electrochemical detection device, detection system, and detection method for detecting target analytes based on enzyme inhibition. Background Technology

[0002] Electrochemical biosensing technology is characterized by high efficiency, simplicity, sensitivity, and speed. Furthermore, compared to commonly used optical detection technologies, electrochemical detection devices are lighter, cheaper, less energy-intensive, and easier to miniaturize and integrate, making them suitable for on-site detection. Therefore, electrochemical biosensing technology is considered one of the preferred technologies for biological detection in situations with high limitations on timeliness and cost. Simultaneously, the rapid development of printing technology has greatly facilitated the development of low-cost, compact, disposable electrode chips, making it possible to integrate electrochemical detection modules with electronic, mechanical systems, and chips to achieve portable, real-time on-site detection of biochemical molecules.

[0003] In recent years, electrochemical detection methods based on the principle of enzyme inhibition have attracted attention. The principle of target analyte analysis using enzyme inhibition is as follows: the target analyte inhibits the enzyme, affecting the reaction between the enzyme and the corresponding substrate. This affects the corresponding signal output through substrate changes, thus allowing the deduction of the presence or amount of the target analyte. This method is widely used for the detection of organophosphorus or carbamate pesticides, and can also be used for the determination of benzoic acid (a preservative in food) or L-cysteine. Theoretically, once the enzyme inhibition system and the corresponding enzyme-substrate reaction system are developed, new application scenarios may be opened up. In these scenarios, a two-step reaction is essential, with the enzyme inhibition reaction occurring first, followed by the reaction of the remaining enzyme with the substrate to output a signal.

[0004] Since the two-step reaction must proceed sequentially, the second reaction can only proceed after the first reaction has reached a certain level of reaction. However, existing technologies are cumbersome to implement. For example, Chinese Patent Publication No. CN101587092A discloses a portable electrochemical biosensor for rapid detection of organophosphorus pesticide residues. The embodiments disclose a method for contacting a liquid with an enzyme electrode: a reaction cell is fixed on a screen-printed electrode immobilized with acetylcholinesterase; 10 μL of control solution or sample extract is first added to the reaction cell and left for at least 15 minutes; then, 2 μL of substrate solution is added dropwise to the reaction cell, and the response current signal is read after 2 minutes. In this method, the two samples need to be prepared separately and added sequentially with precise time intervals, which makes the final experimental operation cumbersome and inconvenient.

[0005] Therefore, there is still a need in this field to develop detection equipment, detection systems, and detection methods that are simple to operate, portable, fast, and quantitative. Summary of the Invention

[0006] The applicant has previously invented an electrochemical detection chip based on enzyme inhibition (application number CN202010896038.7). In conjunction with that invention, the present invention addresses the above-mentioned problems by providing a new electrochemical detection device, detection system, and detection control method based on enzyme inhibition for the detection of target analytes, thereby overcoming the shortcomings of existing technologies in this field.

[0007] The two-step reaction for electrochemical detection of target analytes based on enzyme inhibition is defined as follows:

[0008] The first reaction: the reaction between the target analyte and the enzyme. If the sample extract contains the target analyte, this first reaction will inhibit the enzyme.

[0009] The second reaction: the reaction between the substrate and the enzyme. This reaction alters the substrate, and the resulting product is electrochemically active, allowing for the collection of corresponding electrical signals. This reaction only begins after the first reaction has progressed to a certain extent; therefore, it is referred to here as the second reaction.

[0010] The second reaction is initiated at any point between 20% and 90% of the first reaction's progress. Experiments have verified that initiating the second reaction within this reaction range ensures both detection sensitivity and rapid detection. More preferably, the second reaction is initiated at any point between 30% and 80%. In this invention, the extent of the first reaction at a given inhibition time is defined as: the inhibition rate I at that time. R Divide by the highest inhibition rate I RMAX The percentage obtained.

[0011] Based on the above principles, the first objective of this invention is to provide an electrochemical detection device based on enzyme inhibition, which is connected to an electrochemical detection chip. When the electrochemical detection chip performs a sample addition reaction, the reaction signal is captured and analyzed in real time, and the detection result is obtained immediately, enabling on-site terminal test operations.

[0012] The second objective of this invention is to provide an electrochemical detection system based on enzyme inhibition, which can realize one-stop detection of sampling, sample addition, detection and result analysis.

[0013] The third objective of this invention is to provide an electrochemical detection method based on enzyme inhibition, enabling real-time analysis and processing of detection results.

[0014] In a first aspect, the present invention provides an electrochemical detection device based on an enzyme inhibition method, which is connected to a single-sample electrochemical detection chip also based on an enzyme inhibition method, receives the detection signal from the electrochemical detection chip, and processes the signal, and has the following technical features: including an electrochemical detection chip interface, a chip insertion and removal detection module, a sample addition detection module, an electrochemical detection module, and a microprocessor.

[0015] The electrochemical detection chip interface is used for the installation and communication connection of the electrochemical detection chip, preferably in the form of a USB interface; the chip insertion and removal detection module senses the installation of the electrochemical detection chip; the sample addition detection module senses whether the sample has been added; the electrochemical detection module acquires the current signal at different time points or preset time points in real time during the sample detection process.

[0016] The microprocessor receives the detection signals from the above modules. After receiving the sample addition signal from the sample addition detection module, it outputs the current signal collected by the electrochemical detection module according to the timing instruction and after the set time interval is reached.

[0017] A preferred approach is that during the execution of the aforementioned timing command, the electrochemical detection module continuously detects the current signal. When the set time interval is reached, the instantaneous current signal is captured, or the average value of the current signal over a short period at the end of the time interval is taken as the output current signal.

[0018] Another preferred approach is to use a timing command followed by a current signal acquisition command. The timing command starts when the sample addition detection module detects that the sample addition area (25) has received a liquid sample. When the set time interval is reached, the electrochemical detection module executes the current signal acquisition command and outputs a current signal after the acquisition is completed.

[0019] The timing can be either forward or countdown, with countdown being the preferred method.

[0020] The timing can be started by receiving an immediate command from the user, or automatically by the sample addition detection module when the circuit is activated after the liquid is added. Automatic start is preferred, as it enables "foolproof" operation and contributes to consistent detection results.

[0021] The timing instructions can be built into the microprocessor's software or installed in the corresponding control software of the smart terminal.

[0022] Optionally, the electrochemical detection device is also equipped with additional modules: including an input module for selecting target analytes, a display module for showing detection results, power status or operating status indicator lights, and a timing module; the input module can be a screen display input module for user input or a barcode scanning module for identifying the identification code attached to the electrochemical detection chip, and both can coexist. Preferably, the corresponding program within the detection device is started by scanning the identification code attached to the chip using the barcode scanning module. In this invention, the identification code is a barcode or a QR code, preferably a QR code.

[0023] The timing module can be either independent of or integrated into the microprocessor. After the sample addition detection module receives the sample addition signal, it starts the timing command. This command can be started by accepting an immediate command from the user or automatically by the sample addition detection module when it detects that the circuit is turned on after the liquid is added. After the set time interval is reached, it sends a command to the microprocessor to collect the current signal at the corresponding moment.

[0024] It may also include a local storage module that stores standard curves, which are available for use in calculating test results.

[0025] It may also include a printing module for printing out the test results.

[0026] It may also include a power management module.

[0027] It may also include a communication module that connects to a remote server or smart terminal.

[0028] Different electrochemical detection chips correspond to different target analytes, and the substrates and enzymes set in the chip are determined by the target analyte. For example, when the target analyte is an organophosphorus or carbamate pesticide, the substrate is thioacetylcholine chloride or thioacetylcholine iodide, and the enzyme is acetylcholinesterase; or the substrate is α-naphthyl acetate, and the enzyme is wheat bran esterase; when the target analyte is benzoic acid or L-cysteine, the substrate is catechol, and the enzyme is polyphenol oxidase.

[0029] Different enzymes react with target analytes and substrates at different rates, and the time interval t1 between the first and second reactions, as well as the appropriate time t2 for the second reaction to proceed, may vary. Therefore, the analytical programs corresponding to electrochemical detection chips for detecting different target analytes will differ.

[0030] Preferably, the electrochemical detection module includes a digital signal generation module for providing a signal source, a constant potential module for maintaining the potential at a set control potential, a signal processing circuit for converting the current detection signal into a standard signal, an analog-to-digital conversion circuit for converting the standard signal into a digital signal, and a data acquisition and processing circuit for rectifying, amplifying, and filtering the digital signal, thereby realizing the acquisition and processing of current signal values ​​at different time points during the detection process.

[0031] Preferably, the electrochemical detection device communicates with a remote server or a smart terminal via a wired or wireless communication module. The remote server or smart terminal stores standard curves that the electrochemical detection device can access.

[0032] The smart terminal is a computer, smartphone, or tablet, on which software is installed to control the electrochemical detection device. The software includes a timing module, a communication module, and a control module. It may also include the input module and the display module that originally set on the electrochemical detection device to display the detection results.

[0033] The smart terminal then communicates with a remote server, which stores standard curves for later use. The software is, for example, an electrochemical detection app or mini-program installed on a smartphone. The software transmits control commands from the smart terminal to the detection device via wireless or wired data transmission circuitry; the detected data is then transmitted from the smart terminal to the remote server; the calculation results from the remote server are transmitted to the smart terminal and displayed thereon, or transmitted from the smart terminal to the detection device and displayed on its screen. The advantages of this implementation scheme are that it utilizes smart terminals such as smartphones, which have powerful data transmission capabilities; the software operates flexibly; the detection device can be made more compact and portable; and, with the cooperation of other programs, the smart terminal can easily process and store data, and perform various data processing tasks.

[0034] In summary, the data processing obtained from the detection can be achieved through the following three methods:

[0035] The first method involves a built-in local standard curve library in the microprocessor of the detection device. The corresponding standard curve of the detection chip is called through the corresponding program to perform the corresponding calculations and obtain the quantitative data of the target analyte.

[0036] The second method involves transmitting data to a remote server via a wired or wireless communication module contained in the testing equipment. The remote server receives the transmitted signal data, calls the corresponding standard curve in the remote server, performs the corresponding calculations, and returns the quantitative data calculation results of the target analyte to the testing instrument.

[0037] The third method involves establishing communication with a smart terminal through a wired or wireless communication module contained in the detection equipment. The smart terminal then transmits data to a remote server. The remote server receives the transmitted signal data, calls the corresponding standard curve in the remote server, performs the corresponding calculations, and returns the quantitative data calculation results of the target analyte to the detection instrument.

[0038] A second aspect of the present invention provides an electrochemical detection system for detecting target analytes based on enzyme inhibition, comprising a single-sample-addition type electrochemical detection chip; a buffer solution, extraction solution or standard required for detection and analysis; and an electrochemical detection device connected to the electrochemical detection chip for processing the electrochemical detection signal.

[0039] The single-sample electrochemical detection chip requires only one liquid sample addition, including:

[0040] An electrode chip (1) is provided, comprising at least a working electrode (11) and a counter electrode (12); at least one enzyme for electrochemical detection is immobilized on the working electrode (11).

[0041] A test strip (2) is mounted on the electrode chip (1). The test strip (2) has a sample application area (25), a detection area (26), and a substrate area (24) located outside the sample application area (25) and the detection area (26). The sample application area (25) is located in front of or directly above the working electrode (11). The detection area (26) is located directly above the working electrode (11). The sample application area (25) and the detection area (26) are adjacent, partially overlap, or completely overlap. The leads of the electrode chip (1) extend beyond the test strip (2).

[0042] The substrate pre-prepared in the substrate region (24) can be dissolved by the sample liquid and migrate to the detection region (26) for reaction with the enzyme to output an electrical signal.

[0043] The structure of the chemical detection equipment is as described above, including an electrochemical detection chip interface, a chip insertion and removal detection module, a power management module, a sample addition detection module, an electrochemical detection module, and a microprocessor self-addition area (25). When the microprocessor receives a liquid sample, it starts to execute a timing command. After the set time interval is reached, it outputs the current signal collected by the electrochemical detection module.

[0044] In the single-sample electrochemical detection chip of this invention, there are various ways to pre-prepare the substrate on the test strip. For example, the substrate can be directly pre-prepare in the substrate region, or a substrate strip containing the substrate can be laminated onto the substrate region. Direct pre-prepare can be achieved by methods such as substrate solution spraying or injection to adsorb the substrate or by other dissociable methods to fix it onto the substrate region of the test strip. As for the scheme of laminating a substrate strip containing the substrate onto the substrate region, as long as the substrate strip and the test strip are fluidly connected, the substrate can dissolve and migrate to the test strip.

[0045] There are various methods for immobilizing enzymes on the working electrode, including but not limited to coating, embedding, and deposition. Any known method can be used as long as it does not affect the enzyme's reactivity.

[0046] Since the working electrode is fixed with an enzyme for electrochemical detection, the area of ​​the working electrode where the enzyme is located, which is the location where the current signal is generated, corresponds to the detection area (26) on the test strip (2). In this invention, the substrate area is located outside the sample application area and the detection area. When a sufficient amount of liquid sample is injected into the test strip, the liquid diffuses along the test strip, and the substrate area and the detection area can be fluidly connected.

[0047] When the above-mentioned chip is used for detection, when the extract of the target analyte is injected into the sample application area of ​​the chip, the enzyme first combines with the target analyte in the extract to carry out a first reaction. After the substrate pre-made on the test strip diffuses to the detection area (26), the substrate and the enzyme carry out a second reaction to output an electrical signal.

[0048] In a preferred embodiment, the distance between the substrate area (24) and the detection area (26) on the test strip can be set to control the interval (t1) between the first and second reactions. In this embodiment, the distance between the substrate area and the detection area affects the migration time of the substrate on the test strip after it has been immersed and dissolved in the solution, thus reserving a corresponding reaction time for the first reaction. This ensures that the second reaction only begins after the enzyme has been inhibited to a certain extent during the first reaction. After the second reaction has proceeded for an appropriate time (t2), the current signal is collected and can be used as the output signal for detecting the target analyte based on the enzyme inhibition method. In the timing command executed by the detection device, the timing duration is t1 + t2.

[0049] For different enzyme inhibition test systems, the optimal t1 and t2 values ​​can be obtained experimentally and then set in the timing instructions of the control program.

[0050] The preferred method for the substrate to migrate to the detection zone (26) after dissolution is directional migration. To achieve directional migration, our research team has conducted numerous experiments and selected a preferred test strip structure consisting of three materials: the test strip (2) includes a sample pad (21), a chromatography membrane (22), and an absorbent pad (23); the substrate zone (24) is located at the front end of the sample pad (21); the sample application zone (25) is located in the middle or rear of the sample pad (21); the detection zone (26) is perpendicular to the working electrode chip (11) and overlaps with or is located at the rear of the sample application zone (25); the sample pad (21) overlaps with the front end of the chromatography membrane (22), leaving the middle of the chromatography membrane (22) empty, and the absorbent pad (23) overlaps with the rear end of the chromatography membrane (22). The absorbent pad attracts the solution, guiding the solution containing the dissolved substrate to migrate directionally from the front end of the sample pad to the detection zone. With the middle of the chromatographic membrane left empty, the chromatographic control of the membrane can effectively control the migration progress and uniformity of the dissolved substrate on the test strip. Since both the sample pad and the chromatographic membrane are hydrophilic and have a permeable bottom surface, the detection area is preferably located on the sample pad. The sample application area (25) on the test strip is preferably located directly above the working electrode (11). In this case, the sample application area (25) coincides with the detection area (26) and is the same region.

[0051] Experiments have shown that starting the second reaction when the first reaction reaches 15% or more of its completion yields better detection sensitivity. Specifically, the distance between the substrate region (24) and the detection region (26) is set such that the second reaction begins at any point after the first reaction reaches 15% of its completion. Preferably, the second reaction begins at any point between 20% and 90% of the first reaction completion. Experiments have verified that starting the second reaction within this range ensures both detection sensitivity and rapid detection. More preferably, the second reaction begins at any point between 30% and 80%. Of course, starting the second reaction when the first reaction reaches 100% completion yields even higher sensitivity, but the substrate region distance needs to be greater, and the total detection time also needs to be extended accordingly.

[0052] In this invention, the extent of the first reaction occurring at a certain inhibition time is defined as: the inhibition rate I at that time. R Divide by the highest inhibition rate I RMAX The percentage obtained.

[0053] In the single-sample-addition implementation scheme, the test strip (2) only accepts one liquid sample addition. The liquid sample is the extract of the target analyte. The sample is added to the addition area (25). After the liquid is injected, the target analyte diffuses to the working electrode (11) and first reacts with the enzyme. When the liquid diffuses to the substrate area (24) which is slightly further away, the substrate area (24) and the detection area (26) are in fluid communication. After the substrate dissolves, it migrates along the test strip to the detection area (26) and comes into contact with the enzyme to undergo a second reaction. The distance between the substrate area (24) and the detection area (26) is set to be the time when the substrate dissolves from the substrate area (24) and migrates to the front edge of the detection area (26) to start the second reaction at any point after the first reaction has progressed to 60%.

[0054] In a preferred embodiment, in order to better achieve the penetration and diffusion of the solution, the test strip can be designed as necessary, including but not limited to selecting suitable materials, selecting the length and width of the materials, or selecting a combination of different materials, as long as the above-mentioned corresponding effects can be achieved.

[0055] In this preferred embodiment, the sample pad (21) material, since it needs to receive liquid samples and release pre-adsorbed substrates, should meet certain requirements for hydrophilicity, absorbency, rapid rewetting, sufficient release of adsorbates, and permeability. This includes, but is not limited to, fabrics or meshes made of cotton, nylon, polyester, polypropylene, polyethylene, etc.; filter paper, such as Whatman 1C, 2C, etc.; glass cellulose membranes, such as Whatman GFA or GFD; polyester cellulose membranes, such as Ahlstrom 6613, etc.; plastic fibers, or any hydrophilic synthetic membrane; absorbent porous plastics, etc. Glass cellulose membranes and polyester cellulose membranes are preferred; glass cellulose membranes are more preferred.

[0056] The chromatographic membrane (22) material needs to control the chromatographic speed and the uniformity of the chromatographic lines on the test strip. Nitrocellulose membrane and Predator membrane are preferred; nitrocellulose membrane is even more preferred.

[0057] The absorbent pad (23) material needs to have the function of absorbing and storing water, guiding the chromatography to the rear end, and storing excess fluid. Filter paper, absorbent paper, and absorbent cotton are preferred; absorbent paper is even more preferred.

[0058] In a preferred embodiment, the sample pad (21) is a glass cellulose membrane, the chromatography membrane (22) is a nitrocellulose membrane, and the absorbent pad (23) is absorbent paper. Testing has shown that this combination provides excellent chromatography performance. The glass cellulose membrane ensures sufficient liquid sample absorption, allowing for rapid diffusion when applied. The absorbent paper ensures the liquid flows along the test strip towards the rear end, particularly guiding the buffer solution containing the dissolved substrate towards the rear end. The nitrocellulose membrane ensures that the substrate, after dissolution, migrates relatively slowly along the glass cellulose membrane, and the substrate front moves neatly, reaching the detection zone at a higher concentration, thus increasing detection sensitivity and ensuring the uniformity and repeatability of the detection.

[0059] In another preferred embodiment, a substrate strip pre-formed with substrate is coated on the substrate area of ​​the sample pad, for dissolving into the substrate and migrating along the test strip to the detection area after fluid communication is established. The substrate strip is made of a polyester cellulose membrane. The substrate strip is preferably adhered tightly to the substrate area of ​​the sample pad.

[0060] In designing the test strip, it is crucial to ensure that the time it takes for the substrate to dissolve in the substrate zone (24) and then chromatographically advance towards the detection zone (26) allows the first reaction to proceed to an appropriate extent. For example, the substrate should only reach the detection zone and begin the second reaction after the first reaction has progressed to any point between 15% and 100%. This chromatographic time can be affected by various factors, such as the total volume of solution received in the sample application zone, the specific selection of each component material in the test strip, the overall width of the test strip, the distance between the substrate zone and the detection zone on the sample pad, the length of the chromatographic membrane, the length and absorbency of the absorbent pad, etc. Through multiple experiments, various schemes that can achieve the desired result can be screened. A preferred scheme is to adjust the overall width of the test strip, the distance between the substrate zone and the detection zone on the sample pad, the length of the chromatographic membrane, and the length of the absorbent pad according to the total volume of solution added to the test strip (2), so that the second reaction occurs after the first reaction has progressed to any point between 15% and 100%.

[0061] In a preferred embodiment, the absorbent pad in the test strip is designed, for example, by controlling the length and width of the absorbent pad, so that the peak position of the substrate in the substrate dissolution solution flowing towards the rear end of the chromatography is just stopped in the detection area where the enzyme is located, and does not continue to move towards the rear end, thereby improving the detection sensitivity.

[0062] In some implementation schemes for detecting pesticide residues using the acetylcholinesterase-chlorothiazide acetylcholine system, when the sample pad (21) is a glass cellulose membrane, the chromatography membrane (22) is an unbacked nitrocellulose membrane, and the absorbent pad (23) is absorbent paper, the substrate chlorothiazide acetylcholine is pre-prepared in the substrate area of ​​the sample pad by spraying. For a test strip with an overall width of 10 mm, in a one-step sample addition detection mode, when the liquid sample volume received by the sample addition area (25) at one time is 50-300 μL, the distance between the rear edge of the substrate area (24) and the front edge of the detection area (26) is 1 mm to 12 mm; the non-covered length of the chromatography membrane (22) connecting the sample pad (21) and the absorbent pad (23) between the rear edge of the sample pad (21) and the front edge of the absorbent pad (23) is 8 mm to 15 mm; and the length of the absorbent pad (23) is 10 mm to 30 mm. The substrate reaches the detection zone when the first reaction has progressed to between 15% and 100%.

[0063] Particularly preferably, when the liquid sample volume received by the sample loading zone (25) is 150-200 μL, the distance between the rear edge of the substrate zone (24) and the front edge of the detection zone (26) is 8 mm to 9 mm; the non-covered length of the chromatography membrane (22) connecting the sample pad (21) and the absorbent pad (23) between the rear edge of the sample pad (21) and the front edge of the absorbent pad (23) is 9 mm to 11 mm; and the length of the absorbent pad (23) is 15 mm to 25 mm. The substrate reaches the detection zone when the first reaction has progressed to 30% to 100%.

[0064] In other preferred embodiments for detecting pesticide residues using the acetylcholinesterase-chlorothioacetylcholine system, in the case where the sample pad (21) is a glass cellulose membrane, the chromatography membrane (22) is an unbacked nitrocellulose membrane, and the absorbent pad (23) is absorbent paper, the substrate chlorothioacetylcholine is pre-prepared on a polyester cellulose membrane as a substrate strip, and the 5 mm long substrate strip is attached to the front substrate area of ​​the sample pad.

[0065] The single-sample electrochemical detection chip of this invention is composed of a test strip (2) and an electrode chip (1). The leads of the electrode chip can extend from the absorbent pad end of the test strip (2), the sample pad end of the test strip (2), or the side of the test strip, as long as the liquid on the test strip does not leak to the leads and cause a short circuit. Preferably, the leads extend from the sample pad end of the test strip (2).

[0066] As a preferred embodiment, based on the aforementioned electrochemical detection chip, a housing may also be included to enclose the detection structure formed by the aforementioned test strip and electrode chip. The housing (3) has a sample application hole (321) corresponding to the sample application area (25) of the test strip (2) for adding liquid samples containing the target analyte. The front end of the housing (3) has an opening (33), from which the electrode leads of the electrode chip (2) extend. The housing may include a detachable lower cover (31) and an upper cover (32); the sample application hole (321) is located at the corresponding position on the upper cover (32).

[0067] The outer casing (3) may also have drainage holes corresponding to the position of the absorbent pad, for example, at the corresponding position of the upper cover (32), to allow for additional drainage when necessary, such as by inserting absorbent material into the drainage hole to contact the absorbent pad. Necessary situations include, but are not limited to, situations where a large amount of liquid is added, or situations where it is necessary to guide the chromatography front position. Absorbent cotton strips are preferred as the absorbent material.

[0068] When the electrode lead extends from one end of the absorbent pad, the rear end of the outer casing opens accordingly, while the front end closes.

[0069] Electrochemical detection equipment, systems, and methods based on enzyme inhibition are applicable in various scenarios. As long as an enzyme inhibition system and the corresponding enzyme-substrate reaction system are developed, this method can be used. Examples include substrate-enzyme systems for organophosphorus or carbamate pesticides mentioned in the background section, and substrate-enzyme systems for benzoic acid or L-cysteine.

[0070] The following are some enzyme-substrate pairings for detecting target analytes based on enzyme inhibition methods:

[0071] In some implementation schemes, when detecting organophosphorus pesticides or carbamate pesticides, the enzyme systems that can be used include, but are not limited to, acetylcholinesterase systems, butyrylcholinesterase systems, tyrosinase systems, and type B plant esterase systems.

[0072] When the enzyme immobilized on the working electrode is acetylcholinesterase, the substrate can be selected from thioacetylcholine chloride or thioacetylcholine iodide; when the enzyme immobilized on the working electrode is butyrylcholinesterase, the substrate can be selected from thiobutyrylcholine chloride or thiobutyrylcholine iodide; when the enzyme immobilized on the working electrode is a type B plant esterase, such as wheat bran esterase, the substrate is α-naphthyl acetate; when the enzyme immobilized on the working electrode is a tyrosinase, the substrate can be selected from catechol or 1,2-naphthoquinone-4-sulfonic acid.

[0073] 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.

[0074] In some embodiments, an electron mediator material is also immobilized on the working electrode (11). The electron mediator can reduce the oxidation potential of the products generated by the enzyme-catalyzed hydrolysis of the substrate, thereby improving the electrode's anti-interference capability and detection sensitivity. Simultaneously, some electron mediators oxidize very rapidly on the electrode surface, significantly shortening the detection time. Examples include Prussian blue or potassium ferricyanide.

[0075] In a preferred embodiment, the target analyte is an organophosphorus or carbamate pesticide; the substrate is thioacetylcholine chloride; the corresponding enzyme immobilized on the working electrode is acetylcholinesterase; and the electron mediator material is Prussian blue. Prussian blue can significantly reduce the oxidation potential of thiocholine chloride produced by the hydrolysis of thioacetylcholine chloride catalyzed by acetylcholinesterase, and produces a good peak shape and high peak current, effectively improving the detection sensitivity.

[0076] In some implementations, when the target analyte is benzoic acid or L-cysteine, the available enzyme systems include, but are not limited to, polyphenol oxidase systems. When using polyphenol oxidase, the substrate is catechol.

[0077] A third aspect of the present invention provides a method for electrochemically detecting a target analyte using an electrochemical detection system, comprising the following steps:

[0078] Step 1: Insert the mounting end of the single-sample electrochemical detection chip into the electrochemical detection chip interface to establish an electrical connection;

[0079] Step 2: When the sample addition detection module detects the addition of sample, the microprocessor starts the electrochemical test process according to the preset electrochemical parameters corresponding to the chip, and starts timing according to the set time.

[0080] Step 3: When the set time arrives, the microprocessor controls the electrochemical test to stop. The electrochemical test module processes the electrical signals collected on the electrochemical detection chip to obtain the raw electrochemical detection data.

[0081] Step 4: The microprocessor further processes the raw electrochemical detection data and transmits the processed detection data to a remote server or calls the local storage database. The detection results are obtained by comparing with the standard curve and displayed on the detection device or smart terminal.

[0082] Since different enzyme-inhibition-based electrochemical detection chips have different timing durations, different programs can be used to embed the time intervals. Each program can be invoked by recognizing the identification code attached to the electrochemical detection chip. The identification code can be a QR code, barcode, etc.

[0083] A preferred detection method, for situations where the detection equipment and the smart terminal are separate, includes:

[0084] (1) Install an electrochemical detection APP or electrochemical detection mini-program on a smart terminal. Smart phones are preferred as smart terminals.

[0085] (2) The testing equipment is powered on and waits to connect to the smart terminal;

[0086] (3) Launch the above-mentioned APP-software or mini-program to establish a wireless or wired connection between the smart terminal and the detection equipment, preferably a Bluetooth wireless connection;

[0087] (4) Insert the electrochemical detection chip into the detection device;

[0088] (5) Use a smart terminal to scan and identify the identification code on the electrochemical detection chip, retrieve and start the corresponding program, preferably a QR code;

[0089] (6) Inject the test solution into the sample application area (25) of the electrochemical detection chip;

[0090] (7) After the detection equipment detects the sample liquid, it starts the electrochemical test process according to the preset electrochemical parameters corresponding to the chip, and starts timing according to the set time;

[0091] (8) When the set time arrives, the detection equipment stops the electrochemical test and processes the electrical signals collected on the electrochemical detection chip to obtain the raw electrochemical detection data;

[0092] (9) The detection equipment further processes the raw electrochemical detection data and transmits the processed detection data to the smart terminal.

[0093] (10) The smart terminal calls the standard curve in the local or remote database to perform comparison calculations and outputs the calculation results.

[0094] When the sample extract does not contain the target analyte or contains a target analyte below the detection limit, a negative result or specific data will be output; when the sample extract contains a target analyte above the detection limit, a positive result or specific data will be output.

[0095] In the above steps, the order of steps (4) and (5) can be interchanged.

[0096] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0097] Pre-prepared substrate test strips are combined with enzyme-immobilized electrode chips to form a single-addition electrochemical detection chip. This chip can detect multiple target analytes based on the principle of enzyme inhibition, overcoming the inconvenience of additional preparation and addition of substrate solutions, reducing substrate usage, and the single-addition technology enhances the convenience and stability of operation, which is conducive to rapid on-site detection.

[0098] The detection equipment is equipped with a single-sample-addition electrochemical detection chip. It uses an automatic start timing to ensure that the interval between the second and first reaction steps matches the detection structure of the electrochemical detection chip. After the timing ends, the electrochemical signal is automatically collected without manual intervention, which can ensure the consistency of detection and is beneficial for on-site detection.

[0099] The testing equipment can also include commonly used smart terminals, which can be used to control the equipment and transmit data, increasing the applicability and convenience of the application. The testing system can also include a remote server to store a standard curve library. Users do not need to create standard curves on-site; based on the identification information such as the QR code on the chip, the testing system automatically retrieves the corresponding standard curve, facilitating user-friendly operation and making it more suitable for home use and on-site testing. Attached Figure Description

[0100] Figure 1 This is a schematic diagram of the electrochemical detection chip structure of the present invention, wherein (a) is a schematic diagram of the test strip structure and (b) is a schematic diagram of the electrode chip structure;

[0101] Figure 2 for Figure 1 The cross-sectional structural diagrams are shown, where (a) is a cross-sectional structural diagram of the test strip and (b) is a cross-sectional structural diagram of the electrode chip.

[0102] Figure 3 This is a schematic diagram of the structure of the encapsulated electrochemical detection chip of the present invention, wherein (a) is the upper cover, (b) is the electrochemical detection chip, and (c) is the lower cover;

[0103] Figure 4 This is a framework diagram of the electrochemical detection device of the present invention;

[0104] Figure 5 This is a framework diagram of the electrochemical detection device and intelligent terminal of the present invention;

[0105] Figure 6 The electrochemical detection process of this invention Figure 1 ;

[0106] Figure 7 The electrochemical detection process of this invention Figure 2 ;

[0107] Figure 8 This is a flowchart of the control process during the detection process of the present invention.

[0108] Figure Labels

[0109] A. Chip front end; B. Chip back end

[0110] 1. Electrode chip: 11. Working electrode; 12. Counter electrode; 13. Reference electrode

[0111] 2. Test strip: 21. Sample pad; 22. Chromatography membrane; 23. Absorbent pad; 24. Substrate zone; 25. Sample loading zone; 26. Detection zone; 27. Substrate strip

[0112] 3. Outer shell: 31. Lower cover; 32. Upper cover; 321. Sample inlet; 322. Drainage hole; 33. Front opening Detailed Implementation

[0113] Example 1: Electrochemical Detection Chip Structure

[0114] Figure 1 This is a schematic diagram of the electrochemical detection chip structure one of the present invention. Figure 2 for Figure 1 A cross-sectional schematic diagram of the first structure of the electrochemical detection chip.

[0115] like Figure 1 and Figure 2 As shown, the detection chip includes an electrode chip 1 and a test strip 2 superimposed on it. The electrode chip 1 includes a working electrode 11 and a counter electrode 12 and a reference electrode 13 located on both sides of the working electrode 11, respectively. The three electrodes are fabricated on a substrate using a screen printing process to form the electrode chip. The leads of the three electrodes are gathered at the front end of the chip and extend from the front end of the test strip. An enzyme for electrochemical detection is fixed on the working electrode 11, therefore the working electrode is also where the enzyme region is located.

[0116] The test strip 2 is attached tightly to the electrode chip 1 and consists of three parts, from front to back: a sample pad 21, a chromatography membrane 22, and an absorbent pad 23. The sample pad 21 has substrate adsorbed at its front substrate area 24, containing the enzyme substrate for the electrochemical reaction. The middle section is the sample application area 25 (shown as a dashed box in the figure), used to receive liquid samples containing the target analyte. Its rear end overlaps and contacts the front end of the chromatography membrane 22, which is located below the sample pad 21. The rear end of the chromatography membrane 22 overlaps and contacts the absorbent pad 23, which is also located below it. The chromatography membrane 22 has a partially uncovered area in the middle.

[0117] When the entire test strip 2 is tightly attached to the electrode chip 1, the sample application area 25 on the test strip is located directly above the working electrode 11. Since the detection area 26 is located on the sample pad 21 corresponding to the working electrode 11, in this embodiment, the detection area 26 and the sample application area 25 coincide and are the same area.

[0118] In other designs, the sample application area 25 can be offset from the detection area 26, partially overlapping, and located at the front of the detection area 26. However, regardless of the arrangement, the substrate area 24 should always be outside the sample application area 25 and the detection area 26, and at a certain distance.

[0119] In this embodiment, the sample application area 25 only accepts one liquid sample application. After the target analyte extract is injected into the sample application area (25), the target analyte diffuses to the working electrode (11) and reacts with the enzyme. When the liquid diffuses to the substrate area (24) which is slightly further away, the substrate area (24) and the detection area (26) are in fluid communication. After the substrate dissolves, it migrates along the test strip to the detection area (26) and comes into contact with the enzyme to undergo a second reaction.

[0120] Example 2: Electrochemical Detection Chip Packaging Structure

[0121] Figure 3 The packaging structure of the electrochemical detection chip encapsulated in Example 1 is shown; it is a single-sample-addition type. Figure 5 As shown, a housing 3 is provided outside the chip. The housing 3 includes a detachable lower cover 31 and an upper cover 32. The detection chip, which consists of the electrode chip 1 and the test strip 2, is fixed inside the housing 3. The upper cover 32 is provided with a sample application hole 321 at the sample application area 25 of the test strip 2 for adding liquid samples containing the target analyte. The front end of the housing 3 has an opening 33, from which the electrode leads of the electrode chip 1 extend.

[0122] Example 3: Fabrication of screen-printed electrode chips

[0123] The working and counter electrodes of the screen-printed electrode are carbon electrodes, and the reference electrode is an Ag / AgCl electrode. It can be fabricated using conventional screen printing techniques. For example, the following fabrication steps can be employed:

[0124] (1) Customize a screen with a suitable aperture, clean it thoroughly, pour an appropriate amount of silver paste into the screen frame, place the PET substrate on the positioned machine table, lower the screen frame, and control the distance between the printing plate and the substrate to be between 5-8mm. Adjust the height of the back-ink blade to be horizontal with the screen plate, and adjust the height of the squeegee to maintain an angle of 15-20° with the screen plate. Set the printing parameters on the machine, place the PET substrate, and the back-ink blade spreads the silver paste evenly to print the conductors and contacts, which are then printed onto the PET substrate through the mesh openings of the screen plate's cutout graphic portion. After the squeegee scrapes, the screen springs back and separates from the PET substrate. Lift the screen frame and remove the PET substrate from the machine table; bake at 120℃ for 10-15 minutes.

[0125] (2) Replace the screen, add insulating ink to the screen frame, align the screen, print a layer of insulating ink on the PET substrate, and bake at 120°C for 15 minutes.

[0126] (3) After the insulating ink dries, a layer of carbon paste is printed on the working electrode and counter electrode areas and baked at 120°C for 10-15 minutes.

[0127] (4) Overprint silver chloride paste onto the reference electrode area. Bake for 15 minutes to complete the printing of the entire electrode.

[0128] Example 4: Immobilization of Prussian blue and acetylcholinesterase on the working electrode

[0129] To further enhance the electrical signal, a layer of electron mediator material—Prussian blue or potassium ferricyanide—can be pre-immobilized on the working electrode before immobilizing acetylcholinesterase. Prussian blue can significantly reduce the oxidation potential of thiocholine chloride produced by the hydrolysis of thiocholine chloride catalyzed by acetylcholinesterase, and it also produces a good peak shape and a large peak current, effectively improving the detection sensitivity.

[0130] Prussian blue was deposited onto the surface of a screen-printed working electrode using an electrodeposition method. The specific method is as follows: an electrodeposition solution was dropped onto the working electrode surface, and Prussian blue was electrodeposited using a time-current curve (IT). After deposition, the electrodeposition solution was removed, forming a Prussian blue film on the working electrode surface. This film was then further activated using cyclic voltammetry in a mixed solution of HCl and KCl. The activated electrode was gently rinsed once with purified water and then baked in an oven at 100°C for 1 hour. The electrodeposition solution (1.5 mL system) was prepared as follows: 150 μL of 20 mM FeCl3·6H2O, 150 μL of 20 mM K3[Fe(CN)6], 750 μL of 0.1 M HCl, 250 μL of 0.3 M KCl, and 200 μL of purified water. The activation solution was prepared by mixing 0.1 M KCl solution and 10 mM HCl solution in equal proportions.

[0131] The method for immobilizing acetylcholinesterase is as follows:

[0132] 0.2% Nafion 117, 0.1 mg / mL carboxylated multi-walled carbon nanotubes, and 8 mg / mL acetylcholinesterase were mixed in a volume ratio of 1:1:3. 5 μL of this mixture was then added dropwise to the surface of the working electrode and dried at 25°C and 55%-65% humidity. The electrode chip with the immobilized enzyme was stored at 4°C.

[0133] Nafion 117 was purchased from Sigma-Aldrich; acetylcholinesterase was purchased from Sigma-Aldrich or prepared using conventional methods; multi-walled carbon nanotubes (MWCNTs) were purchased from Shenzhen Nanoport Co., Ltd. Carboxylated MWCNTs were prepared in-house using the following method: MWCNTs were soaked in concentrated hydrochloric acid solution for 2 days, filtered, washed multiple times with 0.1M NaOH, then washed multiple times with ultrapure water, dried, and 1g of MWCNTs was dissolved in 90mL of concentrated sulfuric acid, ultrasonically dispersed, and then 30mL of concentrated nitric acid was added. The mixture was incubated in an oil bath at 60-80°C for 10 hours. The mixture was centrifuged at 4000rpm for 10-20 minutes, 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 the mixture was reacted at 70°C for 30 minutes. The mixture was centrifuged at 4000rpm for 10 minutes, the supernatant was discarded, and the mixture was washed several times with ultrapure water and dried. Using carboxylated multi-walled carbon nanotubes can effectively load enzymes, increase the amount of enzyme immobilized, and thus improve the enzyme's catalytic performance. On the other hand, it provides three-dimensional space for enzyme molecules, which can better maintain their biological functions and also helps to enhance electrical conductivity.

[0134] Example 5: Fabrication of a single-sample-addition type electrochemical detection chip

[0135] test strips such as Figure 1 The structure was designed with glass cellulose membrane (purchased from Shanghai Jieyi Biotechnology Co., Ltd., product name: GL0145) as the sample pad material, nitrocellulose membrane (purchased from Waterman, model: whatmanAE99, without backing) as the chromatography membrane material, and absorbent paper (purchased from Shanghai Jieyi Biotechnology Co., Ltd., product model: H5015) as the absorbent pad material.

[0136] (1) Cut the glass cellulose membrane into strips with a width of 10 mm; cut the nitrocellulose membrane into strips with a length of 25 mm and a width of 10 mm; cut the absorbent paper into strips with a length of 17 mm and a width of 10 mm.

[0137] (2) Spray a PBS buffer solution of substrate-thioacetylcholine chloride (substrate concentration of 50mM) onto one end of the glass cellulose membrane by spraying. This end is defined as the front end of the test strip. Let it air dry at room temperature to form a strip-shaped substrate area with a width of about 5mm.

[0138] (3) Cut the glass cellulose membrane to a length that ensures an 8.5 mm gap between the rear edge of the substrate area and the front edge of the detection area. The detection area is also the sample loading area, which covers the working electrode of the electrode chip. The length of the detection area is 6 mm, and the remaining 5.5 mm after the rear edge of the detection area covers the nitrocellulose membrane.

[0139] (3) The front 5.5mm of the nitrocellulose membrane is covered under the glass cellulose membrane, the middle 10mm is left as an uncovered area, and the rear 3.5mm is covered under the absorbent paper.

[0140] (4) The front 3.5mm of the absorbent paper covers the nitrocellulose membrane, and the back 13.5mm is left blank.

[0141] (5) The three connected parts are glued to the back sides of the double-sided tape to form a complete test strip. The total length is 68mm.

[0142] (6) Attach the test strip to the 10mm wide electrode chip using double-sided adhesive. The electrode chip is as follows: Figure 1 As shown, the electrode is located in the middle, with some blank space on both sides to allow the test strip to be pasted without affecting the electrode. The working electrode, which is fixed with acetylcholinesterase, is located directly below the detection area 26 (which is also the sample application area 25) of the test strip and is covered by the detection area.

[0143] (7) The electrode leads of the electrode chip extend from the front end of the sample pad portion of the test strip, forming... Figure 1 The electrochemical detection chip shown.

[0144] The fabricated detection chip is installed in a detachable housing. A sample application hole 321 is provided on the top cover of the housing corresponding to the sample application area 25. The electrode leads of the screen-printed electrode extend from the front end of the housing, forming... Figure 3 The single-sample electrochemical detection chip shown is shown.

[0145] Example 6: Simulated Detection of Vegetable Samples

[0146] use Figure 4 or Figure 5 The electrochemical detection device shown works in conjunction with the aforementioned single-sample electrochemical detection chip to perform simulated detection. Figure 4 The structure of an electrochemical detection device capable of performing detection independently is shown. Figure 5 The block diagram structure of an electrochemical detection device adapted to a smart terminal is shown.

[0147] according to Figure 4 The electrochemical detection device 4 includes an electrochemical detection chip interface 401, a chip insertion and removal detection module 402, a sample addition detection module 403, an electrochemical detection module 404, a power management module 405, and a microprocessor 406. It also includes additional functional modules: an LED indicator 407, a display module 408, a printing module 409, a communication module 410, a memory 411, a user input module 412, and a barcode scanning module 413.

[0148] according to Figure 5The electrochemical detection device 5 is wirelessly connected to the smart terminal 6 via Bluetooth, including an electrochemical detection chip interface 501, a chip insertion and removal detection module 502, a sample addition detection module 503, an electrochemical detection module 504, a power management module 505, and a microprocessor 506. It also includes additional functional modules: a working indicator light 507, a button 508, a wireless communication module 509, and a memory 510.

[0149] The following uses the detection of dichlorvos in vegetables as an example to illustrate the detection procedures of two types of electrochemical detection equipment. The detection procedure using independent electrochemical detection equipment is as follows: Figure 6 As shown, the detection process using electrochemical detection equipment and a smart terminal is as follows: Figure 7 As shown.

[0150] according to Figure 6 The detection process, which uses an independent electrochemical detection device 4 connected to an electrochemical detection chip, is as follows:

[0151] S1-1: Spray a certain amount of dichlorvos pesticide on a clean rapeseed leaf, let it air dry at room temperature for 1-2 days, cut the rapeseed leaf into pieces about 1cm in size, take 1g of sample, put it into an extraction bottle, add 5mL of PBS buffer solution, shake for 1-2 minutes, pour out the extract, let it stand for 3-5 minutes, and set aside for later use.

[0152] S1-2, turn on the testing equipment, the equipment working indicator light 507 and display module 408 will light up.

[0153] S1-3, the scanning module 413 scans the QR code on the electrochemical detection chip to enter the detection program corresponding to the QR code. At the same time, the target analyte name is displayed on the display module 408, or the target analyte can be selected through the input module.

[0154] S1-4, Insert the electrochemical detection chip into the electrochemical detection chip interface 401, and the display module 408 will show that the insertion and connection are successful.

[0155] S1-5, Take 180 μL of the vegetable sample extract prepared in step S1-1 and add it to the sample well of the detection chip.

[0156] S1-6 After the sample addition detection module 403 detects the addition of sample, the microprocessor 406 starts the electrochemical test process according to the preset electrochemical parameters corresponding to the chip, and starts a 660s countdown according to the set time.

[0157] The single-sample electrochemical detection chip fabricated in Example 5, due to its specific length and structural arrangement, allows for a suitable sampling time of 660 seconds, ensuring that both the first and second reactions proceed sufficiently. Other length or structural settings require experimental determination of their optimal sampling times.

[0158] S1-7, the electrochemical detection module 404 performs a duration-current curve test as soon as the countdown begins. The test potential is 0.13V, and the current value at the end of the 660s countdown is taken as the detection signal.

[0159] S1-8, the microprocessor 406 further processes the raw electrochemical detection data and transmits the processed detection data to a remote server or calls a local database. The remote server or local database compares the data with the standard curve to perform calculations, obtains the detection results, and displays them on the display module. Alternatively, the final detection results can be printed out through the printing module 409.

[0160] according to Figure 7 The detection process, which uses electrochemical detection equipment 5 and intelligent terminal 6 connected to the electrochemical detection chip, is as follows:

[0161] S2-1: Spray a certain amount of dichlorvos pesticide on a clean rapeseed leaf, let it air dry at room temperature for 1-2 days, cut the rapeseed leaf into pieces about 1cm in size, take 1g of sample, put it into an extraction bottle, add 5mL of PBS buffer solution, shake for 1-2 minutes, pour out the extract, let it stand for 3-5 minutes, and set aside for later use.

[0162] S2-2, Scan the QR code on the electrochemical detection chip packaging with your mobile phone and follow the instructions to download the electrochemical detection APP software.

[0163] S2-3, the testing equipment is powered on, the indicator light is on, and the mobile electrochemical testing APP shows that it has been connected to the testing equipment via Bluetooth.

[0164] S2-4. Take one of the above electrochemical detection chips, scan the QR code displayed on the electrochemical detection chip with your mobile phone, and the APP will display the QR code recognition to enter the detection program corresponding to the QR code.

[0165] S2-5, following the testing procedure, the user inserts the testing chip, and the APP displays that the insertion was successful.

[0166] S2-6, Take 180 μL of the vegetable sample extract prepared in step S2-1 and add it to the sample well of the detection chip.

[0167] S2-7 After the sample addition detection module 503 detects the addition of sample, the microprocessor 506 starts the electrochemical test process according to the preset electrochemical parameters corresponding to the chip, and starts a 660s countdown according to the set time.

[0168] S2-8, the electrochemical detection module 504 performs a duration-current curve test as soon as the countdown begins. The test potential is 0.13V, and the current value at the end of the 660s countdown is taken as the detection signal.

[0169] S2-9, the microprocessor 406 further processes the raw electrochemical detection data and transmits the processed detection data to the APP. The APP automatically transmits the processed data to the remote server according to the detection program, and the APP interface displays the waiting detection results.

[0170] S2-10: The remote server processes the received data, such as retrieving standard curve libraries and performing calculations. The processing result is quantitative pesticide concentration data, which is transmitted to the APP via the network and displayed on the APP. In this simulation test, the pesticide concentration was measured to be 10 ng / mL, and the APP displays: Pesticide concentration 10 ng / mL, positive. This data can also be displayed in accordance with national standards, converted to mg / kg data using a certain formula.

[0171] In the two processes described above, the 660s countdown can also be divided into two segments. The first 600s is for timing only, without time-current curve testing. After 600s, a duration-current curve test is performed for 60s. The test potential is 0.13V. The current value at the end of the 660s countdown is taken as the detection signal.

[0172] The data in the standard database on the remote server is created and updated regularly by the experimenters, eliminating the need for on-site creation by testing personnel, thus enabling rapid testing.

[0173] If the measured data is below the detection limit, the app will display a negative result.

[0174] Regardless of whether a standalone electrochemical detection device or a combination of electrochemical detection device and smart terminal is used for detection, the control process remains the same. (See [link to relevant documentation]). Figure 8 The details are as follows:

[0175] S3-1, Before testing, the microprocessor controls the connection of the electrochemical testing equipment and executes the instructions of the electrochemical testing equipment;

[0176] S3-2, the microprocessor controls the operation prompt to scan the QR code on the chip;

[0177] S3-3, microprocessor controls the execution of instructions to connect to the electrochemical detection chip;

[0178] S3-4, microprocessor-controlled operation prompts for sample addition instructions;

[0179] S3-5, After the sample is added, the microprocessor controls the execution of the timing command;

[0180] S3-6: Take the current value at the end of the 660s countdown as the detection signal and output the signal value;

[0181] S3-7, the microprocessor executes instructions to transmit the electrochemical signal to a remote server or control terminal;

[0182] S3-8: After the remote server finishes analyzing the data, the microprocessor controls the operation to receive the data instructions returned by the remote server and controls the display of the analysis results.

[0183] The undescribed parts of this invention are the same as or implemented using existing technology. The applicant declares that this invention is illustrated through the above embodiments, but the invention is not limited to the above detailed methods, i.e., it does not mean that the invention must rely on the above detailed methods to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. An electrochemical detection device based on enzyme inhibition, connected to a single-sample electrochemical detection chip also based on enzyme inhibition, receiving detection signals from the electrochemical detection chip and processing the signals, characterized in that, It includes an electrochemical detection chip interface, a chip insertion / removal detection module, a power supply module, a sample dispensing detection module, an electrochemical detection module, a microprocessor, and a timing module. The single-sample electrochemical detection chip only requires one liquid sample addition and its structure includes: an electrode chip (1), which includes at least a working electrode (11) and a counter electrode (12); the working electrode (11) has at least an enzyme for electrochemical detection fixed on it; A test strip (2) is mounted on the electrode chip (1). The test strip (2) has a sample application area (25), a detection area (26), and a substrate area (24) located outside the sample application area (25) and the detection area (26). The sample application area (25) is located in front of or directly above the working electrode (11). The detection area (26) is located directly above the working electrode (11). The sample application area (25) and the detection area (26) are adjacent, partially overlap, or completely overlap. The leads of the electrode chip (1) extend beyond the test strip (2). The substrate pre-prepared in the substrate region (24) can be dissolved by the sample liquid and migrate to the detection region (26) for a second reaction with the enzyme to output an electrical signal. The distance between the substrate region (24) and the detection region (26) is set such that the time for the substrate to dissolve from the substrate region (24) and migrate to the front edge of the detection region (26) to initiate the second reaction is any point after the first reaction has progressed to 15%. The electrochemical detection chip interface is used for mounting and communicating with the electrochemical detection chip; the chip insertion / removal detection module senses the installation of the electrochemical detection chip; the sample addition detection module senses whether a sample has been added; during sample detection, the electrochemical detection module acquires current signals at different or preset time points in real time; the microprocessor receives detection signals from the chip insertion / removal detection module, the sample addition detection module, and the electrochemical detection module. Upon receiving the sample addition signal from the sample addition detection module, the microprocessor outputs the current signal acquired by the electrochemical detection module after the set time interval is reached, according to the timing command. The timing module is either independent of or integrated into the microprocessor. After the sample addition detection module receives the sample addition signal, it starts the timing command. This command can be started by accepting an immediate command from the user or automatically started when the sample addition detection module detects that the circuit is turned on after the liquid is added. After the set time interval is reached, it sends a command to the microprocessor to collect the current signal at the corresponding time.

2. The electrochemical detection device based on enzyme inhibition method according to claim 1, characterized in that: in, The electrochemical detection module includes a digital signal generation module for providing a signal source, a constant potential module for maintaining the potential at a set control potential, a signal processing circuit for converting the current detection signal into a standard signal, an analog-to-digital conversion circuit for converting the standard signal into a digital signal, and a data acquisition and processing circuit for rectifying, amplifying, and filtering the digital signal.

3. The electrochemical detection device based on enzyme inhibition method according to claim 1, characterized in that: in, The electrochemical detection device also includes an input module for selecting target analytes, a display module for showing the detection results, and indicator lights for power status or operating status. The input module can be a screen display input module that allows users to input information themselves, or a barcode scanning module that identifies the identification code attached to the electrochemical detection chip.

4. The electrochemical detection device based on enzyme inhibition method according to claim 3, characterized in that: in, The electrochemical detection device also includes a local storage module containing standard curves that can be retrieved to calculate detection results.

5. The electrochemical detection device based on enzyme inhibition method according to claim 1, characterized in that: in, The electrochemical detection device further includes a communication module and an input module for selecting target analytes. The communication module is used to communicate with a remote server or smart terminal, which stores standard curves for the electrochemical detection device to access. The input module can be a screen display input module that allows users to input information themselves, or a barcode scanning module that identifies the identification code attached to the electrochemical detection chip. The smart terminal is a computer, smartphone, or tablet, and is equipped with a timing module. After the sample addition detection module receives the sample addition signal, it starts the timing command. This command can be started by accepting an immediate command from the user or by the sample addition detection module detecting that the circuit is turned on after the liquid is added. After the set time interval is reached, it sends a command to the microprocessor to collect the current signal at the corresponding time.

6. The electrochemical detection device based on enzyme inhibition method according to claim 1, characterized in that: in, The electrochemical detection device also includes a communication module, which is used to communicate with a smart terminal. The intelligent terminal is equipped with a data analysis device, including an input module for selecting target analytes, a display module for displaying detection results, a timing module, a communication module, and a control module. The input module can be a screen display input module that allows users to input information themselves, or a barcode scanning module that identifies the identification code attached to the electrochemical detection chip. Upon receiving a sample addition signal from the sample addition detection module, the timing module initiates a timing command. This command can be initiated either by receiving an immediate user command or automatically by the sample addition detection module detecting circuit continuity after liquid addition. Once the set time interval is reached, the timing module sends a command to the microprocessor via the communication and control modules to collect the corresponding current signal. The smart terminal is connected to a remote server, which stores standard curves for use.

7. An electrochemical detection system based on enzyme inhibition, characterized in that, include: Single-sample electrochemical detection chip; buffer solutions, extraction solutions, or standards required for detection and analysis; An electrochemical detection device that is connected to the electrochemical detection chip and processes the electrochemical detection signal. The single-sample electrochemical detection chip only requires one liquid sample addition, and its structure is as described in claim 1. The electrochemical detection device is as described in any one of claims 1 to 6.

8. A method for electrochemical detection using the electrochemical detection system of claim 7, characterized in that, The steps include the following: Step 1: Insert the mounting end of the single-sample electrochemical detection chip into the electrochemical detection chip interface to establish an electrical connection; Step 2: When the sample addition detection module detects the addition of sample, the timing starts, and the microprocessor starts the electrochemical test process according to the preset process parameters corresponding to the chip. Step 3: When the set time reaches the end, the microprocessor controls the electrochemical test to stop. The electrochemical test module processes the electrical signals collected on the electrochemical detection chip to obtain the raw electrochemical detection data. Step 4: The microprocessor further processes the raw electrochemical detection data and transmits the processed detection data to a remote server or calls the local storage database. The detection results are obtained by comparing with the standard curve and displayed on the detection device or smart terminal.

9. The method for electrochemical detection using the electrochemical detection system according to claim 8, characterized in that: in, In step 2, the electrochemical detection process corresponds one-to-one with the target analyte; the identification code attached to the electrochemical detection chip is scanned by the scanning module in the detection device or smart terminal to start the corresponding program for that chip.

10. A software product for controlling the electrochemical detection device based on enzyme inhibition method as described in claims 1-6, comprising a computer-readable medium having executable code embedded therein; the code, when executed, includes the following steps: after the sample addition detection module in the electrochemical detection device receives a sample addition signal, it initiates a timing command, which is initiated by receiving an immediate command from the user or automatically initiated when the sample addition detection module detects that the circuit is turned on after the liquid is added; after the set time interval is reached, it continues to issue a command to output the acquired current signal.

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