Agricultural product microbial contamination visual detection system and detection method

Abstract

The present application relates to the technical field of food safety detection, and particularly relates to a system and method for visual detection of microbial contamination of agricultural products, comprising a sampling reaction module, a biological anode module, an isolated differential channel module, a passive display control module and a gradient visual display module. The sampling reaction module receives sample extract and provides microbial metabolic substrate and electron mediator, the biological anode module accelerates electron transfer and charge accumulation, the isolated differential channel module realizes sample diversion and abiotic background elimination, the passive display control module regularly turns on the circuit, and the gradient visual display module drives the pixels to change color one by one through charge accumulation. In the present application, the detection paradigm combining microbial electricity generation and electrochromic display is adopted, so that on-site rapid detection can be realized without external power instruments and professional personnel, the result is presented in the form of the number of digital pixels, and the differential detection mechanism is used to eliminate complex matrix interference, so as to ensure the accuracy and consistency of the detection result.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, and in particular to a visual detection system and method for detecting microbial contamination in agricultural products. Background Technology

[0002] Microbial contamination of agricultural products is one of the main causes of foodborne illnesses, and timely and accurate detection of microbial contamination is a crucial link in ensuring food safety. Currently, microbial testing of agricultural products is mainly divided into two categories: laboratory testing and on-site rapid testing. Laboratory testing, as the gold standard, provides reliable results, but it has a long testing cycle, relies on large, sophisticated instruments and professional operators, and cannot meet the needs of on-site, real-time testing at all stages of production, distribution, and sales. While existing on-site rapid testing technologies have shortened the testing time, most still require portable reading devices, and the results largely depend on subjective judgment of color intensity, leading to significant human error.

[0003] Currently, there is no technology that can simultaneously achieve completely passive detection of microbial contamination in agricultural products, requiring no external power supply, instruments, or professional personnel, and providing objective and accurate results. This technological deficiency makes it impossible to conduct large-scale, routine rapid screening for microbial contamination in scenarios lacking laboratory facilities, such as farmers' markets, fields, and cold chain transportation nodes, severely impacting the early detection and timely handling of food safety risks. Summary of the Invention

[0004] To overcome the above shortcomings, this invention provides a visual detection system and method for detecting microbial contamination in agricultural products, aiming to improve the shortcomings of existing agricultural product microbial contamination detection technologies that cannot simultaneously achieve complete passive operation, require no external power supply or professional personnel, and provide objective and accurate interpretation of detection results.

[0005] In a first aspect, the present invention provides the following technical solution: a visual detection system for microbial contamination in agricultural products, comprising:

[0006] The sampling reaction module is used to receive the sample extract, provide the substrates and electron mediators required for microbial metabolism, convert microbial metabolic activities into electron output, and simultaneously output the sample extract in a split stream.

[0007] The bioanode module, connected to the sampling reaction module, is used to receive electrons output by the sampling reaction module and accelerate electron transfer and charge accumulation through its fixed electrocatalytic amplification seed layer.

[0008] The isolation differential channel module is connected to the sampling reaction module. It is used to receive the sample extract output by the sampling reaction module, split it into a test liquid stream and a reference liquid stream, and perform microbial activity inhibition treatment on the reference liquid stream.

[0009] The passive start-up control module is connected to the bioanode module and the isolation differential channel module respectively. It is used to conduct at regular intervals after absorbing the test liquid flow and reference liquid flow output by the isolation differential channel module, so as to control the transfer of the charge accumulated in the bioanode module to the subsequent modules.

[0010] The gradient visualization display module is connected to the passive display control module. It is used to receive the charge output by the bioanodine module after the passive display control module is turned on, and drive multiple pixels to change color sequentially through charge accumulation to form a visual gradient indication corresponding to the degree of microbial contamination.

[0011] By adopting the above technical solutions: This invention employs a detection paradigm that combines microbial metabolic electricity generation with electrochromic display, enabling rapid on-site detection without the need for external power supplies, instruments, or professional personnel. Charge accumulation drives the stepped area pixels to change color sequentially, presenting the results as a digital count of lit pixels, eliminating human interpretation errors. An isolated differential channel module is set up, using the difference in the number of lit pixels from two paths for interpretation, fundamentally eliminating interference from complex matrices. The electrocatalytic seed layer on the surface of the bioanode module accelerates electron transfer and charge accumulation, shortening detection time. The dissolution kinetics of a soluble hydrogel film achieve passive, timed conduction, ensuring consistent detection results. Each electrochromic pixel is equipped with an ion storage balance unit, maintaining the pixel's color-changing state and enabling long-term memory of the detection results.

[0012] Preferably, the sampling response module includes:

[0013] Hydrophilic microwell units are exposed to the outside to receive sample extracts;

[0014] The substrate-mediator composite unit is freeze-dried and fixed at the bottom of the hydrophilic microwell unit and connected to the hydrophilic microwell unit. It is used to release the freeze-dried nutrient substrate and artificial electron mediator after the sample extract is added, so that the microorganisms in the sample can transfer electrons to the bioanode module through the artificial electron mediator during metabolism.

[0015] Preferably, the bioanode module includes:

[0016] The composite conductive substrate unit, composed of a composite conductive nanopaper of carbon nanotubes and bacterial cellulose, is connected to the sampling reaction module and the passive display control module, respectively, to provide a continuous conductive path for electron transmission.

[0017] The electrocatalytic seed layer unit, fixed on the surface of the composite conductive substrate unit, is composed of cell lysates of non-pathogenic environmental microorganisms. These cell lysates are rich in cytochromes and flavin redox active components, which are used to rapidly initiate and amplify electron transfer received from the sampling reaction module, thereby accelerating charge accumulation on the composite conductive substrate unit.

[0018] Preferably, the isolated differential channel module includes:

[0019] The capillary splitting unit, connected to the sampling reaction module, is used to split the sample extract output from the sampling reaction module into a test liquid flow along the test channel and a reference liquid flow along the reference channel.

[0020] A bioactivity inhibition unit, located on the inner wall of the reference channel, contains a coating of a broad-spectrum antibiotic mixture to selectively inhibit the metabolic activity of microorganisms in the reference liquid stream;

[0021] The liquid storage tank unit is located at the end of the test channel and the reference channel, respectively, to temporarily store the test liquid flow and the reference liquid flow. The bottom of the liquid storage tank unit is in direct contact with the passive display control module, so that the liquid flow can wet the passive display control module.

[0022] Preferably, the gradient visualization display module includes:

[0023] The stepped area pixel array unit is composed of multiple electrochromic pixels with stepped increasing area connected in series. They are connected to the passive display control module and the ion storage balance unit respectively. During charge injection, the pixels are arranged in order of increasing area to complete the state transition from colorless to deep blue-purple.

[0024] An ion storage balancing unit is set up one-to-one with each electrochromic pixel to store and release ions to balance the charge during the electrochromic reaction process and to maintain the color-changing state of the pixel in the open circuit state.

[0025] Secondly, the present invention provides the following technical solution: a method for visually detecting microbial contamination in agricultural products, comprising the following steps:

[0026] S1. Sample pretreatment: Collect microbial samples from the surface of agricultural products and prepare them into a homogeneous sample extract.

[0027] S2, Sample Addition and Splitting: The sample extract is added dropwise to the hydrophilic microwell unit of the sampling reaction module, so that the sample extract dissolves the nutrient substrate and artificial electron mediator in the substrate-mediator complex unit, and at the same time, it is split into the test channel and reference channel of the isolation differential channel module.

[0028] S3, Metabolic Initiation and Activity Inhibition: Microorganisms in the test channel utilize nutrient substrates for respiratory metabolism and transfer electrons outward through artificial electron mediators; microorganisms in the control channel have their metabolic activity inhibited by the bioactivity inhibition unit, producing only abiotic background electrons.

[0029] S4. Accelerated Bioelectrocatalysis: Electrons generated in the test channel are transferred to the bioanode module, amplified by the electrocatalytic seed layer unit, and continuously accumulated on the composite conductive substrate unit.

[0030] S5, Passive Delayed Activation: The test liquid flow and the reference liquid flow flow into the corresponding storage tank unit respectively, wetting the passive activation control module; after a delay determined by the dissolution kinetics, the passive activation control module is turned on, and the charge accumulated in the bioanode module is injected into the gradient visualization display module.

[0031] S6. Gradient Display and Interpretation: The injected charge drives multiple pixels in the gradient visualization display module whose area changes with a gradient, changing color sequentially in ascending order; the difference in the number of fully lit pixels in the corresponding pixel arrays of the test channel and the reference channel is compared to determine the microbial contamination level.

[0032] Preferably, in step S1, the sample pretreatment specifically involves: wiping a specified area of ​​the surface of the agricultural product to be tested with a sterile swab, immersing the swab head in a quantitative amount of sterile physiological saline, and thoroughly shaking and eluting to obtain a uniform sample extract.

[0033] Preferably, in step S3, electrons generated by the respiratory metabolism of microorganisms in the test channel are transferred to the surface of the composite conductive substrate unit of the bioanode module via an artificial electronic mediator, and then captured and relayed by the electrocatalytic seed layer unit.

[0034] Preferably, in step S5, the passive delay activation specifically involves: the passive activation control module being electronically insulated in a dry state, and beginning to dissolve after contacting the test liquid flow and reference liquid flow in the storage tank unit; during the dissolution process, electrons continuously accumulate on the composite conductive substrate unit of the bioanode module; when the passive activation control module is completely dissolved and turned on, the accumulated electrons are released in a concentrated manner and injected into the gradient visualization display module through the conductive path.

[0035] Preferably, in step S6, the gradient display and interpretation specifically involves: observing the first pixel array driven by the test liquid flow and the second pixel array driven by the reference liquid flow, subtracting the number of fully lit pixels in the second pixel array from the number of fully lit pixels in the first pixel array, and the resulting difference directly corresponds to the preset microbial contamination level.

[0036] The present invention has the following beneficial effects:

[0037] 1. This invention employs a detection paradigm that combines microbial metabolic electricity generation with electrochromic display, eliminating the need for any external power supply, instruments, or professional personnel, thus achieving completely passive, rapid on-site detection. Charge accumulation drives the stepped area pixels to change color sequentially, and the result is presented as a digital count of the number of lit pixels, eliminating human interpretation errors.

[0038] 2. In this invention, an isolation differential channel module is set up to split the sample liquid into a test liquid flow and a reference liquid flow. The bioactivity inhibition unit of the reference channel eliminates microbial metabolic signals, retaining only non-biological background signals. The difference in the number of illuminated pixels in the two channels is used for interpretation, thus eliminating interference from complex matrices in principle.

[0039] 3. In this invention, the surface of the bioanode module is immobilized with cell lysates of non-pathogenic environmental microorganisms as an electrocatalytic seed layer, which is rich in redox active components. This allows the bioanode module to quickly capture and relay electrons transferred by artificial electron mediators, thereby accelerating electron transfer and charge accumulation processes and shortening the detection time.

[0040] 4. In this invention, a passive start-up control module is used to automatically control the reaction time. The circuit is timed to activate based on the dissolution kinetics of the soluble hydrogel film, eliminating the need for an external electronic timer. During dissolution, charge continuously accumulates and is released in a concentrated manner upon activation, driving the colorimetric reaction and ensuring consistent detection results.

[0041] 5. In this invention, each electrochromic pixel is equipped with an ion storage balancing unit, which can balance the charge during the electrochromic reaction and maintain the color-changing state of the pixel in an open-circuit state, thus achieving long-term memory of the detection results. A series-connected stepped-area pixel array is used, making the color development process intuitive and orderly. Attached Figure Description

[0042] Figure 1 This is a framework diagram of a visual detection system for microbial contamination in agricultural products proposed in this invention;

[0043] Figure 2 This is a flowchart of a visual detection method for microbial contamination in agricultural products proposed in this invention. Detailed Implementation

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Example 1: In the first embodiment of this invention, a visual detection system for microbial contamination in agricultural products is provided. This system has a disposable card-like structure, composed of five functional films vertically stacked from top to bottom and bonded together using a hot-pressing process. The stacking order is as follows: sampling reaction module, isolation differential channel module, bioanodine module, passive activation and display control module, and gradient visualization display module. The layers are bonded together with double-sided pressure-sensitive adhesive to ensure structural stability and unobstructed flow of liquid and electrons. Figure 1As shown, it includes:

[0046] The sampling reaction module is used to receive the sample extract, provide the substrates and electron mediators required for microbial metabolism, convert microbial metabolic activities into electron output, and simultaneously output the sample extract in a split stream.

[0047] Furthermore, the sampling response module includes:

[0048] Hydrophilic microwell units are exposed to the outside to receive sample extracts;

[0049] The substrate-mediator composite unit is freeze-dried and fixed at the bottom of the hydrophilic microwell unit and connected to the hydrophilic microwell unit. It is used to release the freeze-dried nutrient substrate and artificial electron mediator after the sample extract is added, so that the microorganisms in the sample can transfer electrons to the bioanode module through the artificial electron mediator during metabolism.

[0050] Specifically, the sampling reaction module uses a 100μm thick transparent polyethylene terephthalate film as a substrate. Circular hydrophilic microwell units with a diameter of 5mm are fabricated on the substrate surface using photolithography. A 1mm wide hydrophobic isolation zone is formed around each microwell unit through plasma hydrophobization to prevent sample extract from overflowing. The substrate-mediator composite unit is precisely added to the bottom of the hydrophilic microwell unit using a micropipette. After addition, it is placed in a vacuum freeze dryer for lyophilization to form a uniform vitrified film. The mixture of the substrate-mediator composite unit comprises three parts: a nutrient substrate, an artificial electron mediator, and a lyophilization protectant. The nutrient substrate provides carbon and nitrogen sources for microbial growth. The artificial electron mediator acts as an electron shuttle, transferring electrons generated by intracellular metabolism of microorganisms to the extracellular bioanode module. The lyophilization protectant uses a trehalose and sucrose compound system to maintain the bioactivity of the nutrient substrate and electron mediator during lyophilization, preventing their denaturation and inactivation. The hydrophilic microwell unit has a 0.5mm diameter liquid outlet at its bottom, which connects to the capillary effluent unit inlet of the isolation differential channel module via a microchannel. After freeze-drying, a peelable aluminum foil protective layer is applied to the surface of the hydrophilic microwell unit to isolate it from air and moisture, extending the shelf life of the detection system.

[0051] The bioanode module, connected to the sampling reaction module, is used to receive electrons output by the sampling reaction module and accelerate electron transfer and charge accumulation through its fixed electrocatalytic amplification seed layer.

[0052] Furthermore, the bioanode module includes:

[0053] The composite conductive substrate unit, composed of a composite conductive nanopaper of carbon nanotubes and bacterial cellulose, is connected to the sampling reaction module and the passive display control module, respectively, to provide a continuous conductive path for electron transmission.

[0054] The electrocatalytic seed layer unit, fixed on the surface of the composite conductive substrate unit, is composed of cell lysates of non-pathogenic environmental microorganisms. These cell lysates are rich in cytochromes and flavin redox active components, which are used to rapidly initiate and amplify electron transfer received from the sampling reaction module, thereby accelerating charge accumulation on the composite conductive substrate unit.

[0055] Specifically, the bioanode module comprises two independent and structurally identical anode units, corresponding to the bottom positions of the test channel and the reference channel, respectively. Each anode unit consists of a composite conductive substrate unit and an electrocatalytic seed layer unit. The composite conductive substrate unit is prepared using a vacuum filtration method to create a composite conductive nanopaper of carbon nanotubes and bacterial cellulose. Its top surface is in direct contact with the bottom of the corresponding channel, and its bottom surface is connected to the corresponding area of ​​the passive activation control module, providing a continuous conductive path for electron transport. The electrocatalytic seed layer unit is fixed to the top surface of the composite conductive substrate unit using a drop-coating method. First, the non-pathogenic environmental microorganism *Rhodopseudomonas palustris* is inoculated into a liquid culture medium for expansion culture. After reaching the logarithmic growth phase, the bacterial cells are collected by centrifugation, washed multiple times with phosphate buffer solution, and then a cell lysate is prepared using ultrasonic disruption. After centrifugation to remove cell debris, the supernatant is obtained and drop-coated onto the surface of the composite conductive substrate unit. After vacuum freeze-drying, the electrocatalytic seed layer unit is formed. The electrocatalytic seed layer unit is rich in cytochrome c and flavin redox active components, which can rapidly capture electrons transferred by artificial electron mediators in the channel and perform relay amplification, significantly improving electron transfer efficiency.

[0056] The current gain in the electrocatalytic amplification process can be described by the following formula:

[0057] I = G·I0;

[0058] Where I is the actual output current of the bioanode module, I0 is the base output current without the electrocatalytic seed layer unit, and G is the current gain coefficient. The current gain coefficient G is positively correlated with the concentration of redox active components in the electrocatalytic seed layer unit, and its magnitude can be controlled by adjusting the concentration of the cell lysis buffer. The output current I directly determines the charge accumulation rate on the bioanode module, and the relationship between the charge accumulation rate and the output current is as follows:

[0059] ;

[0060] Where Q represents the amount of charge accumulated on the bio-anode module, and t represents the charge accumulation time. The accumulated charge Q directly determines the number of pixels that can be lit up in the subsequent gradient visualization display module.

[0061] The isolation differential channel module is connected to the sampling reaction module. It is used to receive the sample extract output by the sampling reaction module, split it into a test liquid stream and a reference liquid stream, and perform microbial activity inhibition treatment on the reference liquid stream.

[0062] Furthermore, the isolated differential channel module includes:

[0063] The capillary splitting unit, connected to the sampling reaction module, is used to split the sample extract output from the sampling reaction module into a test liquid flow along the test channel and a reference liquid flow along the reference channel.

[0064] A bioactivity inhibition unit, located on the inner wall of the reference channel, contains a coating of a broad-spectrum antibiotic mixture to selectively inhibit the metabolic activity of microorganisms in the reference liquid stream;

[0065] The liquid storage tank unit is located at the end of the test channel and the reference channel, respectively, to temporarily store the test liquid flow and the reference liquid flow. The bottom of the liquid storage tank unit is in direct contact with the passive display control module, so that the liquid flow can wet the passive display control module.

[0066] Specifically, the isolation differential channel module uses a wax printing process to prepare a hydrophobic pattern on the same PET substrate as the sampling reaction module, forming a capillary flow unit, a test channel, a reference channel, and a reservoir unit. The capillary flow unit is designed with a Y-shaped structure, which can evenly split the sample extract flowing from the sampling reaction module into two streams of equal volume, which enter the test channel and the reference channel respectively. The inner wall of the test channel is made of pure PET material without any modification, ensuring that the microorganisms in the sample can maintain complete metabolic activity. The inner wall of the reference channel is prepared with a bioactivity inhibition unit by spraying. The bioactivity inhibition unit is a mixed coating of ciprofloxacin and actinomycete ketone. Ciprofloxacin can inhibit the DNA gyrase activity of Gram-positive and Gram-negative bacteria, while actinomycete ketone can inhibit the protein synthesis of fungi. The synergistic effect of the two can selectively inhibit the metabolic activity of almost all viable bacteria in the reference stream without affecting the redox properties of non-biological reducing substances. The liquid storage tank unit is designed as a cylindrical structure, located at the ends of both the test and reference channels. The volume of the liquid storage tank unit is larger than the total liquid flow volume within the channels, fully accommodating the test and reference liquid flows in from the channels. The bottom of the liquid storage tank unit is laser-drilled to create multiple micropores with a diameter of 10μm, allowing the liquid flow to permeate the passive display control module below.

[0067] The passive start-up control module is connected to the bioanode module and the isolation differential channel module respectively. It is used to conduct at regular intervals after absorbing the test liquid flow and reference liquid flow output by the isolation differential channel module, so as to control the transfer of the charge accumulated in the bioanode module to the subsequent modules.

[0068] Specifically, the passive start-up control module is a physically cross-linked hydrogel film formed by low-polymerization degree polyvinyl alcohol and borax, prepared by casting. First, the low-polymerization degree polyvinyl alcohol is dissolved in deionized water, heated and stirred until completely dissolved. Then, a borax solution is added, and the mixture is quickly stirred until homogeneous before being poured into a polytetrafluoroethylene mold and allowed to dry naturally at room temperature. The dried hydrogel film is a solid insulating membrane with good mechanical strength and chemical stability. In its dry state, it can completely isolate the bioanode module and the gradient visualization display module, blocking electron transmission between them. When the hydrogel film comes into contact with the liquid flow from the storage tank unit, it begins to absorb water, swells, and gradually dissolves. During the dissolution process, the insulation performance of the film gradually decreases. When the film is completely dissolved, a continuous ion conduction path is formed between the bioanode module and the gradient visualization display module, and the circuit is instantly turned on.

[0069] The passive start-up control module comprises two independent hydrogel film regions of equal area, corresponding to the reservoir units of the test channel and reference channel, respectively, and the corresponding bioanode unit and display unit below them. The hydrogel films in both regions have identical composition and thickness, ensuring simultaneous dissolution and conduction of both pathways.

[0070] The dissolution kinetics of the passive display control module can be described by the following formula:

[0071] α = 1 - exp(-k·t);

[0072] Where α represents the degree of dissolution of the hydrogel film, ranging from 0 to 1, k is the dissolution rate constant, and t is the contact time between the liquid flow and the film. The dissolution rate constant k is determined by the thickness of the hydrogel film, the degree of polymerization of polyvinyl alcohol, and the concentration of borax. By adjusting these parameters, the dissolution time of the film can be precisely controlled, thereby achieving precise control over the charge accumulation time. For the test channel and the reference channel, there are corresponding total charge amounts Q. T and Q R , are the integral values ​​of the current in the two bioanode units during the charge accumulation time, respectively. When the degree of dissolution α reaches 1, both hydrogel films dissolve completely simultaneously, Q T and Q R Simultaneously, the first pixel array and the second pixel array are injected respectively.

[0073] The gradient visualization display module is connected to the passive display control module. It is used to receive the charge output by the bioanodine module after the passive display control module is turned on, and drive multiple pixels to change color sequentially through charge accumulation to form a visual gradient indication corresponding to the degree of microbial contamination.

[0074] Furthermore, the gradient visualization display module includes:

[0075] The stepped area pixel array unit is composed of multiple electrochromic pixels with stepped increasing area connected in series. They are connected to the passive display control module and the ion storage balance unit respectively. During charge injection, the pixels are arranged in order of increasing area to complete the state transition from colorless to deep blue-purple.

[0076] An ion storage balancing unit is set up one-to-one with each electrochromic pixel to store and release ions to balance the charge during the electrochromic reaction process and to maintain the color-changing state of the pixel in the open circuit state.

[0077] Specifically, the gradient visualization display module comprises two independent and structurally identical display units, corresponding to the test channel and the reference channel, respectively, namely the first pixel array and the second pixel array. Each display unit uses a transparent conductive PET film as a substrate, and interdigitated transparent conductive electrodes are fabricated using a screen printing process. Then, a nanoporous titanium dioxide-violetin derivative composite electrochromic layer is coated on the electrode surface. Finally, a nickel oxide nanosheet ion storage layer is deposited on the surface of the electrochromic layer to form a single electrochromic pixel. The stepped area pixel array unit consists of 8 independent electrochromic pixels connected in series. The area of ​​each pixel increases in a geometric progression, and adjacent pixels are connected by printed carbon resistive lines. The series connection method is as follows: the input terminal of the smallest pixel is connected to the corresponding output terminal of the passive display control module, the output terminal of each pixel is connected to the input terminal of the next larger pixel, and the output terminal of the largest pixel is connected to the common ground terminal. When charge is injected into the stepped-area pixel array unit, the smallest pixel reaches the color-changing threshold voltage first, undergoing a reversible reduction reaction from colorless to deep blue-violet. As the amount of injected charge increases, the larger pixels successively reach the color-changing threshold voltage and undergo the color-changing reaction in sequence. An ion storage balance unit is configured one-to-one with each electrochromic pixel. During the electrochromic reaction, the ion storage balance unit releases hydrogen ions to balance the negative charge in the electrochromic layer, ensuring the smooth progress of the electrochromic reaction. Simultaneously, the ion storage balance unit can maintain charge balance in an open-circuit state, keeping the colored pixels in a deep blue-violet state, thus achieving long-term memory of the detection results.

[0078] Example 2: There is a need for rapid detection of microbial contamination in agricultural product transactions at farmers' markets. Existing technologies cannot achieve real-time on-site detection, and reducing substances such as lycopene and vitamin C on the surface of tomatoes can severely interfere with test results, leading to false positives. To solve these problems, the visual detection method for microbial contamination of agricultural products provided by this invention is used for on-site detection. To solve the above problems, this invention provides a visual detection method for microbial contamination of agricultural products, such as... Figure 2 As shown. Includes the following steps:

[0079] S1. Sample pretreatment: Collect microbial samples from the surface of agricultural products and prepare them into a homogeneous sample extract.

[0080] Furthermore, the sample pretreatment specifically involves wiping a specified area of ​​the surface of the agricultural product to be tested with a sterile swab, immersing the swab head in a quantitative amount of sterile physiological saline, and thoroughly shaking and eluting to obtain a homogeneous sample extract.

[0081] Specifically, microbial samples are collected from the surface of agricultural products and prepared into a homogeneous sample extract. Specifically, a sterile polyester fiber swab is used to cover a designated area of ​​the tomato surface to be tested in a unidirectional wiping motion. During the wiping process, the swab is held at a 45-degree angle to the tomato surface and rotated moderately to ensure sufficient collection of surface microorganisms. The swab head is then completely immersed in a centrifuge tube containing a quantitative amount of sterile physiological saline, and the tube is vortexed to elute the microorganisms, ensuring thorough transfer of the microorganisms from the swab to the physiological saline, resulting in a homogeneous sample extract. For example, when testing a batch of tomatoes for sale, three tomatoes are randomly selected, and the equatorial region of each tomato is swab-sampled to prepare the corresponding sample extract.

[0082] S2, Sample Addition and Splitting: The sample extract is added dropwise to the hydrophilic microwell unit of the sampling reaction module, so that the sample extract dissolves the nutrient substrate and artificial electron mediator in the substrate-mediator complex unit, and at the same time, it is split into the test channel and reference channel of the isolation differential channel module.

[0083] Specifically, the sample extract is dropwise added to the hydrophilic microwell unit of the sampling reaction module, allowing the sample extract to dissolve the nutrient substrate and artificial electron mediator in the substrate-mediator complex unit, while simultaneously diverting it into the test and reference channels of the isolation differential channel module. Specifically, a fixed volume of sample extract is drawn using a disposable micropipette and vertically dropwise added to the center of the hydrophilic microwell unit. Upon contact with the lyophilized substrate-mediator complex unit, the sample extract rapidly re-dissolves, releasing nutrient substrates such as glucose and beef extract peptone, as well as artificial electron mediators such as neutral red and 2-hydroxy-1,4-naphthoquinone, forming a homogeneous reaction system. Simultaneously, driven by capillary forces, the reaction system is uniformly diverted into two equal-volume streams through a Y-shaped capillary dispersing unit, which enter the test and reference channels respectively.

[0084] S3, Metabolic Initiation and Activity Inhibition: Microorganisms in the test channel utilize nutrient substrates for respiratory metabolism and transfer electrons outward through artificial electron mediators; microorganisms in the control channel have their metabolic activity inhibited by the bioactivity inhibition unit, producing only abiotic background electrons.

[0085] Furthermore, electrons generated by the respiratory metabolism of microorganisms in the test channel are transferred to the surface of the composite conductive substrate unit of the bioanode module via an artificial electronic mediator, and then captured and relayed by the electrocatalytic seed layer unit.

[0086] Specifically, microorganisms in the test channel utilize nutrient substrates for respiratory metabolism and transfer electrons outward through an artificial electron mediator; microorganisms in the reference channel have their metabolic activity inhibited by a bioactivity inhibition unit, producing only abiotic background electrons. Specifically, under suitable nutritional conditions, microorganisms in the test channel initiate aerobic respiration metabolism, breaking down glucose to produce energy and metabolic products, while simultaneously transferring electrons to the artificial electron mediator. The artificial electron mediator, acting as an electron shuttle, can penetrate the microbial cell membrane, transferring intracellular electrons to the extracellular bioanolyte module. When the liquid flow in the reference channel passes through the bioactivity inhibition unit, ciprofloxacin and actinomycete ketone rapidly dissolve in the liquid flow, inhibiting bacterial DNA replication and fungal protein synthesis, respectively, causing all viable bacteria to lose metabolic activity, retaining only the redox activity of abiotic reducing substances such as lycopene and vitamin C in the sample.

[0087] S4. Accelerated Bioelectrocatalysis: Electrons generated in the test channel are transferred to the bioanode module, amplified by the electrocatalytic seed layer unit, and continuously accumulated on the composite conductive substrate unit.

[0088] Specifically, electrons generated within the test channel are transferred to the test anode unit below the test channel, while abiotic background electrons generated within the reference channel are transferred to the reference anode unit below the reference channel. After being relayed and amplified by their respective electrocatalytic seed layer units, the electrons independently and continuously accumulate on the two composite conductive substrate units. Specifically, electrons carried by the artificial electron mediator are rapidly captured by cytochrome c and flavin redox active components in the electrocatalytic seed layer unit after being transferred to the surface of the composite conductive substrate unit. These active components, acting as electron relay stations, significantly reduce the energy barrier for electron transfer, accelerating electron transport and accumulation on the composite conductive substrate unit. The current gain during the electrocatalytic amplification process follows the formula described in Example 1.

[0089] S5, Passive Delayed Activation: The test liquid flow and the reference liquid flow flow into the corresponding storage tank unit respectively, wetting the passive activation control module; after a delay determined by the dissolution kinetics, the passive activation control module is turned on, and the charge accumulated in the bioanode module is injected into the gradient visualization display module.

[0090] Furthermore, the passive delay-activated display is specifically as follows: the passive activation control module is electronically insulated in a dry state, and begins to dissolve after it comes into contact with the test liquid flow and the reference liquid flow in the storage tank unit; during the dissolution process, electrons continuously accumulate on the composite conductive substrate unit of the bioanode module; when the passive activation control module is completely dissolved and turned on, the accumulated electrons are released in a concentrated manner and injected into the gradient visualization display module through the conductive path.

[0091] Specifically, the test liquid flow and the reference liquid flow flow into their respective reservoir units, wetting the passive display control module. After a delay determined by dissolution kinetics, the passive display control module is activated, and the charge accumulated in the bioanode module is injected into the gradient visualization display module. Specifically, the test liquid flow and the reference liquid flow flow along the channel driven by capillary force, eventually entering the reservoir unit at the end. Micropores at the bottom of the reservoir unit allow the liquid flow to slowly permeate, wetting the passive display control module below. The passive display control module is a low-polymerization-degree polyvinyl alcohol-borax hydrogel film, which is in an insulating state in the dry state, completely blocking electron transfer between the bioanode module and the gradient visualization display module. When the film absorbs moisture, it begins to gradually dissolve, and its insulation performance decreases with increasing degree of dissolution. The dissolution kinetics of the passive display control module follow the formula described in Example 1. The passive display control module regions corresponding to the test channel and the reference channel have exactly the same dissolution kinetics, therefore both paths are activated simultaneously, and the total charge Q accumulated in the test anode unit... T The total charge Q accumulated in the reference anode unit R Simultaneously, the first pixel array and the second pixel array are injected respectively.

[0092] S6. Gradient Display and Interpretation: The injected charge drives multiple pixels in the gradient visualization display module whose area changes with a gradient, changing color sequentially in ascending order; the difference in the number of fully lit pixels in the corresponding pixel arrays of the test channel and the reference channel is compared to determine the microbial contamination level.

[0093] Furthermore, the gradient display and interpretation specifically involves: observing the first pixel array driven by the test liquid flow and the second pixel array driven by the reference liquid flow, subtracting the number of fully lit pixels in the second pixel array from the number of fully lit pixels in the first pixel array, and the resulting difference directly corresponds to the preset microbial contamination level.

[0094] Specifically, the injected charge drives multiple pixels with gradient-changing areas in the gradient visualization module to change color sequentially in ascending order. The difference in the number of fully illuminated pixels in the corresponding pixel arrays of the test channel and the reference channel is compared to determine the microbial contamination level. Specifically, the injected charge first enters the stepped area pixel array unit. Since the smallest pixel requires the least amount of charge to reach the color-changing threshold, it undergoes the reduction reaction from colorless to deep blue-purple first. As the injected charge increases, subsequent pixels with progressively larger areas reach the color-changing threshold one by one and undergo the color-changing reaction. The ion storage balance unit releases hydrogen ions during the reaction to balance the negative charge in the electrochromic layer, while maintaining the color state of the already colored pixels. The preset contamination level classification standard is pre-defined based on the detection sensitivity requirements. Different difference ranges correspond to different contamination levels and can be adjusted according to the safety standards of different agricultural products. For example, in the detection of a tomato sample, the first pixel array corresponding to the test channel illuminates 5 pixels, while the second pixel array corresponding to the reference channel illuminates 1 pixel, with a difference of 4. According to the preset contamination level classification standard, the tomato sample is determined to have moderate microbial contamination.

[0095] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A visual detection system for microbial contamination in agricultural products, characterized in that, include: The sampling reaction module is used to receive the sample extract, provide the substrates and electron mediators required for microbial metabolism, convert microbial metabolic activities into electron output, and simultaneously output the sample extract in a split stream. The bioanode module, connected to the sampling reaction module, is used to receive electrons output by the sampling reaction module and accelerate electron transfer and charge accumulation through its fixed electrocatalytic amplification seed layer. The isolation differential channel module is connected to the sampling reaction module. It is used to receive the sample extract output by the sampling reaction module, split it into a test liquid stream and a reference liquid stream, and perform microbial activity inhibition treatment on the reference liquid stream. The passive start-up control module is connected to the bioanode module and the isolation differential channel module respectively. It is used to conduct at regular intervals after absorbing the test liquid flow and reference liquid flow output by the isolation differential channel module, so as to control the transfer of the charge accumulated in the bioanode module to the subsequent modules. The gradient visualization display module is connected to the passive display control module. It is used to receive the charge output by the bioanodine module after the passive display control module is turned on, and drive multiple pixels to change color sequentially through charge accumulation to form a visual gradient indication corresponding to the degree of microbial contamination.

2. The visual detection system for microbial contamination of agricultural products according to claim 1, characterized in that, The sampling reaction module includes: Hydrophilic microwell units are exposed to the outside to receive sample extracts; The substrate-mediator composite unit is freeze-dried and fixed at the bottom of the hydrophilic microwell unit and connected to the hydrophilic microwell unit. It is used to release the freeze-dried nutrient substrate and artificial electron mediator after the sample extract is added, so that the microorganisms in the sample can transfer electrons to the bioanode module through the artificial electron mediator during metabolism.

3. The visual detection system for microbial contamination of agricultural products according to claim 1, characterized in that, The bioanode module includes: The composite conductive substrate unit, composed of a composite conductive nanopaper of carbon nanotubes and bacterial cellulose, is connected to the sampling reaction module and the passive display control module, respectively, to provide a continuous conductive path for electron transmission. The electrocatalytic seed layer unit, fixed on the surface of the composite conductive substrate unit, is composed of cell lysates of non-pathogenic environmental microorganisms. These cell lysates are rich in cytochromes and flavin redox active components, which are used to rapidly initiate and amplify electron transfer received from the sampling reaction module, thereby accelerating charge accumulation on the composite conductive substrate unit.

4. The visual detection system for microbial contamination of agricultural products according to claim 1, characterized in that, The isolation differential channel module includes: The capillary splitting unit, connected to the sampling reaction module, is used to split the sample extract output from the sampling reaction module into a test liquid flow along the test channel and a reference liquid flow along the reference channel. A bioactivity inhibition unit, located on the inner wall of the reference channel, contains a coating of a broad-spectrum antibiotic mixture to selectively inhibit the metabolic activity of microorganisms in the reference liquid stream; The liquid storage tank unit is located at the end of the test channel and the reference channel, respectively, to temporarily store the test liquid flow and the reference liquid flow. The bottom of the liquid storage tank unit is in direct contact with the passive display control module, so that the liquid flow can wet the passive display control module.

5. The visual detection system for microbial contamination of agricultural products according to claim 1, characterized in that, The gradient visualization display module includes: The stepped area pixel array unit is composed of multiple electrochromic pixels with stepped increasing area connected in series. They are connected to the passive display control module and the ion storage balance unit respectively. During charge injection, the pixels are arranged in order of increasing area to complete the state transition from colorless to deep blue-purple. An ion storage balancing unit is set up one-to-one with each electrochromic pixel to store and release ions to balance the charge during the electrochromic reaction process and to maintain the color-changing state of the pixel in the open circuit state.

6. A visual detection method for microbial contamination in agricultural products, characterized in that, A visual detection system for microbial contamination of agricultural products according to any one of claims 1-5 includes the following steps: S1. Sample pretreatment: Collect microbial samples from the surface of agricultural products and prepare them into a homogeneous sample extract. S2, Sample Addition and Splitting: The sample extract is added dropwise to the hydrophilic microwell unit of the sampling reaction module, so that the sample extract dissolves the nutrient substrate and artificial electron mediator in the substrate-mediator complex unit, and at the same time, it is split into the test channel and reference channel of the isolation differential channel module. S3, Metabolic Initiation and Activity Inhibition: Microorganisms in the test channel utilize nutrient substrates for respiratory metabolism and transfer electrons outward through artificial electron mediators; microorganisms in the control channel have their metabolic activity inhibited by the bioactivity inhibition unit, producing only abiotic background electrons. S4. Accelerated Bioelectrocatalysis: Electrons generated in the test channel are transferred to the bioanode module, amplified by the electrocatalytic seed layer unit, and continuously accumulated on the composite conductive substrate unit. S5, Passive Delayed Activation: The test liquid flow and the reference liquid flow flow into the corresponding storage tank unit respectively, wetting the passive activation control module; after a delay determined by the dissolution kinetics, the passive activation control module is turned on, and the charge accumulated in the bioanode module is injected into the gradient visualization display module. S6. Gradient Display and Interpretation: The injected charge drives multiple pixels in the gradient visualization display module whose area changes with a gradient, changing color sequentially in ascending order; the difference in the number of fully lit pixels in the corresponding pixel arrays of the test channel and the reference channel is compared to determine the microbial contamination level.

7. The method for visually detecting microbial contamination in agricultural products according to claim 6, characterized in that, In step S1, the sample pretreatment specifically involves wiping a specified area of ​​the surface of the agricultural product to be tested with a sterile swab, immersing the swab head in a quantitative amount of sterile physiological saline, and thoroughly shaking and eluting to obtain a homogeneous sample extract.

8. The method for visually detecting microbial contamination in agricultural products according to claim 6, characterized in that, In step S3, electrons generated by the respiratory metabolism of microorganisms in the test channel are transferred to the surface of the composite conductive substrate unit of the bioanode module via an artificial electronic mediator, and then captured and relayed by the electrocatalytic seed layer unit.

9. The method for visually detecting microbial contamination in agricultural products according to claim 6, characterized in that, In step S5, the passive delay activation is specifically as follows: the passive activation control module is electronically insulated in a dry state, and begins to dissolve after it comes into contact with the test liquid flow and the reference liquid flow in the storage tank unit; during the dissolution process, electrons continuously accumulate on the composite conductive substrate unit of the bioanode module. Once the passive display control module is completely dissolved and activated, the accumulated electrons are released and injected into the gradient visualization display module through the activated pathway.

10. A method for visually detecting microbial contamination in agricultural products according to claim 6, characterized in that, In step S6, the gradient display and interpretation specifically involves: observing the first pixel array driven by the test liquid flow and the second pixel array driven by the reference liquid flow, subtracting the number of fully lit pixels in the second pixel array from the number of fully lit pixels in the first pixel array, and the resulting difference directly corresponds to the preset microbial contamination level.

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