Test paper for measuring content of escherichia coli in food

Abstract

The invention relates to the technical field of microbiological detection, and provides test paper for determining the content of escherichia coli in food, which comprises a substrate made of an insulating material such as polyethylene glycol terephthalate or polyimide. A working electrode, a counter electrode and a reference electrode are arranged at the top of the substrate, a silver chloride material layer is arranged at the top of the reference electrode, and three groups of circuits communicated with the working electrode, the counter electrode and the reference electrode are respectively arranged at the top of the substrate; through use of inorganic nanoparticles, a polymer film and a sulfhydrylated Escherichia coli aptamer in the composite material layer, high-sensitivity and specific detection of Escherichia coli is realized, interference of other microorganisms is effectively avoided, the accuracy and reliability of detection are improved, and in addition, a hydrophilic film layer is arranged on the periphery of the composite material layer, so that the detection accuracy and reliability are improved. And a to-be-detected sample is quickly absorbed and wetted by utilizing good wettability and water absorption, so that the contact between escherichia coli and an aptamer is promoted, and the detection efficiency and sensitivity are improved.

Description

Technical Field

[0001] The invention relates to the technical field of microbial detection, and in particular to a test paper for determining the content of Escherichia coli in food. Background Art

[0002] At present, E. coli, as a highly pathogenic foodborne pathogen, has attracted widespread attention. This bacterium can cause a variety of serious diseases in humans, including acute diarrhea, severe vomiting, high fever and sepsis, posing a major threat to public health and food safety. Therefore, it is particularly important to develop a technology that can quickly and accurately detect the content of E. coli.

[0003] However, conventional E. coli detection methods in the prior art often have many limitations. These methods usually require the collected samples to be cultured for a long time in order to allow the bacteria to fully multiply to a detectable level. However, this culture process is not only time-consuming and lengthy, but also cannot achieve real-time detection, resulting in a significant lag in the test results. This lag may not only delay the diagnosis of the disease, but also affect the timely handling of food safety incidents, thereby posing potential risks to public health.

[0004] In view of the above situation, we urgently need a new test paper that can quickly detect the content of E. coli. This test paper should have significant advantages such as simple operation, rapid response, and accurate results. It can effectively detect E. coli in samples in a short time, thereby providing timely and strong support for medical diagnosis and food safety monitoring. Summary of the invention

[0005] The present invention provides a test paper for determining the content of Escherichia coli in food, which solves the problems in the related art that samples need to be cultured for a long time and are difficult to preserve.

[0006] The technical solution of the present invention is as follows:

[0007] To achieve the above object, the present invention provides the following technical solution: a test paper for determining the content of Escherichia coli in food, comprising a substrate, the substrate being made of insulating materials such as polyethylene terephthalate or polyimide, the top of the substrate being respectively provided with a working electrode, a counter electrode and a reference electrode, the top of the substrate being respectively provided with three groups of circuits connected to the working electrode, the counter electrode and the reference electrode, the top of the working electrode being provided with a composite material layer, the top of the reference electrode being provided with a silver chloride material layer;

[0008] The composite material layer includes a polymer film, inorganic nanoparticles dispersed and adhered in the polymer film, and Escherichia coli aptamers loaded on the surface of the inorganic nanoparticles. The Escherichia coli aptamers can specifically adsorb Escherichia coli in the test liquid on the material surface and generate a current response linearly related to the concentration of the bacterial liquid.

[0009] Preferably, the inorganic nanoparticles may be selected from one or more of nano-silicon wire, nano-nickel hydroxide, nano-copper, nano-copper oxide, nano-nickel, nano-platinum, nano-palladium, or nano-gold, as well as composite materials formed based on the above materials.

[0010] Preferably, the material of the polymer membrane is one or more of polytetrafluoroethylene, polyvinylidene fluoride, polyvinyl alcohol, polycyclodextrin, polythiophene, polysulfide, polyolefins, perfluorosulfonic acid resin, and aniline. The above materials are easy to carry out surface modification, so that inorganic nanoparticles and Escherichia coli aptamers can be loaded onto the membrane. The Escherichia coli aptamer can be firmly loaded on the surface of the inorganic nanoparticles through physical adsorption, covalent bonding and other methods, thereby forming a composite material layer.

[0011] Preferably, the E. coli aptamer can be loaded on the surface of inorganic nanoparticles by physical adsorption, covalent binding and other methods, which can fully utilize the excellent performance of inorganic nanoparticles and the high specificity of E. coli aptamers to improve the sensitivity, specificity and stability of E. coli detection.

[0012] Preferably, the silver chloride material layer is arranged on the surface of the reference electrode by screen printing, coating and other techniques, and the thickness of the silver chloride material layer is 10 to 500 microns. Selecting a composite material layer of this thickness can ensure that when the test paper is used for detection, the sensitivity and response speed can be improved while ensuring the strength, thereby speeding up the time required for detection and improving the detection efficiency.

[0013] Preferably, a hydrophilic film layer is provided on the outer peripheral surface of the composite material layer, covering the composite material layer, the silver chloride material layer and a part of the counter electrode. The thickness of the composite material layer is 10 to 500 microns, and its function is to provide a stable potential as a reference for the electrochemical test system. The hydrophilic film layer has good wettability and water absorption, and can quickly absorb and wet the sample to be tested, so that the Escherichia coli in the sample is more likely to contact the Escherichia coli aptamer on the composite material layer, thereby improving the efficiency and sensitivity of the detection.

[0014] The working principle and beneficial effects of the present invention are:

[0015] 1. The test paper of the present invention adopts a dry reagent structure, which does not require on-site reagent preparation, simplifies the detection process, and improves the portability and stability of the test paper.

[0016] 2. The present invention performs cyclic voltammetry measurement on the test paper through an electrochemical workstation, records and analyzes the current response at different potentials, selects the oxidation current at a specific potential as the output signal, and realizes the quantitative analysis of the Escherichia coli content. In addition, during the electrochemical detection process, the change of the current response can be monitored in real time to ensure the accuracy and reliability of the detection results.

[0017] 3. The present invention adopts insulating materials such as polyethylene terephthalate or polyimide to make the substrate. The test paper is small in size and adopts a dry reagent structure, which is easy to transport, carry and store. It is particularly suitable for on-site and field detection. At the same time, the stability and durability of the test paper are also significantly improved, and it can maintain stable detection performance under different environments.

[0018] 4. The present invention achieves high-sensitivity and specific detection of Escherichia coli through the use of inorganic nanoparticles, polymer membranes and thiol-modified Escherichia coli aptamers in the composite material layer, effectively avoids interference from other microorganisms, and improves the accuracy and reliability of detection. In addition, a hydrophilic membrane layer is arranged on the periphery of the composite material layer, and its good wettability and water absorption are used to quickly absorb and wet the sample to be tested, promote the contact between Escherichia coli and the aptamer, improve the efficiency and sensitivity of detection, and also achieve the goal of eliminating the need to culture Escherichia coli during the entire detection process, greatly reducing the time required for the entire detection, further increasing the efficiency of the entire detection, and simultaneously detecting multiple samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0020] Figure 1 It is a schematic diagram of the overall structure of the present invention;

[0021] Figure 2 An exploded view of the present invention;

[0022] Figure 3 This is the test result of the test paper on Escherichia coli in the embodiment of the present invention.

[0023] In the figure: 1, substrate; 2, working electrode; 3, counter electrode; 4, reference electrode; 5, circuit; 6, composite material layer; 7, hydrophilic film layer; 8, silver chloride material layer. DETAILED DESCRIPTION

[0024] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] like Figure 1 to Figure 3 As shown, this embodiment proposes a test paper for determining the content of Escherichia coli in food, including a substrate 1, the substrate 1 is made of insulating materials such as polyethylene terephthalate or polyimide, a working electrode 2, a counter electrode 3 and a reference electrode 4 are respectively arranged on the top of the substrate 1, and three groups of circuits 5 connected to the working electrode 2, the counter electrode 3 and the reference electrode 4 are also respectively arranged on the top of the substrate 1, a composite material layer 6 is arranged on the top of the working electrode 2, and a silver chloride material layer 8 is arranged on the top of the reference electrode 4;

[0026] The composite material layer 6 includes a polymer film, inorganic nanoparticles dispersed and adhered in the polymer film, and E. coli aptamers loaded on the surface of the inorganic nanoparticles. The E. coli aptamers can specifically adsorb E. coli in the test solution to the material surface and generate a current response linearly related to the concentration of the bacterial solution.

[0027] like Figure 1 to Figure 3 As shown, the inorganic nanoparticles may be selected from one or more of nano-silicon wires, nano-nickel hydroxide, nano-copper, nano-copper oxide, nano-nickel, nano-platinum, nano-palladium, or nano-gold, as well as composite materials formed based on the above materials.

[0028] like Figure 1 to Figure 3 As shown, the material of the polymer membrane is one or more of polytetrafluoroethylene, polyvinylidene fluoride, polyvinyl alcohol, polycyclodextrin, polythiophene, polysulfide, polyolefins, perfluorosulfonic acid resin, and aniline. The use of the above materials is easy to carry out surface modification, and it is convenient to load inorganic nanoparticles and Escherichia coli aptamers onto the membrane. The Escherichia coli aptamer can be firmly loaded on the surface of the inorganic nanoparticles through physical adsorption, covalent bonding and other methods, thereby forming a composite material layer 6.

[0029] like Figure 1 to Figure 3 As shown, the E. coli aptamer can be loaded on the surface of inorganic nanoparticles by physical adsorption, covalent binding and other methods, which can make full use of the excellent performance of inorganic nanoparticles and the high specificity of the E. coli aptamer to improve the sensitivity, specificity and stability of E. coli detection. The E. coli aptamer loaded on the surface of inorganic nanoparticles is a thiol-modified E. coli aptamer, which can be obtained commercially.

[0030] like Figure 1 to Figure 3 As shown, the silver chloride material layer 8 is arranged on the surface of the reference electrode 4 by screen printing, coating and other techniques. The thickness of the silver chloride material layer 8 is 10 to 500 microns. The preferred thickness of the silver chloride material layer 8 is 300 microns. The composite material layer 6 of this thickness can ensure that when the test paper is used for detection, the sensitivity and response speed can be improved while ensuring the strength, thereby speeding up the time required for detection and improving the detection efficiency.

[0031] like Figure 1 to Figure 3 As shown, a hydrophilic film layer 7 is provided on the outer peripheral surface of the composite material layer 6, covering the composite material layer 6 and the silver chloride material layer 8 as well as a portion of the counter electrode 3. The thickness of the composite material layer 6 is 10 to 500 microns, and the preferred thickness of the composite material layer 6 is 250 microns. The function of the composite material layer 6 is to provide a stable potential as a reference for the electrochemical test system. The hydrophilic film layer 7 has good wettability and water absorption, and can quickly absorb and wet the sample to be tested, so that the Escherichia coli in the sample is more likely to contact with the Escherichia coli aptamer on the composite material layer 6, thereby improving the efficiency and sensitivity of the detection.

[0032] Example:

[0033] First, prepare the calibration solution: under sterile conditions, draw 100 μL of the original test solution and slowly inject it into a sterile centrifuge tube containing 900 μL of sterile saline, shake it to mix it thoroughly, and prepare a 1:10 sample solution;

[0034] Repeat the previous steps to perform ten-fold concentration gradient dilution. Ensure strict sterility during the operation. Select plates of different concentrations for counting. Pipette 100 μL of E. coli solution and inject it on LB solid plate culture medium to spread the plate. Spread three plates in parallel for each concentration and culture them at 37°C to prepare calibration solutions of different concentrations. Observe and count them, record the dilution multiple and the corresponding number of colonies. Use colony-forming units (cfu) to represent the total number of colonies. When counting, select plates with a colony count between 30 and 300 cfu and no spreading colony growth. Calculate the concentration of the original bacterial solution based on the dilution multiple and colony count, and convert it into a logarithmic value for subsequent comparison with the output signal of electrochemical detection.

[0035] The test paper was incubated in each calibration solution with different E. coli content for 30 minutes, and then the test paper was transferred into a phosphate buffer solution with pH 6.8 containing 0.1 mol / L magnesium chloride. The circuit 5 on the test paper was connected to the electrochemical workstation;

[0036] Cyclic voltammetry was performed using a three-electrode system, and the oxidation current at a potential of 0.4 V was taken as the output signal to detect the calibration solutions of different concentrations prepared in the embodiment. The results are as follows: Figure 3 As shown (it should be noted that the E. coli aptamer can specifically adsorb the E. coli in the test solution to the surface of the material and affect the electron migration efficiency due to the steric hindrance effect, thereby affecting the current signal, so that currents of different sizes can be detected. In addition, the test paper is disposable and a new test paper needs to be replaced when testing a new sample);

[0037] like Figure 3The output signal of the E. coli test paper is plotted against the logarithm of the E. coli concentration in the test solution. The output signal is linearly related to the logarithm of the E. coli concentration (Y = -1.92X + 20.08). The result of the linear fitting is R 2 is 0.98, which reflects the accuracy of this method. It should be noted that Figure 3 The vertical axis is the current value generated by the sensor, the unit is Current (μA), the horizontal axis is the logarithm of the corresponding bacterial concentration, the unit is lg CE.coli represents the logarithm of the concentration of Escherichia coli (E.coli). In this representation, "lg" represents the logarithm base 10, and "CE.coli" represents the concentration of E. coli.

[0038] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A test paper for determining the content of Escherichia coli in food, characterized in that: The invention comprises a substrate (1), wherein the substrate (1) is made of an insulating material such as polyethylene terephthalate or polyimide, a working electrode (2), a counter electrode (3) and a reference electrode (4) are arranged on the top of the substrate (1), three groups of circuits (5) connected to the working electrode (2), the counter electrode (3) and the reference electrode (4) are also arranged on the top of the substrate (1), a composite material layer (6) is arranged on the top of the working electrode (2), and a silver chloride material layer (8) is arranged on the top of the reference electrode (4); The composite material layer (6) comprises a polymer film, inorganic nanoparticles dispersed in and adhered to the polymer film, and Escherichia coli aptamers loaded on the surface of the inorganic nanoparticles.

2. A test paper for determining the content of Escherichia coli in food according to claim 1, characterized in that: The inorganic nanoparticles may be selected from one or more of nano-silicon wires, nano-nickel hydroxide, nano-copper, nano-copper oxide, nano-nickel, nano-platinum, nano-palladium, or nano-gold, as well as composite materials formed based on the above materials.

3. A test paper for determining the content of Escherichia coli in food according to claim 2, characterized in that: The material of the polymer membrane is one or more of polytetrafluoroethylene, polyvinylidene fluoride, polyvinyl alcohol, polycyclodextrin, polythiophene, polysulfide, polyolefins, perfluorosulfonic acid resin, and aniline.

4. A test paper for determining the content of Escherichia coli in food according to claim 3, characterized in that: The E. coli aptamer can be loaded on the surface of inorganic nanoparticles by physical adsorption, covalent binding and other methods.

5. A test paper for determining the content of Escherichia coli in food according to claim 3, characterized in that: The silver chloride material layer (8) is arranged on the surface of the reference electrode (4) by using screen printing, coating or other techniques, and the thickness of the silver chloride material layer (8) is 10 to 500 micrometers.

6. A test paper for determining the content of Escherichia coli in food according to claim 4, characterized in that: A hydrophilic film layer (7) is provided on the outer peripheral surface of the composite material layer (6), covering the composite material layer (6), the silver chloride material layer (8) and a portion of the counter electrode (3), and the thickness of the composite material layer (6) is 10 to 500 micrometers.

Images