Immune micro-fluidic chip for efficiently enriching and detecting escherichia coli O157: H7 and application thereof

Abstract

The invention discloses an immune micro-fluidic chip for efficiently enriching and detecting escherichia coli O157: H7 and application of the immune micro-fluidic chip, and belongs to the field of food-borne pathogenic bacterium detection. The immune micro-fluidic chip is constructed by seven layers of plates. The seven layers of plates structurally and sequentially comprise a top plate, a first immune fiber channel layer, a first isolation layer, a second immune fiber channel layer, a second isolation layer, a third immune fiber channel layer and a bottom plate from top to bottom. Coupling the amination modified glass fiber membrane with a monoclonal antibody to successfully prepare an immune fiber membrane; the immune fiber membrane is arranged in a chip channel, and the escherichia coli O157: H7 in food can be rapidly detected on the basis of a double-antibody sandwich and an enzymatic reaction principle. The chip preparation process is simplified, the manufacturing cost is reduced, the time-consuming and tedious pre-enrichment step is avoided through large-volume sample injection, and the innovation is expected to provide important technical support in the fields of food quality control and safety detection such as food production and first-line supervision.

Description

Technical Field

[0001] The present invention relates to the field of food pathogenic bacteria detection, and particularly to an immunomicrofluidic chip for efficiently enriching and detecting Escherichia coli O157:H7 and its application. Background Art

[0002] Escherichia coli O157:H7 is Escherichia coli, which can cause hemorrhagic diarrhea and enteritis in humans and is a common pathogenic bacterium. There are many detection methods for this pathogenic bacterium, but they all have certain defects:

[0003] (1) The traditional culture and identification method has always been the "gold standard" for detecting Escherichia coli O157:H7. This method mainly relies on selective media (such as Sorbitol-MacConkey agar) to isolate Escherichia coli O157:H7 strains, and then combines biochemical tests for further identification. This method is a food standard enforced by the Food Safety Law of the People's Republic of China, but the enrichment time is too long, which is still the key problem to be solved for improving the detection efficiency of Escherichia coli O157:H7. Through the analysis of the detection time of 51 national and industrial standards for 8 foodborne pathogenic bacteria such as Escherichia coli O157:H7, the average total detection time of 16 biochemical identification standards is 114.7 hours; the average total detection time of 35 rapid detection standards is 42.1 hours, and the enrichment time accounts for 76.2% of all detections. And it is a common feature of all standards to draw several microliters or milliliters of solution from the enrichment broth for detection. Therefore, solving the problem of long enrichment time is the key to the current traditional culture and identification method.

[0004] (2) Detection techniques based on the specific binding principle of immunological antigen-antibody are rapid detection methods for Escherichia coli O157:H7. Common immunological detection techniques include enzyme-linked immunosorbent assay (ELISA), immunochromatographic strip (ICS), and immunomagnetic bead (IMB) techniques, etc. (1) ELISA is an immunological method widely used in the detection of Escherichia coli O157:H7. Its detection principle is based on the specific binding between antibodies and antigens, and then a visible color change is produced through an enzymatic reaction, thereby achieving qualitative or quantitative detection. The main advantages of ELISA are high sensitivity, simple operation, and suitability for rapid detection of a large number of samples. In recent years, researchers have made several improvements to the ELISA method to improve its sensitivity and specificity. For example, Shan et al. combined double-antibody sandwich ELISA with competitive ELISA to establish a method for signal amplification detection of Escherichia coli O157:H7, with a detection sensitivity of 20 CFU / mL, which is thousands of times higher than that of traditional ELISA. However, this method still has the problems of cumbersome detection steps and long time consumption. In another study, Pang et al. developed a paper-based ELISA detection technique, which achieved rapid and convenient detection through the principle of indirect ELISA, and its sensitivity could reach 10 4 CFU / mL. This method is low-cost and suitable for on-site detection, but interference factors in food matrices may affect the detection results and need further optimization. (2) ICS is a convenient immunological detection tool. Its principle is that the sample flows on the test paper through capillary action and reacts with the pre-coated antibody, thus achieving rapid detection. The advantages of ICS are simple operation, rapid results, and low cost, especially suitable for on-site detection and large-scale screening. However, this method has relatively poor detection sensitivity compared with molecular detection techniques such as PCR. (3) Immunomagnetic bead technology combines the advantages of immunology and magnetic separation. It captures the target antigen through magnetic particles and uses a magnetic field for separation. After combining this technology with ELISA, it can significantly improve the detection sensitivity and efficiency. For example, Wang et al. developed a magnetic bead-based electrochemical impedance immunosensor with a sensitivity of 10 2 CFU / mL, which is thousands of times higher than that of traditional ELISA. However, the detection equipment of IMB technology is relatively complex, and the sample processing time is relatively long, so it is limited in practical applications.

[0005] (III) Molecular biology detection techniques mainly rely on nucleic acid amplification techniques, such as polymerase chain reaction (PCR) and real-time fluorescence quantitative PCR (qPCR), to detect the specific gene sequences of Escherichia coli O157:H7. With its high sensitivity and specificity, PCR technology can detect trace amounts of pathogen DNA in a short time, so it is widely used in the field of food safety detection. However, these techniques rely on professional technicians and equipment and are difficult to be widely applied to rapid detection at the grass-roots level.

[0006] (IV) Biosensor technology has gradually attracted attention in recent years. This technology combines biomolecules (such as antibodies, enzymes, DNA fragments) with physicochemical signal transduction devices and can achieve highly sensitive detection of target substances. As one of the biosensor technologies, microfluidic chips have developed rapidly due to their high throughput, low sample reagent volume, rapid detection, integration and other characteristics. Microfluidic chips realize the overall miniaturization, automation, integration and portability of the chemical analysis system from sample processing to detection through the intersection of disciplines such as analytical chemistry, computer science, electronics, biology, and medicine.

[0007] The manufacturing methods of microfluidic technology under different actions are different. Generally, wet etching is carried out with potassium hydroxide and tetramethylammonium hydroxide. Silicon-based microfluidic chips prepared from materials such as silicon wafers, glass or quartz achieve the immobilization of antibodies by changing the surface properties. The most prominent advantage of using quartz as the immunochip matrix is its good optical properties, high precision, good thermal conductivity, high capture rate, etc. However, chip manufacturing requires high precision. Generally, expensive lithography machines are needed for ultraviolet exposure, and then a series of subsequent processing is carried out. The manufacturing process is cumbersome and the operation difficulty of mass production is relatively large. Paper-based microfluidic chips can be fabricated through simple techniques such as wax printing, inkjet printing, 3D printing, or even drawing, and have higher affordability, easy miniaturization, biocompatibility, and environmental friendliness. However, the liquid in this kind of chip flows passively due to capillary action, the liquid is difficult to control, and the reagent or sample loading is limited, so it is not suitable for solving problems with this technology. Summary of the Invention

[0008] The purpose of the present invention is to provide an immunomicrofluidic chip for efficient enrichment and detection of Escherichia coli O157:H7 and its application, so as to solve the problems existing in the above-mentioned prior art. By optimizing the design of the microfluidic chip and the novel curing method of monoclonal antibodies, the immunofiber membrane is loaded into the chip structure with multi-layer channels, realizing the efficient enrichment of target pathogenic bacteria, significantly shortening the detection time at the microscale, and facilitating the rapid on-site detection of Escherichia coli O157:H7.

[0009] To achieve the above purpose, the present invention provides the following solutions:

[0010] The present invention provides an immune microfluidic chip for efficient enrichment and detection of Escherichia coli O157:H7. The immune microfluidic chip is constructed from seven layers of plates. The structures of the seven layers of plates are, from top to bottom, a top plate, a first immune fiber channel layer, a first isolation layer, a second immune fiber channel layer, a second isolation layer, a third immune fiber channel layer, and a bottom plate;

[0011] Among them, several detection area channels are respectively arranged in parallel in the same direction at the same positions in the first immune fiber channel layer, the second immune fiber channel layer, and the third immune fiber channel layer (the detection area channels on the three layers of plates are arranged corresponding to each other up and down). At both ends of the position of the top plate corresponding to each detection area channel, a sample injection hole and a sample outlet hole are respectively arranged. The sample injection hole communicates with the detection area channel of the first immune fiber channel layer. The detection area channel of the first immune fiber channel layer communicates with the detection area channel on the second immune fiber channel layer through a connection hole arranged in the first isolation layer. The detection area channel on the second immune fiber channel layer communicates with the detection area channel on the third immune fiber channel layer through a connection hole arranged on the second isolation layer. The detection area channel on the third immune fiber channel layer communicates with the sample outlet holes arranged on the second isolation layer, the second immune fiber channel layer, the first isolation layer, the first immune fiber channel layer, and the top plate (that is, on the plates of the 1st - 5th layers, sample outlet holes for liquid connection are arranged at corresponding positions, so that after the liquid reaches the detection area channel on the third immune fiber channel layer, it can flow out through the sample outlet hole). An immune fiber membrane is loaded into the detection area channel, and the immune fiber membrane is used to capture and enrich Escherichia coli O157:H7 in the sample to be tested.

[0012] Optionally, the material of the seven layers of plates is polymethyl methacrylate; and / or each layer of plates is fixed by pasting with an optically grade double - sided tape. The length of each layer of plates is 70 - 80 mm, the width is 60 - 70 mm, the thickness of the top plate and the bottom plate is 0.5 mm, and the thickness of the other five layers of plates is 0.2 mm each.

[0013] Optionally, at least 5 detection area channels are respectively arranged on the first immune fiber channel layer, the second immune fiber channel layer, and the third immune fiber channel layer. The length of the detection area channel is 38 mm, the width is 1 mm, and the interval between two adjacent detection area channels is 12 mm.

[0014] Optionally, the diameters of the sample injection hole, the sample outlet hole, and the connection hole are all set to 2 mm.

[0015] Optionally, the immune fiber membrane is prepared by labeling a glass fiber membrane with an Escherichia coli O157:H7 capture antibody. More preferably, the glass fiber membrane is amino - modified with APTES and then mixed with an Escherichia coli O157:H7 monoclonal antibody to make the immune fiber membrane.

[0016] The present invention also provides a method for detecting Escherichia coli O157:H7 using the immunomicrofluidic chip described above, comprising the following steps:

[0017] Connect the sample outlet to an injection pump, connect the sample inlet to a silica gel tube and insert it into the solution of the sample to be tested, form a negative pressure on the sampling hole in the extraction mode, so that the solution of the sample to be tested enters the detection area channel of the first immunofiber channel layer through the sample inlet, and through immunofiber membrane chromatography, Escherichia coli O157:H7 in the solution of the sample to be tested is bound and fixed by the capture antibody on the immunofiber membrane, and after being sequentially bound and fixed by the capture antibodies in the detection area channels of the second immunofiber channel layer and the third immunofiber channel layer, it is drawn out from the sample outlet;

[0018] After the sample loading is completed, put the silica gel tube at the sample inlet into the PBST solution, and wash the immunofiber membrane in the immunomicrofluidic chip in the extraction mode; after the washing is completed, then put the silica gel tube into the solution of the detection antibody of Escherichia coli O157:H7 labeled with HRP enzyme, and make the solution of the detection antibody of Escherichia coli O157:H7 labeled with HRP enzyme fill the detection area channel in the extraction mode for reaction; after the reaction is completed, wash again with the PBST solution, and then put the silica gel tube into the TMB chromogenic solution, and make the TMB chromogenic solution fill the detection area channel in the extraction mode for color reaction in the dark; after the color reaction is completed, put the silica gel tube into the termination solution, terminate the reaction and collect it in the extraction mode, and aspirate the collected solution to measure OD 450nm According to OD 450nm Judge whether the solution of the sample to be tested contains Escherichia coli O157:H7.

[0019] Optionally, one of the channels in the detection area channel is set as the negative control group, and the other channels are used as the experimental group. When the ratio of OD 450nm of the experimental group to OD 450nm of the negative control group is ≥2.1, the result is determined to be positive, that is, the solution of the sample to be tested contains Escherichia coli O157:H7; when the ratio of OD 450nm of the experimental group to OD 450nm of the negative control group is <2.1, the result is determined to be negative, that is, the solution of the sample to be tested does not contain Escherichia coli O157:H7.

[0020] Optionally, the extraction parameters during sample loading are: the flow rate is 20 min / mL, and the sample is loaded at 37 °C for 4 h;

[0021] The extraction parameters during washing are: the flow rate is 1 min / mL, and the washing time is 3 min;

[0022] The parameters for extracting the HRP enzyme-labeled Escherichia coli O157:H7 detection antibody solution into the detection area channel are as follows: the flow rate is 1 min / mL, the extraction time is 10 s, and the reaction time is 10 min;

[0023] The parameters for extracting the TMB chromogenic solution into the detection area channel are as follows: the flow rate is 1 min / mL, the extraction time is 10 s, and the chromogenic reaction time is 10 min.

[0024] Optionally, the Escherichia coli O157:H7 is derived from artificially contaminated food.

[0025] The present invention also provides the application of the immunomicrofluidic chip in detecting Escherichia coli O157:H7 in food.

[0026] The present invention discloses the following technical effects:

[0027] (1) The present invention has high detection sensitivity for Escherichia coli O157:H7, and the lowest detection limit is 1 CFU / mL; the present invention has high specificity for Escherichia coli O157:H7 and has no cross-reaction with other 23 strains of foodborne pathogenic bacteria, providing a guarantee for the accurate detection of Escherichia coli O157:H7.

[0028] (2) The present invention does not require the pre-enrichment process of Escherichia coli O157:H7, and only 4.5 hours are required to complete all processes from sample processing, sample loading, detection to result determination, while the national standard method requires more than 24 - 36 hours. The present invention is simple to manufacture, low in cost, short in detection time, and simple in operation. In application scenarios such as pollution source tracing, on-site detection, and disease diagnosis, the detection sample throughput can be increased only by increasing the number of parallel channels in the chip, and the detection can be completed faster, improving the timeliness of detection and being suitable for large-scale food safety supervision.

[0029] (3) It is expected that the highly efficient enrichment and detection immunomicrofluidic chip for Escherichia coli O157:H7 prepared by the present invention can better realize the commercial application of highly sensitive and rapid detection of foodborne pathogenic bacteria in the fields of food production, circulation, supervision, environmental monitoring, and disease diagnosis.

[0030] (4) The present invention conforms to the clear requirements put forward in the "Biological Economy Development Plan for the 14th Five-Year Plan": promoting the integrated innovation of biotechnology and information technology, accelerating the development of industries such as biomedicine, bio-breeding, bio-materials, and bio-energy, and strengthening the bio-economy. Through biotechnological innovation, the present invention provides important technical support for food safety detection, helps to promote the application of biotechnology in the field of food safety, improves the efficiency and accuracy of food safety detection, and provides a strong technical guarantee for ensuring the food safety of the people. Description of the Drawings

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0032] Figure 1 Schematic diagram of the high-efficiency enrichment and detection of Escherichia coli O157:H7 immune microfluidic chip in Embodiment 1 of the present invention; 1 is the top plate of the microfluidic chip, 2, 4, and 6 are all immune fiber channel layers, 3 and 5 are isolation layers, 7 is the bottom plate of the microfluidic chip, 8 is the sample injection hole, 9 is the sample outlet hole, and 10 is the detection area;

[0033] Figure 2 Schematic diagram of the detection principle of the high-efficiency enrichment and detection of Escherichia coli O157:H7 immune microfluidic chip in Embodiment 1 of the present invention, HRP is horseradish peroxidase, and TMB is the chromogenic agent 3,3',5,5'-tetramethylbenzidine;

[0034] Figure 3 Results of verifying the HRP enzyme-labeled Escherichia coli O157:H7 detection antibody by the direct ELISA method in Embodiment 1 of the present invention; 500, 1000, 2000 to 32000 are all dilution multiples of the enzyme-labeled antibody, and N is the negative control;

[0035] Figure 4 Results of verifying the glass fiber-coupled Escherichia coli O157:H7 capture antibody by the ELISA method in Embodiment 1 of the present invention; a is the result of the positive and negative absorbance values, b is the microscopic image of the glass fiber before labeling with the Escherichia coli O157:H7 capture antibody, and c is the microscopic image of the glass fiber after labeling with the Escherichia coli O157:H7 capture antibody;

[0036] Figure 5 Physical diagram of the high-efficiency enrichment and detection of Escherichia coli O157:H7 immune microfluidic chip in Embodiment 1 of the present invention;

[0037] Figure 6 Results of optimizing the sample loading time and temperature of the microfluidic chip in Embodiment 1 of the present invention; a is room temperature, b is 37°C, and N is the negative control;

[0038] Figure 7 Results of evaluating the specificity of the microfluidic chip for detecting Escherichia coli O157:H7 in Embodiment 2 of the present invention; 1-26 are 26 experimental strains to be detected, and N is the negative control;

[0039] Figure 8This is the simulated detection result of bacteria-carrying for the high-efficiency enrichment and detection of Escherichia coli O157:H7 immunomicrofluidic chip in Example 3 of the present invention. 1 represents beef, 2 represents Chinese cabbage, 3 represents milk, 4 represents bread, 5 represents chicken, and N represents the negative control. Detailed implementation manners

[0040] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0041] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0042] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0043] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0044] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0045] To solve the problems existing in the detection of Escherichia coli O157:H7 in the prior art, the present invention designs an immunomicrofluidic chip that integrates high-efficiency enrichment and rapid detection, with significant beneficial effects, specifically as follows: (1) The present invention prepares an immunofiber membrane with micron-scale channels by aminating and modifying a glass fiber membrane and coupling the carboxyl group of a monoclonal antibody with the modified amino group. This immunofiber membrane is loaded into the microfluidic chip channel made of low-cost materials (PMMA) and a low-cost laser engraving machine, combined with an optical double-sided adhesive, and a microfluidic chip without processes such as photolithography etching and bonding can achieve rapid detection of the microfluidic chip. This innovative method not only simplifies the chip preparation process, reduces the manufacturing cost, but also enables large-volume sample injection, with the advantages of simple preparation and low cost. This technology is expected to provide important technical support in the fields of food quality control and safety detection such as food production and on-site supervision. (2) Aiming at the problem of the long enrichment time in the detection method of foodborne pathogenic bacteria, the present invention proposes corresponding solutions to improve the timeliness and sensitivity of the detection of foodborne pathogenic bacteria. First, by optimizing the design of the microfluidic chip and the new curing method of monoclonal antibodies, the immunofiber membrane is loaded into the chip structure with multiple layers of channels, realizing the efficient enrichment of target pathogenic bacteria and significantly shortening the detection time at the microscale. Second, based on the principle of double-antibody sandwich and enzymatic reaction, rapid detection is achieved on the new microfluidic chip. Through the enzyme-linked immunosorbent microfluidic chip of immunofibers, trace amounts of Escherichia coli O157:H7 can be effectively captured, enriched, and rapidly detected without the need for a pre-enrichment process. At the same time, detection parameters such as the injection speed and temperature are optimized, and the detection specificity and feasibility of the present invention are evaluated to meet the application requirements in the detection of actual food samples.

[0046] The following specific examples further illustrate the microfluidic chip prepared by the present invention and its scheme for detecting Escherichia coli O157:H7.

[0047] The main experimental materials involved in the following examples:

[0048] 1. Main reagents and consumables

[0049] Ammonium sulfate, ethylene glycol, 3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB), citric acid monohydrate, urea peroxide, and concentrated sulfuric acid were purchased from Sinopharm Chemical Reagent Co., Ltd.; glutaraldehyde was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; horseradish peroxidase (HRP) and 3-aminopropyltriethoxysilane (APTES) were purchased from Sigma; PMMA sheets were purchased from Dongguan Keyu Insulating Materials Co., Ltd.; Luer connector RH-M-(1 / 4-28)N, Teflon tube TFLG00001, and silicone hose were purchased from Nanjing Runze Fluid Control Equipment Co., Ltd.; Peek connector A-1428 was purchased from Anhui Zhiwei Technology Co., Ltd.; optically clear double-sided tape was purchased from Guangdong Fuyin Adhesive Technology Co., Ltd.

[0050] 2. Main Instruments

[0051] CO 2 A laser engraver (VLS2.3) was purchased from Universal Laser Systems, Inc. (USA); a multi-channel syringe pump (TYD02) was purchased from LeiFu Fluid Technology Co., Ltd.; a magnetic heating stirrer was purchased from Suzhou Sains Instruments Co., Ltd.; a combined shaking incubator (ZQPZ 228R) was purchased from Labotery Instrument & Equipment Co., Ltd.; a single-person laminar flow hood (SW-SJ-2D) was purchased from Suzhou Purification; and a microplate reader (SpectraMaxM2) was purchased from Molecular Devices (USA).

[0052] 3. The strains used in the present invention are shown in Table 1.

[0053] Table 1 Experimental Strains

[0054]

[0055]

[0056] Example 1 Establishment of a Microfluidic Chip

[0057] 1. Fabrication of the Microfluidic Chip

[0058] As shown in Figure 1 , the chip pattern was drawn in CAD software and imported into CO 2The laser engraver cuts on a polymethyl methacrylate (PMMA) sheet. The unit of distance annotation in the figure is mm, and the diameter of each hole is 2 mm. The channel within the red square is the detection area channel. This channel is 38 mm long and 1 mm wide. The chip is designed with 5 parallel channels with a spacing of 12 mm to enable the simultaneous detection of 4 samples. The chip is 70 mm long and 66 mm wide. The specific manufacturing steps are as follows: The chip has a total of 7 layers. Among them, the 1st and 7th layers use PMMA sheets with a thickness of 0.5 mm, the cutting power is set to 80, and the cutting rate is set to 30. The 2nd to 6th layers use sheets with a thickness of 0.2 mm, the cutting power is 60, and the cutting rate is 20. Among them, before cutting the PMMA sheets of the 2nd, 4th, and 6th layers with immune fiber channels, optical-grade double-sided tapes are respectively pasted on both sides of the sheets; all the cut sheets are ultrasonically cleaned for 30 min, dried and then assembled; The 7th layer is the bottom layer. Peel off the double-sided tape protective film on the bottom surface of the 6th layer, and paste the 6th layer sheet on the 7th layer; The red square in the figure is the immune fiber channel, and immune fibers are clamped into each channel of the 6th layer; Then peel off the double-sided tape protective film on the top surface of the 6th layer, and align and paste the 5th layer sheet; After pasting, peel off the double-sided tape protective film on the bottom surface of the 4th layer, and paste the 4th layer sheet on the 5th layer; Then clamp immune fibers into each channel, and peel off the double-sided tape protective film on the top surface of the 4th layer. Align and paste the remaining layer sheets and immune fibers in sequence according to the same steps; Finally, paste peek connectors at each hole of the 1st layer for sample injection and sample output.

[0059] 2. Detection principle and result determination of the microfluidic chip

[0060] The detection principle of the present invention is as Figure 2As shown in the figure, it is a microfluidic chip for detecting trace targets in a large-volume sample, with a glass fiber membrane as the stationary phase, combining enzymatic reaction and double antibody sandwich. The detection process is as follows: The sample outlet is connected to an injection pump through a PEEK joint and a Teflon hard tube. The sample inlet is connected to a silica gel tube through a PEEK joint and a Teflon hard tube and inserted into the sample solution to be detected. A negative pressure is formed on the sample outlet according to the optimal sampling parameters in the extraction mode, so that the sample solution to be detected is inhaled into the chip at the set parameters at the optimal temperature; The sample solution to be detected enters the immune fiber channel at the lower red box and undergoes immunochromatography along with the immune fiber. Escherichia coli O157:H7 in the solution will be bound and fixed by the capture antibody labeled on the glass fiber, and after being captured by each layer of immune fiber, it is drawn out from the sample outlet; After the sample loading is completed, the silica gel tube at the sample inlet is placed in the PBST solution, and the immune fiber in the chip is washed at a sampling parameter of 1 min / mL for 3 min; The silica gel tube is placed in the HRP enzyme-labeled Escherichia coli O157:H7 detection antibody solution with the optimal dilution, and aspirated for 10 s at a sampling parameter of 1 min / mL to fill the immune fiber channel with the enzyme-labeled antibody solution, and react for 10 min; Wash again with PBST in the same way for 3 min. The silica gel tube is placed in the TMB chromogenic solution, and aspirated for 10 s at a sampling parameter of 1 min / mL to fill the immune fiber channel with the TMB chromogenic solution, cover with a light-proof box and react for 10 min; After the chromogenic reaction, aspirate the termination solution for 15 s at the same parameter and collect it. Absorb 200 μL of the collected solution and measure its OD 450nm , with the OD of the experimental group 450nm compared to the OD of the negative control group 450nm ≥2.1 is used as the judgment basis for the detection positive result. If the ratio < 2.1, it is judged as negative. In addition, after each extraction, let it stand for 5 s and then take out the silica gel injection tube from the solution to balance the pressure in the chip and each pipeline to prevent air from entering the chip and affecting the detection result.

[0061] 3. HRP enzyme-labeled Escherichia coli O157:H7 detection antibody

[0062] 10 mg of HRP enzyme was dissolved in 1 mL of sodium acetate buffer (0.05 mol / L, pH 5.6), 1 mL of sodium periodate solution (60 mmol / L, freshly prepared) was added, and the mixture was stirred at 4 °C in the dark for 2 h; 1 mL of ethylene glycol-sodium chloride solution was added, mixed well, and stirred at 4 °C in the dark for 30 min; 12 mL of anhydrous ethanol pre-cooled to 4 °C was added and mixed well. After centrifugation at 4000 rpm for 10 min, the supernatant was discarded. The precipitate was resuspended in 5 mL of phosphate buffer (PB), and the pH was adjusted to between 9.0 - 9.5 with carbonate buffer (CB, 0.01 mmol / L, pH 9.6). 20 mg of Escherichia coli O157:H7 monoclonal antibody was added, and then the pH of the solution was immediately adjusted with CB to keep it unchanged. The mixture was stirred at 4 °C in the dark overnight; after overnight labeling, 100 μL of sodium borohydride (10 mg / mL) solution was added to the solution, and the reaction was carried out at 4 °C in the dark for 2 h; the solution was transferred to a dialysis bag and dialyzed with PB at 4 °C in the dark. The dialysis fluid was changed 3 times at intervals of 3 - 4 h; after dialysis, it was transferred to a centrifuge tube and an equal volume of saturated ammonium sulfate solution was added and mixed well. It was allowed to stand at 4 °C in the dark for 30 min; after centrifugation at 12000 rpm for 30 min at 4 °C, the supernatant was discarded. The precipitate was dissolved in 10 mL of PB, and the antibody was precipitated again with saturated ammonium sulfate. After centrifugation, the precipitate was resuspended and dissolved in 10 mL of PB, and dialyzed again at 4 °C in the dark. After changing the solution 3 times, the enzyme-labeled antibody was collected and stored at 4 °C in the dark for standby.

[0063] And the effect of HRP enzyme-labeled Escherichia coli O157:H7 detection antibody was verified by the direct ELISA method. The specific steps were as follows: 100 μL of Escherichia coli O157:H7 bacterial solution cultured to more than 10 8 CFU / mL was pipetted into a 96-well enzyme-linked immunosorbent assay (ELISA) plate and incubated overnight at 4 °C to coat the antigen; the bacterial solution in the ELISA plate was patted dry, washed 3 times with PBST, and patted dry; the prepared enzyme-labeled antibody was serially diluted 500, 1000, 2000 times, etc. to 16000 times with PB, and 100 μL of each gradient antibody solution was added to the ELISA plate and incubated at 37 °C for 1 h. After washing 3 times with PBST, the A and B solutions of the TMB chromogenic solution were mixed in equal volume and 50 μL of the mixture was added to each well of the ELISA plate and developed at room temperature in the dark for 15 min. 50 μL of 0.2 mol / L H 2 SO 4 solution was added to terminate the reaction. The absorbance value at a wavelength of 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The OD 450nm of the experimental group was compared with the OD 450nm of the negative control group. When OD ≥ 2.1, it was used as the judgment basis for the positive detection result.

[0064] The results were as Figure 3 shown. When the enzyme-labeled antibody was diluted 500 to 4000 times, OD 450nmThe value of 0.2349, which is 2.1 times higher than the negative control, indicates that the HRP enzyme has successfully labeled the detection antibody for Escherichia coli O157:H7, and the titer of this enzyme-labeled antibody is 4000.

[0065] 4. Labeling the capture antibody for Escherichia coli O157:H7 on the glass fiber membrane

[0066] The specific steps for labeling the glass fiber with the antibody are as follows: Cut the glass fiber membrane into a size of 3.8 cm × 10 cm. Add 60 mL of absolute ethanol and 4 mL of APTES (γ-aminopropyltriethoxysilane) to the glass fiber with a total area of approximately 380 cm 2 and place it on a magnetic stirrer to stir for 10 h. Then raise the temperature to 60 °C and continue stirring overnight for amination modification. After the amination modification of the glass fiber, wash it 3 times with PB and add 60 mL of PB. Then add 50% glutaraldehyde to a final concentration of 1.25% and place it on a magnetic stirrer to stir for 2 h. Wash the amino-activated glass fiber 3 times with PB. Take out the fiber from the PB, place it on filter paper to absorb the moisture, and then put it into the monoclonal antibody solution of Escherichia coli O157:H7 (20 mL, 0.5 mg / mL) and shake it overnight at 4 °C. After labeling the glass fiber with the antibody, wash it 3 times with PB, put it into a 5% BSA solution and block it at 37 °C for 1 h. After blocking, wash it 3 times with PB, dry it at 37 °C to obtain the immunized fiber. Use a chopper to cut the immunized fiber into a size of 1.1 mm × 38 mm and store it at 4 °C for later use.

[0067] Verify whether the glass fiber is successfully labeled with the antibody according to the method of enzyme-linked immunosorbent assay. The specific steps are as follows: Put the immunized fiber prepared above into a centrifuge tube, and then add 1 mL of Escherichia coli O157:H7 bacterial solution with a concentration of 10 8 CFU / mL or above as the positive control, and use sterile culture medium as the negative control. Let it stand at 37 °C for 1 h. Remove the solution in the centrifuge tube, add 1 mL of PBST, shake for 1 min, remove the washing solution, and wash repeatedly 3 times. Add 500 μL of the enzyme-labeled antibody solution with an appropriate concentration in the above step (1), and react at 37 °C for 1 h. After the reaction, remove the enzyme-labeled antibody solution, wash 3 times with PBST, add 500 μL of TMB chromogenic solution, and develop color at room temperature in the dark for 15 min. Add 100 μL of the termination solution, mix well, and then respectively pipette 200 μL into a 96-well enzyme-labeled plate to measure its OD 450nm , repeat the measurement 3 times. If the average OD 450nm of the positive control group is at least 2.1 times higher than the average OD 450nm of the negative control group (P / N), it indicates that the glass fiber has successfully labeled the capture antibody for Escherichia coli O157:H7.

[0068] The results are as shown in Figure 4 a below. Among the 3 measurement results, the OD 450nmThe average value was 1.7069, which was significantly higher than the OD of the negative control group 450nm The value of 0.599, which was 2.1 times, indicated that the glass fiber successfully labeled the capture antibody of Escherichia coli O157:H7, that is, the immune fiber was successfully prepared. At the same time, as shown in Figure 4 b and c, the glass fiber labeled antibody had no effect on its fiber structure. In summary, the prepared immune fiber could be used for the establishment of a microfluidic chip

[0069] 5. Optimization of the sample aspiration rate of the microfluidic chip

[0070] The microfluidic chip was fabricated according to the aforementioned method steps. As shown in Figure 5 Figure 1 is a physical diagram of the immune microfluidic chip for efficient enrichment and detection of Escherichia coli O157:H7 in the embodiment of the present invention. The red arrow indicates the sample injection port, the blue arrow is the negative control input port, the gray arrow indicates the solution output position and direction, and the red box is the detection area based on the immune fiber channel

[0071] The sample loading rate was optimized through the prepared microfluidic chip. The specific steps were as follows: The overnight cultured Escherichia coli O157:H7 bacterial solution was counted by plate counting. At the same time, the sample aspiration silica gel tube of the microfluidic chip was inserted into 1 mL of the bacterial solution diluted 10 -5 times. The sample was aspirated from the chip outlet hole using an injection pump at the extraction rates of 5, 10, 15, and 20 min / mL, and the bacterial solution captured by the immune fiber was collected. After 1 mL of the bacterial solution was loaded, all the collected bacterial solution was spread on an Escherichia coli O157:H7 chromogenic plate and counted after culturing at 37 °C for 12 h. The capture rate was calculated according to the following formula, and the sample aspiration rate with a high capture rate was selected as the optimal sample loading rate for microfluidic chip detection

[0072] Table 2 Results of the capture rate determination of Escherichia coli O157:H7 by the microfluidic chip

[0073]

[0074]

[0075] As shown in Table 2, when the sample aspiration rate was 20 min / mL, the capture rate was more than 99.5%. When the sample aspiration parameter was larger, the sample loading time on the chip was prolonged more, which was not conducive to rapid detection. Therefore, 20 min / mL was selected as the optimal sample aspiration rate

[0076] 6. Optimization of the sample aspiration time and temperature of the microfluidic chip

[0077] The freshly cultured Escherichia coli O157:H7 bacterial solution was aspirated by plate counting, and approximately 250 CFU of the bacterial solution was added to a sterile enrichment broth medium and shaken well; the injection pump was used to perform detections at room temperature and 37 °C respectively with the optimal sampling parameters, and the sampling times were set to 1 h, 2 h, 3 h, and 4 h respectively; after the sample loading was completed, detections were carried out, and the OD 450nm average value of the experimental group was compared with the OD 450nm average value of the negative control group. If it was ≥ 2.1, it was used as the judgment basis for the positive detection result. The shortest time required to detect a positive result was selected as the optimal sampling time, and at the same time, the environmental temperature was determined; if a positive result was detected at the same sampling time, in order to make the experimental operation simpler, the room temperature environment was selected for detection.

[0078] The results were as Figure 6 shown in a of [reference], which was the detection result of the microfluidic chip with sampling at room temperature. From this result, it could be known that when sampling at room temperature for 1 h to 4 h, the detected values were all lower than 2.1 times the OD 450nm of the negative control. That is, when the Escherichia coli O157:H7 bacterial solution with a final concentration of about 1 CFU / mL was used for the detection of this microfluidic chip, the detection result was a false negative. As Figure 6 shown in b of [reference], as the sampling time extended, its OD 450nm also increased accordingly. When the sampling time was 4 h, the measured OD 450nm was 0.4588, which was higher than the value of 0.3801 that was 2.1 times the negative control value, and the result was judged as positive. Although the detection results of sampling for 1 to 3 h were judged as negative, they were all higher than the OD 450nm of the detection result at room temperature. This might be because the 37 °C environment was more conducive to the antigen-antibody binding and the enzyme-catalyzed chromogenic reaction than the room temperature, or it might be that when sampling at 37 °C for 4 h, the growth and reproduction of Escherichia coli O157:H7 in the sample solution increased the bacterial concentration. In short, by using this simple method of increasing the detection environmental temperature, the occurrence of false negative results could be avoided, thereby achieving the purpose of accurately detecting Escherichia coli O157:H7. To sum up, the microfluidic chip of the present invention could detect Escherichia coli O157:H7 with a concentration of about 1 CFU / mL after 4 h of sample loading in a 37 °C detection environment.

[0079] Example 2 Evaluation of the detection specificity of the microfluidic chip

[0080] All the strains in Table 1 were cultured to above 10 8 CFU / mL, and each bacterial solution was diluted 1000 times with a sterile nutrient broth medium. The diluted solutions of each strain were detected using the microfluidic chip established by the present invention, and the OD 450nm, the specificity of the detection method was evaluated. Since the micro-injection pump can perform 8-channel aspiration and sample loading simultaneously at one time, an 8-channel immunomicrofluidic chip was fabricated in the specificity determination experiment. Among them, 7 channels were used to detect each bacterial solution respectively, and 1 channel was used to load the blank culture medium as a negative control. The results are as Figure 7 shown. When the microfluidic chip of the present invention detected Escherichia coli O157:H7 (ATCC 43889, ATCC 43895, and NCTC12900), the detection result OD 450nm was 2.1 times higher than its negative control, that is, the detection result was determined to be positive; the detection results OD 450nm of the other 23 strains of common foodborne pathogenic bacteria were all less than 2.1 times that of their respective negative controls, and the detection results were negative. The results indicate that the microfluidic chip of the present invention has no cross-reaction with other foodborne pathogenic bacteria and has good specificity for detecting Escherichia coli O157:H7.

[0081] Example 3 Feasibility evaluation of the microfluidic chip for detecting food samples

[0082] The microfluidic chip established by the present invention was used to evaluate the feasibility of obtaining accurate detection results without pre-enrichment in the detection of food samples through a simulated inoculation experiment. The specific steps are as follows: Beef, chicken, milk, bread, and Chinese cabbage samples were purchased from a supermarket. These food samples (25 mL or 25 g) were respectively added to 225 mL of sterile modified EC broth medium. After homogenizing for 2 min, about 250 CFU of Escherichia coli O157:H7 was added to the medium. The sampling silica tube of the microfluidic chip was inserted into the medium, and at an appropriate temperature, sampling and detection were performed with the optimal sampling parameters and sampling time, and OD 450nm was measured. The sterile modified EC broth medium was used as a negative control, and the same sample solution was measured 3 times repeatedly. If the average value of OD 450nm in the experimental group was ≥ 2.1 times that of the negative control group OD 450nm average value, the detection result was determined to be positive. Since 5 samples need to be detected simultaneously in this experiment, 1 additional immunofiber channel was added in parallel when designing and fabricating the chip, that is, a total of 6 channels. Among them, 5 channels were used to simultaneously detect 5 food samples to be detected, and 1 channel was used for negative control. In addition, in order to prevent food samples from clogging the microfluidic chip channels during the sampling process, the silica sampling tube mouth was wrapped with a sterile qualitative filter paper to filter out larger particulate food substances in the sample culture medium solution.

[0083] As Figure 8 shown, the microfluidic chip of the present invention was used to detect foods such as beef, milk, Chinese cabbage, bread, and chicken artificially contaminated with Escherichia coli O157:H7. The OD 450nmThe average values are all higher than 0.3107, which is 2.1 times the value of the negative control, and the detections are all positive results. The microfluidic chip established by the present invention can achieve rapid detection of Escherichia coli O157:H7 in food samples without pre-enrichment.

[0084] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An immunomicrofluidic chip for efficient enrichment and detection of Escherichia coli O157:H7, characterized in that: The immune microfluidic chip is constructed of seven layers of plates, and the structure of the seven layers of plates is, from top to bottom, a top plate, a first immune fiber channel layer, a first isolation layer, a second immune fiber channel layer, a second isolation layer, a third immune fiber channel layer and a bottom plate; wherein the first immune fiber channel layer, the second immune fiber channel layer and the third immune fiber channel layer respectively set a plurality of detection area channels in parallel at the same position and in the same direction, and an injection hole and an outlet hole are respectively set at both ends of the top plate position corresponding to each detection area channel, and the injection hole is connected to the detection area channel of the first immune fiber channel layer, and the first immune fiber channel layer The detection area channel is connected to the detection area channel on the second immune fiber channel layer through the connecting hole arranged on the first isolation layer, the detection area channel on the second immune fiber channel layer is connected to the detection area channel on the third immune fiber channel layer through the connecting hole arranged on the second isolation layer, the detection area channel on the third immune fiber channel layer is connected to the sample outlet holes arranged on the second isolation layer, the second immune fiber channel layer, the first isolation layer, the first immune fiber channel layer and the top plate, and the detection area channel is loaded with an immune fiber membrane, which is used to capture and enrich Escherichia coli O157:H7 in the sample to be tested.

2. The immune microfluidic chip according to claim 1, characterized in that: The material of the seven-layer board is polymethyl methacrylate; and / or each layer of the board is fixed by optical-grade double-sided tape, the length of each layer of the board is 70-80mm, the width is 60-70mm, the thickness of the top plate and the bottom plate is 0.5mm, and the thickness of the other five layers of the board is 0.2mm.

3. The immune microfluidic chip according to claim 1, characterized in that: At least five detection zone channels are respectively arranged on the first immune fiber channel layer, the second immune fiber channel layer and the third immune fiber channel layer. The detection zone channels are 38 mm long and 1 mm wide, and the interval between two adjacent detection zone channels is 12 mm.

4. The immune microfluidic chip according to claim 1, characterized in that: The diameters of the sample inlet, sample outlet and connection holes are all set to 2 mm.

5. The immune microfluidic chip according to claim 1, characterized in that: The immune fiber membrane is prepared by labeling the Escherichia coli O157:H7 capture antibody with a glass fiber membrane.

6. A method for detecting Escherichia coli O157:H7 using the immune microfluidic chip according to any one of claims 1 to 5, characterized in that: The following steps are involved: The sample outlet is connected to a syringe pump, the sample inlet is connected to a silicone tube and inserted into the sample solution to be tested, and a negative pressure is formed on the sampling hole in an extraction mode, so that the sample solution to be tested enters the detection zone channel of the first immune fiber channel layer through the sample inlet, and after immunofiber membrane chromatography, the Escherichia coli O157:H7 in the sample solution to be tested is bound and fixed by the capture antibody on the immune fiber membrane, and is sequentially bound and fixed by the capture antibody in the detection zone channel of the second immune fiber channel layer and the third immune fiber channel layer, and then extracted from the sample outlet; After the sample loading is completed, the silicone tube of the injection hole is placed in a PBST solution to wash the immune fiber membrane in the immune microfluidic chip in an extraction mode; After washing, the silicone tube is placed in the HRP enzyme-labeled Escherichia coli O157:H7 detection antibody solution, and the HRP enzyme-labeled Escherichia coli O157:H7 detection antibody solution is filled in the detection area channel in the extraction mode to react; after the reaction is completed, it is washed again with PBST solution, and then the silicone tube is placed in TMB color development solution, and the TMB color development solution is filled in the detection area channel in the extraction mode, and the color development reaction is carried out in the dark; after the color development is completed, the silicone tube is placed in the stop solution, the reaction is stopped in the extraction mode and collected, and the collected solution is aspirated to measure the OD 450nm , according to OD 450nm Determine whether the sample solution to be tested contains Escherichia coli O157:H7.

7. The method according to claim 6, characterized in that One channel in the detection zone is set as a negative control group, and the other channels are set as experimental groups. 450nm Compared with the negative control group OD 450nm The ratio of ≥2.1 is considered positive, i.e., the sample solution contains Escherichia coli O157:H7; when the experimental group OD 450nm Compared with the negative control group OD 450nm The ratio is less than 2.1, and the result is judged to be negative, that is, the sample solution to be tested does not contain Escherichia coli O157:H7.

8. The method according to claim 6, characterized in that The extraction parameters for loading were: flow rate 20 min / mL, loading at 37 °C for 4 h; The extraction parameters during washing were: flow rate 1 min / mL, washing time 3 min; The parameters for extracting the HRP enzyme-labeled E. coli O157:H7 detection antibody solution into the detection zone channel are: flow rate 1 min / mL, extraction time 10 s, reaction time 10 min; The parameters for extracting TMB color development solution into the detection zone channel are: flow rate is 1 min / mL, extraction time is 10 s, and color development reaction time is 10 min.

9. The method according to claim 6, characterized in that The Escherichia coli O157:H7 originates from artificially contaminated food.

10. Use of the immune microfluidic chip according to any one of claims 1 to 5 in detecting Escherichia coli O157:H7 in food.

Images