Fluorescent paper chip for detecting escherichia coli O157: H7 as well as preparation method and application of fluorescent paper chip
By modifying molecularly imprinted fluorescent nanoparticles with E. coli O157:H7 as the template on the paper chip, and using the change in fluorescence quenching rate for detection, the existing detection methods are solved, and the rapid, high specificity and quantitative detection effects are achieved.
Patent Information
- Application Number
- CN202411818633.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-03-28
AI Technical Summary
The existing E. coli O157:H7 detection method has problems such as long time, high labor consumption, requiring antibodies or aptamers, many interference factors, poor repetition and high price.
Molecularly imprinted fluorescent nanoparticles with E. coli O157:H7 as the template were designed and synthesized by molecular imprinting technology, and these nanoparticles were modified on the surface of paper chips, and quickly identified by changes in fluorescence quenching rate.
The rapid, high specificity and quantitative detection of E. coli O157:H7 detection simplifies the analysis process, reduces costs, improves detection efficiency and sensitivity, and has the advantages of reuse.
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Figure CN119716050A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of food safety detection, and specifically relates to the preparation of a paper chip for rapid food safety detection, and specifically relates to a fluorescent paper chip for detecting Escherichia coli O157:H7, and a preparation method and application thereof. Background Art
[0002] Escherichia coli (E. coli) is a type of facultative anaerobic Gram-negative bacteria that can grow at 8-46°C, with an optimal growth temperature of 37°C. Among them, non-pathogenic E. coli is commonly found in the intestines of humans and animals; pathogenic E. coli can cause various infections and is a common foodborne pathogen. They can easily survive in the environment and spread to more new hosts. Human consumption of contaminated food can cause acute intestinal diseases in humans, and contaminated food chains can even cause outbreaks [Compr. Rev. Food Sci. Food Saf., 22 (2023) 1839-1863]. Pathogenic E. coli O157:H7 can cause contamination of products such as meat, dairy products, and vegetables, thereby endangering human health. After infection, people first show mild watery diarrhea, which turns into bloody diarrhea after a few days, causing bloody colitis or hemolytic uremic syndrome [Analyst., 149 (2024) 1022-1049]. With the outbreak of food safety incidents caused by various foodborne pathogens in the world in recent years, rapid detection of foodborne pathogens including Escherichia coli, Salmonella and Staphylococcus aureus has become a hot topic of research. Escherichia coli O157:H7 is one of the most important foodborne pathogens in the world and is extremely toxic. Nowadays, the main detection methods for Escherichia coli O157:H7 include: traditional culture detection methods, immunological detection methods and molecular biology detection methods. Although most of these methods are relatively mature and have high accuracy, some of them take a long time and consume a lot of manpower, some require antibodies or aptamers, have many interference factors, poor repeatability and high prices [J.Mater.Chem.B.,10(2022)8211-8217]. Therefore, it is necessary to develop a detection method with good specificity, low cost and fast detection speed.
[0003] Molecular imprinting technology (MIT) is a technology that simulates the specific interaction between enzyme-substrate and antigen-antibody, and uses a specific target molecule as a template to synthesize an artificial receptor with specific recognition performance [Chem. Rev., 116 (2016) 11500-11528]. The preparation process of molecular imprinted polymers (MIPs) is that the template and the functional monomer interact in a solution to form a prepolymer complex, and then a cross-linking agent and an initiator are added to polymerize. The template is removed from the polymer through an elution step to obtain a recognition cavity complementary to the template, which can specifically recognize the target. At present, there are many examples of molecular imprinting in scientific research, mostly with small molecules and protein macromolecules as targets or templates. Molecular imprinting technology targeting bacteria is still very challenging. There are two main types of template molecules for bacterial molecular imprinting technology: one is to use the entire bacterium as a template; the other is to use specific macromolecules on the surface of the bacteria as templates, such as surface proteins or lipopolysaccharides.
[0004] The carrier materials of molecular imprinting polymers generally include glass plates, glass balls, micro-nanoparticles, silicon wafers and some biomass materials. Among them, biocellulose materials are not only widely available, inexpensive, lightweight, biodegradable, and easy to operate, but also cellulose has a porous structure, good adsorption properties and multiple hydroxyl groups, etc., which makes it an ideal carrier material for making bacterial molecular imprinting polymers. In addition, cellulose paper can make full use of capillary phenomena and develop into a high-throughput paper chip that does not require additional energy. It has the advantages of high efficiency, portability, simple preparation, easy operation, and low sample consumption. It has been applied in many fields [TrAC-Trends Anal.Chem., 143 (2021) 11637]. Summary of the invention
[0005] Escherichia coli is commonly found in the intestines of humans and animals. Some of them are pathogenic bacteria that can cause various infections and are common foodborne pathogens. Most common detection methods are relatively mature and have high accuracy, but some require a long time and high manpower consumption, and some require antibodies or aptamers, with many interference factors, poor repeatability, and high prices. In order to develop a simple and portable pathogen detection device, the present invention provides a fluorescent paper chip for detecting Escherichia coli O157:H7 and its preparation method and application. Molecular imprinting fluorescent nanoparticles with Escherichia coli O157:H7 as a template are designed and synthesized by molecular imprinting technology. After the molecular imprinting fluorescent nanoparticles are modified on the surface of the paper chip, the Escherichia coli O157:H7 template can be sensitively and quickly identified according to the change of fluorescence quenching rate, thereby preparing a paper chip that can specifically identify Escherichia coli O157:H7 for quantitative detection, which has good application prospects.
[0006] In order to solve the problems of the prior art, the technical solution adopted by the present invention is:
[0007] A method for preparing a fluorescent paper chip for detecting Escherichia coli O157:H7 comprises the following steps:
[0008] Step 1, preparation of molecularly imprinted polymer:
[0009] The Escherichia coli O157:H7 template and functional monomers are added to a phosphate buffer solution and stirred, a fluorescent monomer is added for pre-assembly, and then a cross-linking agent is added and stirred. After the reaction is completed, the nanoparticles are first washed with a phosphate buffer solution, and then amino groups are modified on the surface of the nanoparticles. The template is removed after washing with a methanol / water co-solvent solution of acetic acid to obtain molecularly imprinted fluorescent nanoparticles.
[0010] Step 2: Immobilization of fluorescent molecularly imprinted nanoparticles on the surface of paper chip:
[0011] Taking advantage of the abundant hydroxyl groups on the surface of the paper chip, the amino group and the aldehyde group were modified on the surface of the paper chip in sequence. The molecularly imprinted fluorescent nanoparticles prepared in step 1 were immobilized on the paper chip through the Schiff base reaction to obtain a fluorescent paper chip for detecting Escherichia coli O157:H7.
[0012] As an improvement, the functional monomer reagent in step 1 is obtained by the reaction of 3-aminopropyltriethoxysilane and 3-formylphenylboronic acid, and the fluorescent monomer is obtained by the reaction of 3-aminopropyltriethoxysilane and fluorescein isothiocyanate.
[0013] As an improvement, in step 1, the concentration of the functional monomer is 4.2 mmol / L, the concentration of the fluorescent monomer is 0.2 mmol / L, and the concentration of the bacterial solution of Escherichia coli O157:H7 is 0.5×10 9 CFU / mL.
[0014] Preferably, the concentration of the methanol / water co-solvent solution of acetic acid in step 1 is 100 mmol / L.
[0015] Preferably, the cross-linking agent in step 1 is tetraethoxysilane with a concentration of 351 mmol / L.
[0016] Preferably, the reagent for modifying the amino group in step 1 is a 6% volume fraction 3-aminopropyltriethoxysilane ethanol solution.
[0017] Preferably, the reagent for modifying the aldehyde groups on the surface of the paper chip in step 2 is a glutaraldehyde aqueous solution with a volume fraction of 2.5%.
[0018] The fluorescent paper chip for detecting Escherichia coli O157:H7 is used for quantitative detection and analysis of Escherichia coli O157:H7 in food.
[0019] Preferably, the application utilizes a fluorescent paper chip to selectively adsorb Escherichia coli O157:H7 in a sample, and after washing, determines the fluorescence signal value thereof, and simultaneously determines the blank fluorescence signal value, and uses a standard curve method based on the fluorescence signal quenching rate to perform a quantitative analysis of Escherichia coli O157:H7.
[0020] Beneficial effects:
[0021] Compared with the prior art, the fluorescent paper chip for detecting Escherichia coli O157:H7 of the present invention and its preparation method and application, take Escherichia coli as the research object, design and synthesize molecular imprinting fluorescent nanoparticles with Escherichia coli O157:H7 as the template through molecular imprinting technology, and after the molecular imprinting fluorescent nanoparticles are modified on the surface of the paper chip, the Escherichia coli template can be sensitively and quickly identified according to the change of fluorescence quenching rate, thereby preparing a paper-based array chip that can specifically identify Escherichia coli O157:H7, which has the characteristics of simplicity, low cost, real-time, etc. The specific advantages are as follows:
[0022] 1. The fluorescent paper chip for detecting Escherichia coli O157:H7 prepared by the present invention can not only quickly identify Escherichia coli O157:H7, but also can quantitatively detect it;
[0023] 2. Molecular imprinting technology does not require sample pretreatment for pathogen detection, which simplifies the analysis process, has fast identification and detection speed, and good stability of detection materials;
[0024] 3. The paper chip optical sensor is easy to operate, requires less reagents, and has a shorter analysis time, which greatly improves the efficiency and sensitivity of detection and makes it easy to implement on-site sampling and analysis;
[0025] 4. The paper chip prepared by the present invention has the advantage of being reusable and can be reused for at least 2 cycles without reducing its fluorescence response performance, which is more resource-saving and has good application prospects;
[0026] 5. The surface of the fluorescent molecular imprinted nanoparticles of Escherichia coli O157:H7 prepared by the present invention has a small deviation of the imprinting factor after the amino group is modified, and still has a high selectivity for Escherichia coli O157:H7. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 Schematic diagram of the molecular imprinting fluorescent paper chip of Escherichia coli; (3-aminopropyltriethoxysilane (APTES), triformylphenylboronic acid (PBA), fluorescein isothiocyanate (FITC), functional monomer (PBA-APTES), fluorescent monomer (FITC-APTES));
[0028] Figure 2The fluorescence quenching rate change of fluorescent molecular imprinted nanoparticles of Escherichia coli O157:H7 before and after amino modification;
[0029] Figure 3 This is a selectivity experiment after the fluorescent molecular imprinted nanoparticles of Escherichia coli O157:H7 were immobilized on the surface of paper chip;
[0030] Figure 4 Specificity experiment of fluorescent molecular imprinted nanoparticles of Escherichia coli O157:H7 immobilized on the surface of paper chip
[0031] Figure 5 The fluorescence quenching rate changes of bacteria and paper chip at different incubation times;
[0032] Figure 6 The fluorescence quenching rate changes of bacteria and paper chips at different incubation temperatures;
[0033] Figure 7 This is a selectivity experiment after different concentrations of fluorescent molecularly imprinted nanoparticles were immobilized on the surface of paper chips;
[0034] Figure 8 This is an experiment on the selectivity of paper chips for target bacteria under different pH conditions;
[0035] Fig. 9 The fluorescence quenching rate changes after the paper chip was used three times;
[0036] Fig.10 Fluorescence intensity changes of the paper chip after three repeated uses;
[0037] Fig.11 This is the linear range of fluorescence response of the paper chip to different concentrations of Escherichia coli O157:H7. DETAILED DESCRIPTION
[0038] The present invention is further described below in conjunction with specific embodiments and drawings.
[0039] The experimental methods described in the examples are conventional methods unless otherwise specified; Escherichia coli O157:H7 (CICC21530) and Staphylococcus aureus (ATCC25923) used in the examples of the present invention were kindly provided by the Animal Food Control Laboratory of Nanjing Normal University; Salmonella (ATCC14028) and Shigella (CMCC(B)51592) were kindly provided by Nanjing Product Quality Supervision and Inspection Institute. The reagents and materials described above can be obtained from commercial sources unless otherwise specified.
[0040] Example 1
[0041] Preparation of fluorescent monomers:
[0042] Dissolve fluorescein isothiocyanate (FITC, 6.6 mg, 17 μmol) in 1.5 mL of anhydrous ethanol, then add 3-aminopropyltriethoxysilane (APTES, 4 μL, 17 μmol) and stir at room temperature for 12 h. After the reaction is completed, remove the ethanol to obtain the FITC-APTES product. Take 0.2 mL of FITC-APTES solution for later use.
[0043] Preparation of functional monomers:
[0044] 3-Formylphenylboronic acid (3-FPBA, 75 mg, 0.5 mmol) was dissolved in 10 mL of anhydrous ethanol, and then 3-aminopropyltriethoxysilane (APTES, 120 μL, 0.5 mmol) was added and stirred at room temperature for 12 h. After the reaction was completed, ethanol was removed to obtain a light yellow PBA-APTES product. 20 mg of PBA-APTES was dissolved in 0.2 ml of anhydrous ethanol for later use.
[0045] Example 2
[0046] Preparation of molecularly imprinted fluorescent nanoparticles, the specific synthesis route is as follows Figure 1 As shown:
[0047] Put 0.5mL of 10 9 CFU / mL Escherichia coli O157:H7 bacterial solution was evenly dispersed in 6.55mL PBS (10mM, pH=7.4), and mixed with 0.2mL PBA-APTES solution and 0.2mL FITC-APTES solution in a round-bottom flask for 40min. TEOS (675μL, 3.2mmol) was added, followed by 1mL anhydrous ethanol, and stirred at 1000rpm for 12h. After centrifugation, the polymer was washed twice with phosphate buffer to start modifying the amino group on the polymer surface. The template was then washed with acetic acid methanol / water co-solvent solution to remove the template, and Escherichia coli O157:H7 molecularly imprinted fluorescent nanoparticles FMIP-NH2 were obtained.
[0048] Comparative Example 1
[0049] Non-molecularly imprinted fluorescent nanoparticles (FNIP-NH2) were prepared as a control, and the nanoparticles were generated using the same conditions as the FMIP-NH2 nanoparticles, with specific reference to the preparation of Example 2, except that the template Escherichia coli O157:H7 was omitted.
[0050] Example 3
[0051] Take 100 μL of 1 mg / mL FMIP solution (solvent is PBS, pH 7.4) and FNIP solution (solvent is PBS, pH 7.4), add them to 4 mL fluorescence cuvettes, then add 1.9 mL PBS (10 mM, pH = 7.4), and use a fluorescence spectrophotometer (excitation wavelength 471 nm, emission wavelength 513 nm) to measure the initial fluorescence signal F0. Then add 20 μL, 10 9 E. coli O157:H7 was diluted to a concentration of 10 7 CFU / mL, stirred at room temperature (25°C) for 5 min, and the fluorescence signal F was measured using a fluorescence spectrophotometer to calculate the fluorescence quenching rate. 7 The fluorescence quenching rate after mixing CFU / m of Escherichia coli O157:H7.
[0052] like Figure 2 As shown in the figure, although the fluorescence quenching rate of the fluorescent molecularly imprinted nanoparticles of Escherichia coli O157:H7 changed before and after the modification of the amino group, the imprinting factor was not much different, which was 2.36 and 2.45 respectively. This further proves that the modification group has little effect on the particles.
[0053] Example 4
[0054] Place the paper chip in a 6% (volume fraction) APTES ethanol solution, shake it for 12 hours, take it out, rinse it with ethanol, and dry it; prepare a 2.5% (volume fraction) glutaraldehyde aqueous solution, place the amino-modified paper chip in a centrifuge tube, shake it for 12 hours, take it out, rinse it with deionized water, and dry it; prepare 1 mg / mL FMIP-NH2 solution, soak the paper chip for 12 hours, take it out, and rinse it with PBS.
[0055] Comparative Example 2
[0056] According to the operation of Example 4, a 1 mg / mL FNIP-NH2 solution was prepared, and the paper chip was soaked for 12 hours, then taken out and rinsed with PBS.
[0057] Example 5
[0058] Selectivity test of the fluorescent paper chip for detecting Escherichia coli O157:H7 prepared in Example 4 and the chip prepared in Comparative Example 2
[0059] The initial fluorescence signal F0 of the fluorescent paper chip was measured using a microarray chip scanner. After the measurement, 20 μL of 10 1 , 10 2 , 10 3 , 10 4 , 105 , 10 6 , 10 7 , 10 8 and 10 9 The fluorescence signal F was measured after incubation (temperature was 25°C) for 10 min with a CFU / mL Escherichia coli O157:H7 bacterial solution. A blank sample was measured at the same time to calculate the fluorescence response ratio.
[0060] like Figure 3 Shown 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 and 10 9 The fluorescence response change rate of FMIP@P with CFU / mL concentration was 10 higher than that of their corresponding fluorescent nanoparticles. 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 and 10 9 The CFU / mL concentration of FNIP@P indicated that the fluorescent paper chip had good response selectivity to Escherichia coli O157:H7.
[0061] Example 6
[0062] Specificity test of the fluorescent paper chip for detecting Escherichia coli O157:H7 prepared in Example 4 and the chip prepared in Comparative Example 2
[0063] The initial fluorescence signal F0 of the fluorescent paper chip was measured using a microarray chip scanner, and then 20 μL, 10 9 CFU / mL Escherichia coli O157:H7, Salmonella, Staphylococcus aureus, and Shigella were incubated for 10 minutes (temperature was 25°C), and the fluorescence signal value F was measured to calculate the fluorescence responsiveness ratio (blank samples were measured at the same time).
[0064] like Figure 4 The imprinting factor shown refers to the ratio of the amount of the target adsorbed by MIP or the signal change caused to the amount adsorbed by NIP or the signal change caused, which can be used to indicate the selectivity. The higher the imprinting factor, the higher the selectivity. It can be seen from the figure that the imprinting factors are 3.1, 1.6, 2.1, and 2.7, respectively, indicating that the fluorescent paper chip has good specificity for Escherichia coli O157:H7.
[0065] Example 7
[0066] The reaction time of the fluorescent paper chip for detecting Escherichia coli O157:H7 prepared in Example 4 and the chip prepared in Comparative Example 2 was tested
[0067] The initial fluorescence signal F0 of the fluorescent paper chip was measured using a microarray chip scanner, and 20 μL of Escherichia coli O157:H7 bacterial solution was added and incubated for 0, 3, 5, 8, 10, and 15 min, respectively. F was measured and the fluorescence quenching rate was calculated.
[0068] from Figure 5 It can be seen that the imprinting factor is the largest after the fluorescent paper chip and bacteria are incubated for 5 minutes (temperature is 25°C). The imprinting factors at 8, 10, and 15 minutes after 5 minutes are not much different, indicating that the binding of bacteria and fluorescent molecular imprinted nanoparticles on the fluorescent paper chip has reached saturation at 5 minutes.
[0069] Example 8
[0070] The reaction temperature of the fluorescent paper chip for detecting Escherichia coli O157:H7 prepared in Example 4 and the chip prepared in Comparative Example 2 were tested.
[0071] Use a microarray chip scanner to measure the initial fluorescence signal F0 of the fluorescent paper chip, add 20 μL of Escherichia coli O157:H7 bacterial solution, incubate at 4°C, 10°C, 15°C, 20°C, 25°C, 30°C, 37°C, 45°C, and 55°C for 5 min (temperature is 25°C), measure F, and calculate the fluorescence quenching rate.
[0072] The results are as follows Figure 6 As shown in the figure, it can be seen that at 15°C, the imprinting factor is the largest, which further illustrates that the selectivity is higher at this temperature.
[0073] Example 9
[0074] The polymer concentration was tested on the fluorescent paper chip for detecting Escherichia coli O157:H7 prepared in Example 4 and the chip prepared in Comparative Example 2.
[0075] The paper chip with modified groups was immersed in 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.3 mg / mL, and 0.5 mg / mL FMIP-NH2 and FNIP-NH2 solutions for 12 h, respectively. After being taken out, F0 was determined, 20 μL of Escherichia coli O157:H7 bacterial solution was added, and the mixture was incubated at 15°C for 5 min. F was determined and the fluorescence quenching rate was calculated.
[0076] from Figure 7 It can be seen that the imprinting factor is the largest at a concentration of 0.05 mg / mL.
[0077] Example 10
[0078] The fluorescent paper chip for detecting Escherichia coli O157:H7 prepared in Example 4 and the chip prepared in Comparative Example 2 were tested for pH optimization.
[0079] The initial fluorescence signal F0 of the paper chip was measured using a microarray chip scanner. The E. coli O157:H7 bacterial solution was then washed with PBS at pH = 6, 7.4, and 8.5, and 20 μL of the clean bacterial solution was dripped onto the surface of the paper chip. After 5 minutes, the fluorescence signal F was measured and the fluorescence response ratio was calculated.
[0080] Figure 8 As shown, most non-extreme bacteria can grow in the external pH range of 5.5 to 9.0 and maintain the cytoplasmic pH in the appropriate range of 7.4 to 7.8. It can be seen from the figure that after washing the bacteria with PBS at pH = 7.4, dripping it on the surface of the paper chip and incubating it for 5 minutes (temperature is 15°C), the fluorescence quenching rate is the largest.
[0081] Embodiment 11
[0082] The fluorescent paper chip for detecting Escherichia coli O157:H7 prepared in Example 4 and the chip prepared in Comparative Example 2 were tested for the number of repetitions.
[0083] The initial fluorescence signal F0 of the paper chip was measured using a microarray chip scanner. After the measurement, 20 μL and 10 μL of 9 CFU / mL of E. coli O157:H7 bacterial solution, incubate for 5 min, measure the fluorescence signal F, and calculate the fluorescence response ratio (simultaneously measure the blank sample). After the measurement, the paper chip was washed 6 times with acetic acid methanol / water co-solvent solution, washed twice with PBS, and the above operation was repeated to record the data.
[0084] from Fig. 9 , 10 It can be seen that after 1 to 3 repetitions, the quenching rate of the paper chip for Escherichia coli O157:H7 and the fluorescence intensity of the paper chip itself changed. It can be concluded that the paper chip prepared according to this method can be reused twice.
[0085] Example 12
[0086] Establishment of a standard curve for the fluorescent paper chip for detecting Escherichia coli O157:H7 prepared in Example 4
[0087] The initial fluorescence signal F0 of the fluorescent paper chip was measured using a microarray chip scanner. After the measurement, 20 μL of 10 1 , 10 2 , 10 3 , 104 , 10 5 , 10 6 , 10 7 , 10 8 and 10 9 The fluorescence signal F was measured after incubation (temperature was 15°C) for 5 min with E. coli O157:H7 solution containing CFU / mL, and the fluorescence response ratio was calculated (blank sample was measured at the same time).
[0088] Embodiment 13
[0089] Used for testing actual samples (tap water and Junlebaoyue fresh milk).
[0090] The standard curve was prepared according to the steps of Example 12 to determine the recovery rate of spike addition in this method and calculate the recovery rate of spike addition. First, the initial fluorescence signal F0 of the paper chip was measured using a microarray chip scanner. The bacterial solution was diluted to 10% with tap water. 2 , 10 3 , 10 4 CFU / mL, drop the diluted bacterial solution on the surface of the paper chip, incubate for 5 minutes, measure the fluorescence signal F, and calculate the fluorescence response ratio (simultaneously measure the blank sample). Drop 100μL of bacterial solution into 900μL of milk, and then dilute it 10 times with PBS to make the final concentration of the bacterial solution 10 2 , 10 3 , 10 4 CFU / mL. The treated milk was dripped onto the surface of the paper chip, and the diluted bacterial solution was dripped onto the surface of the paper chip. After incubation for 5 min (temperature was 15°C), the fluorescence signal F was measured and the fluorescence response ratio was calculated (the blank sample was measured at the same time).
[0091] Recovery rate = (sample recovery test value - blank recovery test value) / standard test value * 100%, calculate the recovery rate, the results are shown in Table 1:
[0092] Table 1 Determination of spike recovery
[0093]
[0094] It can be seen from Table 1 that the recovery rate of the spiked samples of the present invention is between 84.31% and 113.27%, which proves the accuracy and reliability of the method.
[0095] In summary, the present invention takes Escherichia coli as the research object, designs and synthesizes molecular imprinting fluorescent nanoparticles with Escherichia coli O157:H7 as the template through molecular imprinting technology, and after the molecular imprinting fluorescent nanoparticles are modified on the surface of the paper chip, the Escherichia coli template can be sensitively and quickly identified according to the change of fluorescence quenching rate, thereby preparing a paper-based array chip that can specifically identify Escherichia coli O157:H7, which has the characteristics of simplicity, low cost, and real-time.
[0096] The present invention and its embodiments are described schematically above, and the description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired by it and designs a structural method and an embodiment similar to the technical solution without creativity without departing from the purpose of the invention, they shall all fall within the protection scope of the present invention.
Claims
1. A method for detecting Escherichia coli O157:H7 The method for preparing a fluorescent paper chip is characterized in that: The following steps are involved: Step 1, preparation of molecularly imprinted polymer: Escherichia coli O157:H7 The template and the functional monomer are added to a phosphate buffer solution and stirred, the fluorescent monomer is added for pre-assembly, and then the cross-linking agent is added and stirred. After the reaction is completed, the nanoparticles are first washed with a phosphate buffer solution, and then the amino groups are modified on the surface of the nanoparticles. The template is removed after washing with a methanol / water co-solvent solution of acetic acid to obtain molecularly imprinted fluorescent nanoparticles. Step 2: Immobilization of fluorescent molecularly imprinted nanoparticles on the surface of paper chip: Using the abundant hydroxyl groups on the surface of the paper chip, the amino group and aldehyde group were modified on the surface of the paper chip in sequence, and the molecularly imprinted fluorescent nanoparticles prepared in step 1 were immobilized on the paper chip through the Schiff base reaction to obtain the detection of Escherichia coli. O157:H7 Fluorescent paper chips.
2. A method for detecting Escherichia coli according to claim 1. O157:H7 The method for preparing a fluorescent paper chip is characterized in that: In step 1, the functional monomer reagent is obtained by reacting 3-aminopropyltriethoxysilane with 3-formylphenylboronic acid, and the fluorescent monomer is obtained by reacting 3-aminopropyltriethoxysilane with fluorescein isothiocyanate.
3. A method for detecting Escherichia coli according to claim 1. O157:H7 The method for preparing a fluorescent paper chip is characterized in that: In step 1, the concentration of the functional monomer was 4.2 mmol / L, the concentration of the fluorescent monomer was 0.2 mmol / L, and the concentration of Escherichia coli was O157:H7 The bacterial concentration was 0.5×10 9 CFU / mL.
4. A method for detecting Escherichia coli according to claim 1. O157:H7 The method for preparing a fluorescent paper chip is characterized in that: The concentration of the methanol / water co-solvent solution of acetic acid in step 1 is 100 mmol / L.
5. A method for detecting Escherichia coli according to claim 1. O157:H7 The method for preparing a fluorescent paper chip is characterized in that: The cross-linking agent in step 1 is tetraethoxysilane with a concentration of 351 mmol / L.
6. A method for detecting Escherichia coli according to claim 1. O157:H7 The method for preparing a fluorescent paper chip is characterized in that: In step 1, the reagent for modifying the amino group is a 6% volume fraction 3-aminopropyltriethoxysilane ethanol solution.
7. A method for detecting Escherichia coli according to claim 1. O157:H7 The method for preparing a fluorescent paper chip is characterized in that: In step 2, the reagent for modifying the aldehyde groups on the surface of the paper chip is a 2.5% volume fraction of glutaraldehyde aqueous solution.
8. The detection Escherichia coli prepared by the preparation method according to any one of claims 1 to 7 O157:H7 Fluorescent paper chip for quantitative detection of Escherichia coli in food O157:H7 Application in.
9. The use according to claim 8, characterized in that: The application uses a fluorescent paper chip to selectively adsorb Escherichia coli in a sample O157:H7 After cleaning, the fluorescence signal value was measured, and the blank fluorescence signal value was measured at the same time. The standard curve method was used according to the fluorescence signal quenching rate, and then the Escherichia coli O157:H7 Quantitative analysis of .