CN113624967B - Magnetic MOF@aptamer and method for detecting food-borne pathogenic bacteria by using same - Google Patents
Magnetic MOF@aptamer and method for detecting food-borne pathogenic bacteria by using same Download PDFInfo
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Abstract
The invention discloses a magnetic MOF@aptamer and a method for detecting food-borne pathogenic bacteria by using high-sensitivity fluorescence thereof, wherein Fe 3O4 @ZIF-8 is prepared mainly by solvothermal method and layer-by-layer autonomous assembly method, fe 3O4 @ZIF-8 is modified by using the aptamer of target bacteria to prepare Fe 3O4 @ZIF-8@aptamer, capturing and separating of the target bacteria are realized, and FITC fluorescent molecules and fluorescence resonance energy transfer on the surface of a Fe 3O4 @ZIF-8 metal group are further realized by using FITC fluorescent labeled antibodies to identify the target bacteria. The research method disclosed by the invention has the advantages of high detection sensitivity, strong specificity, short detection time and the like, and is suitable for rapid detection of food-borne pathogenic bacteria in food samples.
Description
Technical Field
The invention belongs to the technical field of food safety, and relates to a nano technology and a biotechnology. In particular to a magnetic MOF@aptamer (namely Fe 3O4 @ZIF-8@aptamer) and a method for detecting food-borne pathogenic bacteria by using the same, which utilizes the principle of double-recognition fluorescence enhancement effect of the magnetic MOF to detect the food-borne pathogenic bacteria with high sensitivity.
Background
With the rapid development of modern economy, people's demands for high quality foods are growing, and food safety problems caused by food-borne pathogenic bacteria are also becoming more and more interesting. Therefore, the detection of pathogenic bacteria in food is of great importance and is an important task for society, economy and public health. Nanomaterial has been developed rapidly in recent years, and is widely used in fields such as biology and medicine. The nano material has larger specific surface area and special property, and the Fe 3O4 particles have superparamagnetism, thus being applicable to the fields of material recovery and the like; metal Organic Frameworks (MOFs) are a novel class of high porosity materials coordinated by self-assembly of inorganic metal-containing nodes and organic ligands. The MOFs have wide application in the adsorption and sensor fields due to the high specific surface area, the modifiable pore channel structure, various functional parts and the like.
The fluorescent probe technology is widely applied to biological detection, can be used for purposefully carrying out qualitative and quantitative research and characterization, has weak or basically no fluorescence for most biomolecules, and has poor detection sensitivity, so that the application of the fluorescent probe detection technology is objectively possible. The recognition function may label biological macromolecules such as proteins, antigen antibodies, nucleic acids, enzymes, and polymers containing specific genes. When the kit is applied to food-borne pathogenic bacteria detection, the kit has higher detection sensitivity and can realize quantitative detection. The present invention has been made in view of this.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a magnetic MOF@aptamer (Fe 3O4 @ZIF-8@aptamer) and a method for detecting food-borne pathogenic bacteria by using the same.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for preparing a magnetic mof@aptamer comprising the steps of:
1) Adding zinc nitrate hexahydrate and 2-methylimidazole into an anhydrous methanol dispersion liquid of Fe 3O4, uniformly mixing, heating in a water bath at 65-75 ℃ for 15-30 min, cooling to room temperature, washing, and drying to obtain Fe 3O4 @ZIF-8 powder;
2) Washing Fe 3O4 @ZIF-8 with PBS, re-suspending in PBS, adding aptamer, shaking at room temperature for 20-30 min, adding skimmed milk powder for sealing, washing with PBS, and re-suspending in PBS.
Specifically, in step 1), 0.10 to 0.20g of zinc nitrate hexahydrate and 0.20 to 0.30g of 2-methylimidazole are added to an anhydrous methanol dispersion containing 25 mg Fe 3O4.
Further, the step 1) can be repeated for 2 to 4 times to enable the shells of the ZIF-8 to grow on the surface of the Fe 3O4 layer by layer.
Further, in step 1), the Fe 3O4 is obtained by the following treatment: mixing ethylene glycol with ferric trichloride hexahydrate, sodium acetate and sodium citrate, heating at 180-220 deg.c to react 8-12 h, cooling to room temperature, washing and drying. The surface of Fe 3O4 prepared by the method is provided with carboxyl groups, which is more beneficial to the combination with zinc ions in the step 2) and the growth of organic ligands on the surface of the Fe 3O4.
Specifically, in preparing Fe 3O4, 4-5-g ferric trichloride hexahydrate, 7-9g sodium acetate and 0.5-1.0-g sodium citrate can be added into 60-100 mL glycol.
Further, the final concentration of Fe 3O4 @ ZIF-8 and aptamer in step 2) was 2 mg/mL and 2.4. Mu.M, respectively, and the final concentration of skim milk powder added was 2% (W/V).
The invention also provides the magnetic MOF@aptamer prepared by the preparation method.
The invention also provides a method for detecting food-borne pathogenic bacteria by using the magnetic MOF@aptamer, which comprises the following steps:
a) Incubating FITC-labeled streptavidin and biotin-labeled antibody as FITC-antibody recognition agents at room temperature, and preserving at 4 ℃;
b) Placing target food-borne pathogenic bacteria with different concentration gradients and magnetic MOF@aptamer at 37 ℃ to react for 20-40min, washing with PBS, re-suspending in PBS, adding an equal volume of the FITC-antibody recognizing agent prepared in the step a), coupling for 20-40min at 37 ℃, magnetically separating and detecting the fluorescence intensity of supernatant; drawing a standard curve by taking the fluorescence intensity as an ordinate and the concentration of the target food-borne pathogenic bacteria as an abscissa;
c) Placing a sample to be detected and a magnetic MOF@aptamer at 37 ℃ for reaction for 20-40min, washing with PBS, re-suspending in the PBS, adding an equal volume of the FITC-antibody recognition agent prepared in the step a), coupling for 20-40min at 37 ℃, magnetically separating, and detecting the fluorescence intensity of the supernatant; substituting the bacteria concentration into a standard curve, and calculating to obtain the bacteria concentration in the sample to be detected.
Further, in the step b), the food-borne pathogenic bacteria are Listeria monocytogenes, escherichia coli O157: H7, salmonella, staphylococcus aureus, vibrio parahaemolyticus, pseudomonas aeruginosa, and the like.
Specifically, the final concentrations of FITC-labeled streptavidin and biotin-labeled antibody in step a) were 25 μg/mL and 10 μg/mL, respectively. The sample to be detected in the step c) can be a pure culture bacterial liquid or a bacterial-containing food sample, and the volume ratio of the sample to the magnetic MOF@aptamer (5 mg/mL) is 3:2.
In the invention, PBS used in the experiment refers to phosphate buffer solution with the concentration of 0.01M and the pH of 7.4, and needs to be sterilized in advance.
The invention firstly synthesizes and prepares a magnetic MOF@aptamer (Fe 3O4 @ZIF-8@aptamer), and then provides a method for detecting high-sensitivity food-borne pathogenic bacteria by utilizing the principle that fluorescence resonance energy transfer occurs between a fluorescent molecular acceptor and a metal donor to enhance fluorescence intensity. Mainly comprises the following steps: firstly preparing Fe 3O4 by synthesis through a solvothermal method, then growing ZIF-8 layer by layer on the Fe 3O4 to prepare Fe 3O4 @ZIF-8, and then bonding an aptamer on the surface of the Fe 3O4 @ZIF-8@aptamer through adsorption to prepare Fe 3O4 @ZIF-8@aptamer; meanwhile, FITC fluorescence labeled streptavidin is specifically combined with biotin labeled antibody, on one hand, the space binding site of the antibody is increased so as to improve the capture of pathogenic bacteria, and on the other hand, fluorescence signals are provided for quantitative analysis of the method. According to the method, target bacteria are double-identified through Fe 3O4 @ZIF-8@aptamer and FITC-antibody, and fluorescence resonance energy transfer between fluorescent molecules and the surface of a metal group occurs by utilizing the distance between FITC and the surface metal group of Fe 3O4 @ZIF-8 when the target bacteria exist, so that fluorescence enhancement is realized.
The detection method of the invention is that the distance between fluorescent molecules and metal groups is shortened after the target bacteria are added, so that fluorescence resonance energy transfer occurs between the fluorescent molecules and the metal groups, thereby leading to fluorescence enhancement, and along with the increase of the concentration of the target bacteria, more energy is transferred, and the fluorescence intensity is also increased, so that the detection result can have good linear relation within a certain range, and a basis is provided for quantitative analysis. The detection method has high detection sensitivity, and has good linear relation between 1.4X10 1 CFU/mL-1.4×107 CFU/mL for detecting pure culture bacteria; the detection of the bacteria-containing food sample has good linear relation between 6.6X10 1 CFU/mL-6.6×106 CFU/mL; meanwhile, the method has the characteristics of high detection sensitivity, strong specificity and the like.
Compared with other existing traditional detection methods, the invention has the following remarkable characteristics and beneficial effects:
1) The materials and reagents used for detection are stable and simple, and the operation is convenient and quick;
2) The invention has good specificity to the detection result through the dual recognition effect of the aptamer and the antibody;
3) The invention detects through fluorescence enhancement effect, and the fluorescence signal has better linear relation and can realize quantitative detection;
4) The invention has the advantages of quick detection, high sensitivity and detection time within 2 hours, and the detection limit of the bacteria on the pure culture bacteria and bacteria-containing food sample bacteria is lower than 10 CFU/mL.
Drawings
FIG. 1 is a FE-TEM image of Fe 3O4 (a) and Fe 3O4 @ZIF-8 (b) prepared according to the present invention at different magnifications;
FIG. 2 is an XRD pattern of Fe 3O4 and Fe 3O4 @ZIF-8 prepared according to the present invention;
FIG. 3 is a FT-IR spectrum of Fe 3O4 and Fe 3O4 @ZIF-8 prepared by the invention;
FIG. 4 is a VSM hysteresis graph of Fe 3O4 and Fe 3O4 @ ZIF-8 prepared according to the present invention;
FIG. 5 shows XPS measurement spectra of Fe 3O4 @ ZIF-8 (a), zn 2p (b), N1 s (C), C1 s (d) and O1 s (e) prepared by the invention;
FIG. 6 is a zeta potential plot of Fe 3O4 @ ZIF-8 and Fe 3O4 @ ZIF-8@aptamer prepared according to the present invention;
FIG. 7 is a fluorescence spectrum (a) and a standard graph (b) of fluorescence intensity versus concentration of Listeria monocytogenes for detecting pure cultures of different concentrations in the present invention;
FIG. 8 is a graph of fluorescence spectra (a) and a graph of fluorescence intensity versus concentration for listeria monocytogenes in pork samples with varying concentrations according to the present invention;
FIG. 9 is a diagram showing the specificity of detection in the detection of Listeria monocytogenes in the present invention.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that the following examples are intended to illustrate the present invention and are not to be construed as limiting the scope of the invention, and that numerous insubstantial modifications and adaptations can be made by those skilled in the art in light of the foregoing disclosure.
Example 1
A preparation method of a magnetic MOF@aptamer (namely Fe 3O4 @ZIF-8@aptamer) specifically comprises the following steps:
1) Adding 4.32 g ferric trichloride hexahydrate into 80 mL glycol, and magnetically stirring for 10 min; adding 7.94 g sodium acetate, and magnetically stirring 15. 15 min; 0.80 g sodium citrate was added and stirred 5. 5 min. After sonication for 30 min, the resulting solution was transferred to a stainless steel autoclave lined with tetrafluoroethylene at 100 mL, heated at 200 ℃ for reaction 10 h and cooled to room temperature. Washing with absolute methanol for 4 times, and drying at room temperature for 6 h times to obtain Fe 3O4 powder;
2) Taking 25mg Fe 3O4 powder prepared in the step 1), adding into 30mL absolute methanol, carrying out ultrasonic treatment on 5min to uniformly disperse, adding 0.15 g hexahydrate zinc nitrate while stirring 2 min by a glass rod, and adding 0.25 g of 2-methylimidazole and rapidly stirring 1 min. Heating 20 min in water bath at 70 ℃ and cooling to room temperature, washing 3 times with absolute methanol, repeating the operation of the step 2) for 3 times to enable the shells of the ZIF-8 to grow on the surface of Fe 3O4 layer by layer, and finally drying 6 h at room temperature to obtain Fe 3O4 @ZIF-8 powder;
3) 4.8 mg of the Fe 3O4 @ZIF-8 powder prepared in the above step 2) was washed 2 times with PBS (0.01M phosphate buffer solution at pH 7.4), resuspended in 242.4. Mu.L of PBS, and 57.6. Mu.L of 100. Mu.M Listeria monocytogenes aptamer (sequence 5'-TACTATCGCGGAGACAGCGCGGGAGGCACCGGGGA-3', which was custom-manufactured in Huada gene) was added to 10 OD aptamer powder, and 290. Mu.L of ultra-pure water was added to prepare an aptamer solution at a concentration of 100. Mu.M. Shaking 30min at room temperature under mild shaking, magnetically separating to remove supernatant, adding 300 μl of skimmed milk powder with final volume of 2% at 37deg.C, sealing for 30min, washing with PBS for 2 times, and re-suspending in 1.2 mL PBS to obtain Fe 3O4 @ ZIF-8@aptamer, and storing at 4deg.C.
FIG. 1 is a FE-TEM image of Fe 3O4 (a) and Fe 3O4 @ZIF-8 (b) prepared according to the present invention at different magnifications; the FE-TEM image shows: the Fe 3O4 @ZIF-8 prepared by the method is uniformly distributed and shaped, and is a core-shell structure taking Fe 3O4 as an inner core and wrapping the ZIF-8.
FIG. 2 is an XRD pattern of Fe 3O4 and Fe 3O4 @ZIF-8 prepared according to the present invention; the XRD patterns show characteristic peaks of Fe 3O4 @ZIF-8, and the corresponding lattice planes are respectively (011) (002) (112) of ZIF-8 and (222) (220) (311) (400) (511) (440) of Fe 3O4, so that the Fe 3O4 @ZIF-8 prepared by the method is credible from the XRD patterns.
FIG. 3 is a FT-IR spectrum of Fe 3O4 and Fe 3O4 @ZIF-8 prepared by the invention; for the FT-IR spectrum of Fe 3O4, the spectra at 1421 cm -1 and 1626 cm -1 correspond to the Fe 3O4 surface carboxyl groups, and the peak at 584 cm -1 can be attributed to the Fe-O bond; for the FT-IR spectrum of Fe 3O4 @ ZIF-8, except for the peak at 584: 584 cm -1 (belonging to Fe-O bond), all of them belong to ZIF-8, comprising: 3500 The cm -1 - 2250 cm-1 band belongs to the stretching vibration of the-NH-, -CH3 and-OH (Zn-OH) groups, the peaks of 1579 cm -1 and 1678 cm -1 are caused by the bending and stretching of the N-H vibrations in imidazole respectively, the spectrum of 1500 cm -1 - 1350 cm-1 is related to the stretching of its entire ring, and the peak at 420 cm -1 is derived from the stretching pattern of Zn-N. From the above analysis, it is clear that Fe 3O4 @ZIF-8 prepared by the method of the present invention is authentic.
FIG. 4 is a VSM hysteresis graph of Fe 3O4 and Fe 3O4 @ ZIF-8 prepared according to the present invention; VSM hysteresis graph shows: the saturation magnetization of Fe 3O4 and Fe 3O4 @ZIF-8 are 61.8 emu.g -1 and 21.1 emu.g -1 respectively, and the magnetization of Fe 3O4 @ZIF-8 is weaker than that of Fe 3O4, but the magnetic fields still have stronger magnetism, so that the separation requirement can be met.
FIG. 5 is an XPS diagram of Fe 3O4 @ ZIF-8 prepared according to the present invention; wherein (a) the XPS measurement spectrum of Fe 3O4 @ ZIF-8 shows the following corresponding photoelectron peaks: zn 2p (1021.87 eV), fe 2p (711.10 eV), C1 s (284.92 eV), N1 s (399.15 eV) and O1 s (531.31 eV); (b) The Zn 2p XPS spectrum of Fe 3O4 @ ZIF-8 shows that the spin orbits of Zn 2p 1/2 and Zn 2p 3/2 have strong signals at 1045.00 eV and 1022.00 eV, respectively; from the N1 s XPS spectra of (C) Fe 3O4 @ ZIF-8, it can be seen that at 399.10 eV and 400.50 eV represent-N-H and c=n-, respectively; (d) The C1 s XPS spectra of Fe 3O4 @ ZIF-8 show that at 284.80 eV, 285.80 eV and 288.60 eV correspond to C-sp 3, C-O and c=o groups, respectively; (e) The O1 s XPS spectrum of Fe 3O4 @ ZIF-8 shows that the O1 s spectrum signal consists of two peaks at 531.60 eV and 532.66 eV, which can be attributed to zinc ion interacting hydroxyl groups (Zn-OH) and H 2 O, respectively.
FIG. 6 is a zeta potential plot of Fe 3O4 @ ZIF-8 and Fe 3O4 @ ZIF-8@aptamer prepared according to the present invention; zeta potential figures show that the zeta potentials of Fe 3O4 @ ZIF-8 and Fe 3O4 @ ZIF-8@ aptamer are-38.7+ -1.4 mV and-43.37 + -1.2 mV, respectively, and that the aptamer has successfully bound to the surface of Fe 3O4 @ ZIF-8 from the potential change.
Example 2
A method for detecting food-borne pathogenic bacteria by using the magnetic MOF@aptamer (namely Fe 3O4 @ZIF-8@aptamer) specifically comprises the following steps:
a) To 1.19 mL of PBS, 6. Mu.L of FITC-labeled streptavidin (available from Beijing Soy Bao technology Co., ltd., product No. SF068, diluted with PBS) at a concentration of 100. Mu.g/mL and 0.6. Mu.L of biotin-labeled Listeria monocytogenes antibody (available from Ai Bokang (Shanghai) trade Co., product No. ab20766, diluted with PBS) at a concentration of 40. Mu.g/mL were added, and incubated gently at room temperature for 1 h to prepare a FITC-antibody recognition agent, which was stored at 4℃for use;
b) Listeria monocytogenes (ATCC 15313) in the logarithmic growth phase was diluted in a gradient to 1.4×101 CFU/mL、1.4×102 CFU/mL、1.4×103 CFU/mL、1.4×104 CFU/mL、1.4×105 CFU/mL、1.4×106 CFU/mL and 1.4X10 7 CFU/mL under sterile conditions, sterile PBS buffer as a control. And (3) placing the diluted bacterial solutions with different concentrations and Fe 3O4 @ZIF-8@aptamer prepared in the step (3) at a volume ratio of 3:2 at 37 ℃ for reaction 30 min for capturing. The separation was performed with external magnet, the supernatant was discarded, washed 1 time with PBS and resuspended in 50. Mu.L of PBS, and an equal volume of the FITC-antibody recognition reagent prepared in step 4) was added and coupled at 37℃for 30 min. The supernatant was finally magnetically separated and the fluorescence intensity was measured, wherein the excitation wavelength was 495nm and the emission wavelength was 520nm. Drawing a standard curve (see b in fig. 7) with fluorescence intensity as an ordinate and bacterial concentration as an abscissa;
c) Using a listeria monocytogenes bacterial liquid as a sample to be tested and Fe 3O4 @ ZIF-8@aptamer (5 mg/mL) prepared in the step 3) to obtain a sample with the concentration of 3:2, reacting at 37 ℃ for 30 min, magnetically separating and discarding supernatant, washing with PBS for 1 time and re-suspending in 50 mu L of PBS, adding the equal volume of the FITC-antibody recognition agent prepared in the step 4), coupling at 37 ℃ for 30 min, magnetically separating and detecting the fluorescence intensity of the supernatant; substituting the bacteria concentration into a standard curve, and calculating to obtain the bacteria concentration in the sample to be detected.
Analysis of detection results: a standard curve (see b in fig. 7) for detecting listeria monocytogenes is obtained by fitting the relation points of the fluorescence intensity of each concentration at 520 nm and the listeria monocytogenes bacterial liquid of different concentrations, wherein the formula is Y=1030.680+35.439X, R 2 = 0.99043, the detection limit is as low as 0.76 CFU/mL, the relative standard deviation of each concentration gradient detection result is not more than 2%, the detection specificity is good, the relative standard deviation of each pathogenic bacteria detection result is not more than 1.5%, and the detection method has good repeatability and reliability.
The pure bacterial liquid of Listeria monocytogenes is detected, the fluorescence intensity is 1233, and the bacterial amount is 1.4X10 5 CFU/mL. Compared with the method, the traditional plate counting method takes a long time, takes 1-2 days, and can not detect bacteria in a non-culturable state.
FIG. 7 is a fluorescence spectrum (a) and a standard graph (b) of fluorescence intensity versus concentration of Listeria monocytogenes for detecting pure cultures of different concentrations in the present invention; a standard curve for detecting Listeria monocytogenes is obtained by fitting the relationship points of the fluorescence intensity of each concentration at 520 nm and the concentration of different bacterial solutions. Fig. 7 shows: the standard curve equation is y=1030.680+35.439 x, r 2 = 0.99043, the detection limit is as low as 0.76 CFU/mL, and the relative standard deviation of each concentration gradient detection result is not more than 2%.
FIG. 8 is a graph (a) of fluorescence spectrum and a graph (b) of fluorescence intensity versus concentration of bacteria in pork samples containing Listeria monocytogenes at different concentrations according to the present invention. The processing of pork samples is described in GB 4789.30-2016, which is specifically: and adding 25 g pork into a homogenizing bag containing 225 mL PBS, and continuously homogenizing for 1-2 min in a slapping type homogenizer to obtain a homogenized liquid. And respectively adding different concentrations of listeria monocytogenes into the homogeneous solution to prepare a listeria monocytogenes pork sample solution with the final concentration of 6.6×101 CFU/mL,6.6×102 CFU/mL,6.6×103 CFU/mL,6.6×104 CFU/mL,6.6×105 CFU/mL and 6.6X10 6 CFU/mL. A standard curve for detecting artificially contaminated pork samples is obtained by fitting the relationship points of the fluorescence intensity of each concentration at 520 nm and the concentration of different samples. Fig. 8 shows: the standard curve equation is y=859.429+52.987x, r 2 = 0.99953, the detection limit is as low as 2.99 CFU/mL, and the relative standard deviation of each concentration gradient detection result is not more than 2%.
And (3) specificity detection: listeria monocytogenes (L. Monocytogenes), escherichia coli O157:H7 (E. Coli O157:H 7), salmonella typhimurium (S. Typhimurium), staphylococcus aureus (S. Aureus), pseudomonas aeruginosa (P. Aeruginosa) and Vibrio parahaemolyticus (V. Parahemolyticus) at a concentration of 10 7 CFU/mL were taken for detection, and the fluorescence intensities of the respective samples were compared to evaluate the detection specificity of the method of the present invention, with reference to the above step 6.
FIG. 9 is a diagram showing the specificity of detection of pure culture broth according to the present invention. The results of fig. 9 show that: the fluorescence intensity of listeria monocytogenes (l. Monocytogenes) was significantly higher than that of the Blank, compared to the Blank, without any increase in the fluorescence intensity of the other five pathogens. The method has good detection specificity, and the relative standard deviation of detection results of various pathogenic bacteria is not more than 1.5%.
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. A method for detecting food-borne pathogenic bacteria using a magnetic mof@aptamer comprising the steps of:
a) Incubating FITC-labeled streptavidin and biotin-labeled antibody as FITC-antibody recognition agents at room temperature, and preserving at 4 ℃;
b) Placing food-borne pathogenic bacteria with different concentration gradients and a magnetic MOF@aptamer at 37 ℃ to react for 20-40min, washing with PBS, re-suspending in the PBS, adding an equal volume of the FITC-antibody recognizing agent prepared in the step a), coupling for 20-40min at 37 ℃, magnetically separating, and detecting the fluorescence intensity of the supernatant; drawing a standard curve by taking the fluorescence intensity as an ordinate and the concentration of food-borne pathogenic bacteria as an abscissa;
c) Placing a sample to be detected and a magnetic MOF@aptamer at 37 ℃ for reaction for 20-40min, washing with PBS, re-suspending in the PBS, adding an equal volume of the FITC-antibody recognition agent prepared in the step a), coupling for 20-40min at 37 ℃, magnetically separating, and detecting the fluorescence intensity of the supernatant; substituting the bacteria concentration into a standard curve, and calculating to obtain the bacteria concentration in the sample to be detected;
the magnetic MOF@aptamer is prepared by the following steps:
1) Adding zinc nitrate hexahydrate and 2-methylimidazole into an anhydrous methanol dispersion liquid of Fe 3O4, uniformly mixing, heating in a water bath at 65-75 ℃ for 15-30 min, cooling to room temperature, washing, and drying to obtain Fe 3O4 @ZIF-8 powder;
2) Washing Fe 3O4 @ZIF-8 with PBS, re-suspending in PBS, adding aptamer, shaking at room temperature for 20-30 min, adding skimmed milk powder for sealing, washing with PBS, and re-suspending in PBS.
2. The method of claim 1, wherein in step b), the food-borne pathogenic bacteria are listeria monocytogenes, escherichia coli O157: H7, salmonella, staphylococcus aureus, vibrio parahaemolyticus, or pseudomonas aeruginosa.
3. The method for detecting food-borne pathogenic bacteria according to claim 1, wherein in step 1), 0.10 to 0.20g of zinc nitrate hexahydrate and 0.20 to 0.30g of 2-methylimidazole are added to an anhydrous methanol dispersion containing 25 mg Fe 3O4.
4. The method of detecting food-borne pathogenic bacteria of claim 1, wherein the step 1) is repeated 2-4 times to grow the ZIF-8 coat layer by layer on the surface of Fe 3O4.
5. The method of claim 1, wherein in step 1), the Fe 3O4 is obtained by: mixing ethylene glycol with ferric trichloride hexahydrate, sodium acetate and sodium citrate, heating at 180-220 deg.c to react 8-12 h, cooling to room temperature, washing and drying.
6. The method of claim 5, wherein 4-5 g iron trichloride hexahydrate, 7-9g sodium acetate, and 0.5-1.0 g sodium citrate are added to 60-100 mL ethylene glycol.
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