Nanoprobe for identifying listeria monocytogenes and application thereof

Abstract

The invention discloses a nano probe for identifying listeria monocytogenes and application of the nano probe. The nano probe is composed of an aptamer, HRP and PAPP. Wherein the PAPP is obtained by loading Au, Pd and Pt onto PCN-224 through an in-situ synthesis method; the aptamer is A8: AT CCA, TGG, GGC, GGA, GAT, GAG, GGG, GAG, GGG, TAC, CCG, GTT, GAT and the like; the HRP is embedded horse radish peroxidase. Based on vancomycin and aptamer functionalized multi-metal metal organic framework, the method is used for bacterial detection, especially for fluorescence-colorimetric dual-signal detection and inactivation of Listeria monocytogenes, and the method has the characteristics of simplicity and rapidness in operation, high sensitivity and accuracy and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and in particular to a nanoprobe for identifying Listeria monocytogenes and an application thereof. Background Art

[0002] The presence of pathogenic bacteria in food and water environments and the toxins produced by pathogenic bacteria have posed a huge threat to public health and safety. It is well known that even low concentrations of bacteria can threaten human life, but if the bacteria can be detected and inactivated earlier, it can greatly ensure the safety of food and drinking water, thereby reducing the chance of infection. In recent years, many methods have been developed for detecting bacteria, such as fluorescent sensors, electrochemical sensors, chemiluminescence, colorimetry, etc.

[0003] However, these reported bacterial sensors are mainly based on a single signal sensing mode, which may be affected by the environment, equipment and operation. Therefore, a single signal sensing mode may lack the reliability and accuracy of quantitative measurement. In contrast, the dual signal sensing strategy not only retains the inherent characteristics of each sensor, but also provides a large amount of information from the reaction and verifies the results obtained from different sensing modes. In addition, since the dual signal sensor has two optional detection devices, it can flexibly respond to different detection conditions, so it has a wide range of applications. At the same time, increasing the signal output or sensing channel is conducive to improving the sensitivity of detection.

[0004] Although dual-signal detection methods for bacterial detection are already available in the prior art, these detection methods still have shortcomings: first, these detection methods cannot kill bacteria while detecting, which not only causes live bacteria to have a tendency to form biofilms on sensitive components, thereby affecting the performance of the instrument, but also does not control and kill pathogens at the back end of the detection, which is not conducive to ensuring the safety of the detection environment and may even easily cause secondary infection.

[0005] At the same time, existing technologies have designed functional metal-organic frameworks loaded with different components based on functional metal-organic frameworks, but these functional metal-organic frameworks are often loaded with a single component, which results in low sensitivity during detection and ultimately affects their practical applications. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a nanoprobe for identifying Listeria monocytogenes and its application, so as to solve the problems of the prior art such as the single signal output mode and the inability to kill bacteria during detection, thereby posing safety risks and low sensitivity.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A nanoprobe for identifying Listeria monocytogenes, the nanoprobe is composed of aptamer A8, HRP, and PAPP; wherein PAPP is obtained by loading Au, Pd, and Pt onto PCN-224 by an in situ synthesis method; the sequence of the aptamer A8 is as follows:

[0009] AT CCA TGG GGC GGA GAT GAG GGG GAG GAG GGC GGG TAC CCG GTT GAT;

[0010] HRP is embedded horseradish peroxidase.

[0011] Preferably, the nanoprobe is prepared by the following method:

[0012] Step 1: Add HRP and PAPP into water and mix, and incubate at 30-40°C for 1-2 hours to fix HRP on the surface of PAPP to form a complex PAPPH; wherein the volume ratio of HRP to PAPP is 0.04-0.06;

[0013] Step 2: resuspend the complex PAPPH in a buffer solution, add the aptamer A8 solution, and react at 0-4°C for 30-60 minutes; wherein the concentration of the aptamer A8 in the aptamer A8 solution is 1 μmoL / mL, and the amount added is 1-20 μL;

[0014] Step 3: Centrifuge the product after the reaction in step 2 to obtain the nanoprobe PAPPH-Apt.

[0015] Preferably, PAPP is prepared by the following steps:

[0016] PCN-224 is dissolved in water to obtain a mixture; then, at 35-40° C., HAuCl4 aqueous solution, H2PtCl6 aqueous solution and PdCl4 aqueous solution are sequentially added to the mixture at intervals of 30 minutes to form a reaction system; then, a reducing agent NaBH4 is added to the reaction system, and the reaction is continued by stirring for 2-4 hours, and the product after the reaction is washed to obtain PAPP;

[0017] Among them, the volume ratio of HAuCl4 aqueous solution, H2PtCl6 aqueous solution and PdCl4 aqueous solution is (500-1000):(50-100):1; calculated by mass percentage, the concentration of HAuCl4 in the HAuCl4 aqueous solution is 0.01%, the concentration of H2PtCl6 in the H2PtCl6 aqueous solution is 5%; the concentration of PdCl4 in the PdCl4 aqueous solution is 1 moL / mL; the amount of reducing agent NaBH4 added is 10 mg per 1 mL of the reaction system.

[0018] Preferably, PCN-224 is prepared by the following steps:

[0019] H2TCPP, ZrOCl2·8H2O and benzoic acid are added to DMF, stirred to make them uniformly dispersed, then stirred and reacted at 80-100°C for 4-6 hours, cooled and filtered after the reaction, and the collected precipitate is washed several times, and the residue is removed and dried to obtain PCN-224;

[0020] Wherein, the mass ratio of H2TCPP, ZrOCl2·8H2O and benzoic acid is 1:(2-5):28.

[0021] The present invention provides an application of a nanoprobe for identifying Listeria monocytogenes. The nanoprobe is used to detect Listeria monocytogenes in food or drinking water.

[0022] Preferably, the detection steps are as follows:

[0023] S1: adding the sample to be tested to the detection platform and incubating it at 37°C for 20 to 40 minutes, then washing it with PBS buffer for multiple times, adding the nanoprobe and incubating it at 37°C for 10 to 20 minutes; wherein the amount of the nanoprobe added is 80 to 120 μL;

[0024] S2: Collect the supernatant in the detection platform to read the fluorescence signal, so as to determine the concentration of Listeria monocytogenes in the sample to be tested.

[0025] Preferably, the detection platform is a container with a cell culture plate disposed therein, and vancomycin is fixed on the cell culture plate.

[0026] Preferably, vancomycin is immobilized on the cell culture plate by the following steps:

[0027] (1) dissolving vancomycin in an EDC solution, and then adding an NHS solution and a BSA solution to obtain a mixed solution; wherein the amount of vancomycin added is 10 mg of vancomycin per 850 μL of the mixed solution, the concentration of the EDC solution is 20 mg / mL, the concentration of the NHS solution is 1.0 mg / mL, and the concentration of the BSA solution is 1.0 mg / mL;

[0028] (2) incubating the mixed solution obtained in step (1) at 4° C. for at least 12 h, adding a Tris buffer solution to the mixed solution to terminate the chemical reaction, and obtaining a BSA-Van complex;

[0029] (3) adding carbonate buffer to the BSA-Van complex in step (2) to dilute it to obtain a BSA-Van solution, adding the BSA-Van solution to a 96-well plate so that the BSA-Van solution covers the bottom of the 96-well plate, and standing at 4° C. for at least 12 h;

[0030] (4) The 96-well plate treated in step (3) was washed multiple times with a PBST buffer solution composed of PBS and Tween 20, and then a blocking solution was added to the 96-well plate containing BSA-Van so that the blocking solution covered the 96-well plate, and the reaction was carried out at 37° C. for at least 1 hour, and then the 96-well plate was rinsed with a buffer solution to obtain a 96-well plate fixed with vancomycin; wherein, calculated by mass percentage, the blocking solution was a PBS buffer solution containing BSA, and the concentration of BSA in the fixing solution was 5 wt %.

[0031] Preferably, in step S2, the fluorescence signal is read by colorimetry, specifically:

[0032] 80-100 μL NaAc, 5-15 μL TMB and 5-15 μL H2O2 were added to the detection platform in sequence and incubated at 37°C for 15 min; then 40-60 μL H2SO4 with a concentration of 2 moL / L was added to terminate the reaction; the absorbance value A of the solution after the reaction at 450 nm was collected with an ELISA instrument, and a linear equation was established to calculate the concentration of Listeria monocytogenes in the sample to be tested.

[0033] Preferably, in step S2, the fluorescence signal is read using a fluorescence method, specifically:

[0034] The supernatant was added to a fluorescent ELISA plate, followed by 90-110 μL FAM-DNA, mixed and incubated at 37°C; after the incubation, the fluorescence intensity FL of the solution at 518 nm under 495 nm excitation light was collected, and a linear equation was established to detect the concentration of Listeria monocytogenes in the sample to be tested.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The present invention loads catalytically active Au, Pt, Pd and HRP onto PCN-224, which greatly improves the catalytic activity of PCN-224. At the same time, aptamers are further introduced and applied to the detection of Listeria monocytogenes, which greatly improves the sensitivity of detection.

[0037] 2. The present invention innovatively combines fluorescence and colorimetry, and with the introduction of vancomycin, proposes a fluorescence-colorimetry dual-modal detection and inactivation method for Listeria monocytogenes, which improves the sensitivity, accuracy and safety of Listeria monocytogenes detection, can be successfully used for the detection of Listeria monocytogenes in actual samples, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the fluorescence-colorimetry dual-modality detection and inactivation of Listeria monocytogenes of the present invention; a is a schematic diagram of the preparation process of PAPPH-Apt; b is a schematic diagram of the fluorescence-colorimetry dual-modality detection of Listeria monocytogenes.

[0039] Figure 2 It is a characterization diagram of BSA-Van of the present invention; wherein, a is the UV-visible spectrum of Van, BSA, and BSA-Van; b is the UV / visible spectrum of the MTT experiment and its corresponding solution photograph.

[0040] Figure 3 These are experimental photos of inhibition zones of different samples of the present invention.

[0041] Figure 4 In the figure, a is the TEM image of PCN-224; b is the TEM image of PAPPH; c is the XPS image of PCN-224 and PAPPH-Apt; d is the fine spectrum of PCN-224 in the Zr 3d region; hl figure is the XPS fitting spectrum of Zr 3d, Pd 3d, Au 4f, Pt 4f and O1s in PAPPH-Apt.

[0042] Figure 5 It is the absorbance value of TMB reaction catalyzed by materials with different catalytic activities in the present invention.

[0043] Figure 6 In the equation, a is the relationship between the concentration of Listeria monocytogenes and ΔA 450 a is the linear fitting diagram; b is the absorbance value of the colorimetric quantitative analysis strategy of different detection substances; c is the relationship between Listeria monocytogenes concentration and ΔFL 518 d is the linear fitting diagram of the fluorescence intensity value of the fluorescence quantitative analysis strategy of different detection substances. DETAILED DESCRIPTION

[0044] The present invention will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the present invention belong to the scope of protection of the present invention.

[0045] Unless otherwise indicated in specific cases in the present invention, the numerical ranges listed herein include the upper and lower limits, and all integers and fractions within the range, and are not limited to the specific values ​​listed when defining the range.

[0046] 1. A nanoprobe for identifying Listeria monocytogenes

[0047] The nanoprobe of the present invention is composed of aptamer A8, horseradish peroxidase (HRP), and PAPP; wherein PAPP is obtained by loading Au, Pd, and Pt onto PCN-224 through an in situ synthesis method; the sequence of the aptamer A8 is as follows:

[0048] AT CCA TGG GGC GGA GAT GAG GGG GAG GAG GGC GGG TAC CCG GTT GAT;

[0049] HRP is embedded horseradish peroxidase.

[0050] In the prior art, various functional metal organic frameworks have been developed and used in various fields, including energy storage, drug delivery, bioimaging and chemical sensing. This is attributed to the high structural stability, high aperture ratio, large specific surface area and excellent performance provided by metal atoms or ligands. The prior art often loads single-component nanomaterials on functional metal organic frameworks to make them have better catalytic activity. However, the catalytic activity in this case is limited, which makes the functional metal organic framework loaded with single-component nanomaterials not sensitive when used for biological detection. Therefore, the present invention contemplates screening the functional metal organic framework as a carrier, selecting materials with excellent performance as carriers, based on which the present invention uses PCN-224 as a carrier; at the same time, the present invention also contemplates loading metal materials of multiple components thereon, so that it has a higher active specific surface area and simulated enzyme activity. However, in the process of preparing nanoprobes, the present invention found that different metal ions have different affinities and reactivity, which will cause some metal ions to preferentially occupy favorable positions, resulting in uneven loading; moreover, there will be synergistic effects or competitive effects between different metal ions, and the competitive effect will have an adverse effect on the performance of nanoprobes. Therefore, the present invention screens specific metal ions so that they can be uniformly loaded on the carrier PCN-224, which can not only make the nanoprobe have better catalytic activity (such as Figure 5 As shown), the nanoprobe has a lower detection limit for the target bacteria Listeria monocytogenes (can reach 4 CFU / mL), making the nanoprobe have a higher sensitivity.

[0051] In some embodiments of the present invention, the nanoprobe is prepared by the following method:

[0052] Step 1: Add HRP and PAPP into water and mix, and incubate at 30-40°C for 1-2 hours to fix HRP on the surface of PAPP to form a complex PAPPH; wherein the volume ratio of HRP to PAPP is 0.04-0.06;

[0053] Step 2: resuspend the complex PAPPH in a buffer solution, add the aptamer A8 solution, and react at 0-4°C for 30-60 minutes; wherein the concentration of the aptamer A8 in the aptamer A8 solution is 1 μmoL / mL, and the amount added is 1-20 μL;

[0054] Step 3: Centrifuge the product after the reaction in step 2 to obtain the nanoprobe PAPPH-Apt.

[0055] In some embodiments of the present invention, PAPP is prepared by the following steps:

[0056] PCN-224 is dissolved in water to obtain a mixture; then, at 35-40° C., HAuCl4 aqueous solution, H2PtCl6 aqueous solution and PdCl4 aqueous solution are sequentially added to the mixture at intervals of 30 minutes to form a reaction system; then, a reducing agent NaBH4 is added to the reaction system, and the reaction is continued by stirring for 2-4 hours, and the product after the reaction is washed to obtain PAPP;

[0057] Among them, the volume ratio of HAuCl4 aqueous solution, H2PtCl6 aqueous solution and PdCl4 aqueous solution is (500-1000):(50-100):1; calculated by mass percentage, the concentration of HAuCl4 in the HAuCl4 aqueous solution is 0.01%, the concentration of H2PtCl6 in the H2PtCl6 aqueous solution is 5%; the concentration of PdCl4 in the PdCl4 aqueous solution is 1 moL / mL; the amount of reducing agent NaBH4 added is 10 mg per 1 mL of the reaction system.

[0058] In some embodiments of the present invention, PCN-224 is prepared by the following steps:

[0059] H2TCPP, ZrOCl2·8H2O and benzoic acid are added to DMF, stirred to make them evenly dispersed, then stirred and reacted at 80-100°C for 4-6 hours, cooled and filtered after the reaction, the collected precipitate is washed several times, the residue is removed and dried to obtain PCN-224; wherein the mass ratio of H2TCPP, ZrOCl2·8H2O and benzoic acid is 1:(2-5):28.

[0060] 2. Application of a nanoprobe for identifying Listeria monocytogenes

[0061] The nanoprobe of the present invention is used for detecting Listeria monocytogenes in food or drinking water.

[0062] In some embodiments of the present invention, the detection steps are as follows:

[0063] S1: adding the sample to be tested to the detection platform and incubating it at 37°C for 20 to 40 minutes, then washing it with PBS buffer for multiple times, adding the nanoprobe and incubating it at 37°C for 10 to 20 minutes; wherein the amount of the nanoprobe added is 80 to 120 μL; the detection platform is a container with a cell culture plate inside, and vancomycin is fixed on the cell culture plate.

[0064] S2: Collect the supernatant in the detection platform to read the fluorescence signal, so as to determine the concentration of Listeria monocytogenes in the sample to be tested.

[0065] The present invention found when studying the existing methods for detecting Listeria monocytogenes that these methods often need to sterilize the equipment or materials used after the detection is completed, otherwise there will be certain safety risks. The present invention considers whether the sterilization process can be combined with the detection process, and the Listeria monocytogenes is sterilized while being detected, avoiding the trouble of subsequent sterilization and reducing the safety risks in the detection process. Therefore, the present invention considers fixing the material that can play a sterilizing role on the platform used for detection, but this leads to new problems. Although the sterilizing materials (for example, silver ions, copper ions, titanium dioxide and other materials) fixed on the detection platform can play a sterilizing role, these materials have a poor effect on the fixation of target bacteria, resulting in the nanoprobe being unable to detect Listeria monocytogenes with a lower concentration when the sample to be tested is detected. For this reason, the present invention selects vancomycin, because vancomycin can not only play a sterilizing role, but also play a role in fixing the target bacteria, which enables the nanoprobe of the present invention to further improve its overall sensitivity after combining with the detection platform, so that it can detect Listeria monocytogenes with a lower concentration in the sample to be tested.

[0066] In some embodiments of the present invention, how to fix vancomycin on a cell culture plate is studied. Conventional physical adsorption can fix a certain amount of vancomycin on a cell culture plate, but the stability and durability after fixation are not good. Subsequently, the present invention found that vancomycin can be well fixed on a cell culture plate by BSA, the steps are simple and easy to operate, and the effect is very good. Therefore, in the present invention, vancomycin is fixed on a cell culture plate by the following steps:

[0067] (1) dissolving vancomycin in an EDC solution, and then adding an NHS solution and a BSA solution to obtain a mixed solution; wherein the amount of vancomycin added is 10 mg of vancomycin per 850 μL of the mixed solution, the concentration of the EDC solution is 20 mg / mL, the concentration of the NHS solution is 1.0 mg / mL, and the concentration of the BSA solution is 1.0 mg / mL;

[0068] (2) incubating the mixed solution obtained in step (1) at 4° C. for at least 12 h, adding a Tris buffer solution to the mixed solution to terminate the chemical reaction, and obtaining a BSA-Van complex;

[0069] (3) adding carbonate buffer to the BSA-Van complex in step (2) for dilution to obtain a BSA-Van solution, adding the BSA-Van solution to a container having a cell culture plate so that the BSA-Van solution covers one side of the cell culture plate for detecting Listeria monocytogenes, and standing at 4° C. for at least 12 hours;

[0070] (4) washing the cell culture plate treated in step (3) for multiple times with a PBST buffer solution composed of PBS and Tween 20, then adding a fixing solution into a container having the cell culture plate so that the fixing solution covers one side of the cell culture plate used to detect Listeria monocytogenes, reacting at 37° C. for at least 1 hour, and then rinsing the cell culture plate with a buffer solution to obtain a cell culture plate fixed with vancomycin; wherein the fixing solution is a PBS buffer solution containing BSA, and the concentration of BSA in the fixing solution is 5 wt %, calculated by mass percentage.

[0071] In some embodiments of the present invention, in step S2, the fluorescence signal is read using a colorimetric method, specifically:

[0072] NaAc, TMB and H2O2 were added to the detection platform in sequence and incubated at 37°C for 15 min. Then, 40-60 μL of 2 mol / L H2SO4 was added to terminate the reaction. The absorbance value A1 of the solution after the reaction at 450 nm was collected using an ELISA reader, and a linear equation was established to calculate the concentration of Listeria monocytogenes in the sample to be tested.

[0073] In some embodiments of the present invention, in step S2, the fluorescence signal is read using a fluorescence method, specifically:

[0074] The supernatant was added to a fluorescent ELISA plate, followed by 90-110 μL FAM-DNA, mixed and incubated at 37°C; after the incubation, the fluorescence intensity FL of the solution at 518 nm under 490 nm excitation light was collected, and a linear equation was established to detect the concentration of Listeria monocytogenes in the sample to be tested.

[0075] 3. Embodiment

[0076] 1. Synthesis and characterization of BSA-Van complex

[0077] Example 1

[0078] Van (10 mg) was dissolved in 20 mg / mL EDC solution (300 μL), and then 1.0 mg / mL NHS solution (300 μL) and 1.0 mg / mL BSA solution (250 μL) were added. The mixture was incubated overnight at 4 ° C to achieve effective coupling. Subsequently, in order to terminate the chemical reaction, 50mmoLl / L Tris buffer solution (40 μL; pH 7.2) was added to the mixture. Finally, the absorbance of the solution was measured using a UV / visible spectrometer to evaluate the coupling of BSA and Van.

[0079] First, the UV / visible absorption spectra of BSA-Van complex, BSA alone, and vancomycin were recorded to ensure the synthesis of BSA-Van complex. Figure 2 In a, the absorption maximum of vancomycin at 281 nm can be clearly observed. From the absorption spectrum of the BSA-Van complex, its absorption in the range of 200-250 nm overlaps with that of vancomycin, and there is a further absorption peak at 290 nm. The latter can be attributed to the binding of bovine serum albumin to vancomycin hydrochloride, resulting in a red shift in the absorption peak at 281 nm. This red shift phenomenon is direct evidence of the successful immobilization of vancomycin on the BSA carrier.

[0080] In order to investigate the antibacterial activity of the BSA-Van complex, a standard inhibition zone test was used. Figure 3 It can be seen that pure BSA has no antibacterial activity, while pure vancomycin hydrochloride and BSA-Van both have obvious inhibition zones, which proves that vancomycin hydrochloride still has antibacterial activity after being coupled with BSA. This antibacterial activity also ensures that vancomycin hydrochloride can be firmly anchored to Listeria monocytogenes. Although vancomycin hydrochloride is activated by EDS and NHS and coupled to BSA, BSA-Van still retains the activity of vancomycin hydrochloride antibiotics.

[0081] 2. Immobilization of Vancomycin and Characterization of Antibacterial Activity of 96-well Plates Immobilized with Vancomycin

[0082] Example 2

[0083] First, the BSA-Van complex was appropriately diluted with 0.2moL pH 9.6 carbonate buffer to prepare the working solution required for the experiment. Then, the diluted BSA-Van solution was added to a 96-well plate and placed at 4°C overnight to ensure that the complex was evenly adsorbed in the wells. The well plate was then washed with a buffer (PBST) consisting of 10mmoL PBS (pH7.4) and 0.5% (v / v) Tween 20. The washing step was repeated three times to completely remove the unadsorbed complex. Then, a fixative (PBS solution containing 5% BSA) was added to the 96-well plate. The fixation process was reacted at 37°C for 1h to reduce nonspecific binding. Before performing subsequent experiments, the well plate was rinsed three times again with PBS to remove the BSA in the fixative.

[0084] Listeria monocytogenes was incubated with the BSA-Van coated well plate. After the incubation was completed, the 96-well plate was washed. At the same time, PBS was selected as the control group. Then, the color of the solution was observed and its absorbance value was measured using the MTT kit. The results showed ( Figure 2 b), the solution with Listeria monocytogenes added was obviously purple, while the control group was basically colorless. The above phenomenon further shows that the 96-well plate fixed with BSA-Van has bactericidal ability against Listeria monocytogenes.

[0085] 3. Preparation of PCN-224

[0086] Example 3

[0087] H2TCPP (10 mg), ZrOCl2·8H2O (30 mg) and benzoic acid (280 mg) were accurately weighed and mixed in a clean glass sample bottle. DMF (10 mL) was then added and the mixture was magnetically stirred to ensure that the components were evenly dispersed. The glass bottle was then placed on a hot plate stirrer and the reaction mixture was stirred at 300 rpm for 5 h at 90 °C to promote the chemical reaction. After the reaction was completed, the mixture was allowed to cool naturally to room temperature. Subsequently, the mixture was spun in a high-speed centrifuge (15000 rpm, 30 min) to collect the prepared porous organic metal framework material PCN-224. The collected precipitate was rinsed three times with DMF to remove possible residual reaction byproducts and unreacted raw materials. Finally, the clean precipitate was placed in a drying oven to remove the solvent to obtain solid PCN-224 for subsequent experiments.

[0088] 4. Preparation of PCN-224@AuPtPd(PAPP)

[0089] Example 4

[0090] The porous organic metal framework material PAPP doped with multi-metal nanoparticles is assembled by an in-situ synthesis method. First, the PCN-224 (10 mg) prepared in Example 4 was accurately weighed and evenly suspended in ultrapure water (20 mL). At 37 ° C and 200 rpm / min, 0.01% HAuCl4 aqueous solution (1 mL), 5% H2PtCl6 aqueous solution (100 μL) and 1M PdCl4 aqueous solution (2 μL) were added to the PCN-224 suspension at intervals of 30 min. Subsequently, 10 mg / mL of reducing agent NaBH4 solution (1 mL) was added to the reaction system, and stirring was continued for 3 hours under the set conditions to promote the in-situ generation of Au, Pt and Pd nanoparticles on the surface of PCN-224. This step ensures the uniform dispersion and stable fixation of the nanoparticles. Finally, the obtained PAPP was centrifuged and washed three times with ultrapure water to remove possible residual reactants and unreacted precursors, thereby ensuring the purity of the material. The washed PAPP was resuspended in ultrapure water for subsequent experiments and applications.

[0091] 5. Preparation and characterization of nanoprobe PCN-224@AuPtPd@HRP@Aptamer (PAPPH-Apt)

[0092] Example 5

[0093] First, 1 mg / mL HRP solution (50 μL) was co-cultured with PAPP at 37°C for 1 hour to form a complex PAPPH. Then, the PAPPH precipitate was separated by centrifugation. The PAPPH precipitate was then resuspended in a buffer solution, and an appropriate amount of PAPPH solution was taken and 10 μL of 1 μmol / mL aptamer A8 solution was added thereto. The mixed reaction system was reacted at 4°C for 30 minutes to achieve specific binding of the aptamer to PAPPH. Then, the PAPPH-Apt complex was separated by centrifugation at 14,000 rpm for 15 minutes. The supernatant was carefully removed and the PAPPH-Apt complex was washed three times with phosphate buffered saline (PBS) to remove unbound aptamers and other possible impurities.

[0094] TEM( Figure 4 a) shows that PCN-224 nanoparticles are spherical, well dispersed, and have a diameter of about 187 nm. TEM images of Pt NPs, Au NPs, Pd NPs, and HRP coated on the surface of PCN-224 by in situ synthesis method are shown in Figure 1. Figure 4 b, the diameter is about 226nm. XPS was used to explore the surface chemical state and structural details of PCN-224 and PAPPH-Apt. Figure 4c shows the full spectra of the two samples. Compared with PCN-224, the scanning spectrum of PAPPH-Apt shows the presence of Pd 3d, Pt 4f, and Au3d, indicating that Pd, Pt, and Au have successfully functionalized PCN-224. In addition, the XPS fitting spectrum shows that Au 4f has a peak at 83.4 and 87.2 eV ( Figure 4 g), Pd3d at 338.1 and 333.1 eV ( Figure 4 f), Pt 3d at 70.9 and 74.2 eV ( Figure 4 h). These XPS results suggest an excellent in-situ reduction reaction. The O1s energy level spectrum was subjected to curve fitting analysis, and four peaks were observed at 533.1, 532.4, 531.7, and 530.8 eV ( Figure 4 i), where the peak of the Zr-OP bond is located at 531.7 eV. This is consistent with the Zr 3d peaks of PCN-224 at 182.4 eV and 184.82 eV ( Figure 4 d), PAPPH-Apt fitted two new characteristic peaks at 182.8eV and 185.1eV ( Figure 4 e), which is due to the coordination of Aptamer with Zr on PCN-224 to form Zr-OP.

[0095] 6. Detection of Listeria monocytogenes

[0096] Example 6

[0097] First, different concentrations of Listeria monocytogenes were added to the Van-BSA-coated 96-well plate and incubated at 37°C. Then, the plate was washed three times with PBS, and the PAPPH-Apt nanoprobe prepared in Example 5 was added and incubated at 37°C. Finally, the supernatant was collected for reading of the fluorescence signal. At the same time, the 96-well plate was washed with PBS buffer for detection of UV-Vis signals.

[0098] Colorimetric method: NaAc, TMB, and H2O2 were added to a 96-well plate in sequence and incubated at 37°C for 15 minutes. At this time, PAPPH-Apt fixed on the 96-well plate can catalyze H2O2 to oxidize the chromogenic substrate TMB to produce ox TMB. Then, 2 mol H2SO4 was added to terminate the reaction. Finally, the absorbance value (A) of the solution at 450 nm was collected using an ELISA reader and expressed as ΔA. 450 (A-A0, A0 is the absorbance value measured by this method when Listeria monocytogenes is not present) as the ordinate and the logarithm of the bacterial concentration as the abscissa to establish a linear equation. Under the optimal experimental conditions, the fluorescence colorimetry method was used to detect Listeria monocytogenes. The absorbance change (ΔA 450 , A-A0) and the logarithm of Listeria monocytogenes concentration are as follows Figure 6 a. This relationship is 8×10 2 CFU / mL-8×10 7 The linear relationship is in the range of CFU / mL. The linear fit of the calibration is Y=0.12392X~0.17856, and the correlation coefficient R 2 =0.99175. The limit of detection (LOD) value was calculated as 3S / M (S is the standard deviation of the blank group, M is the slope of the linear equation), and the LOD was 11 CFU / mL.

[0099] Fluorescence method: Add the collected supernatant to a fluorescence ELISA plate. Then, add 500mmoL FAM-DNA, mix well, and incubate at 37°C. After the incubation, collect the fluorescence intensity (FL) of the solution at 518nm under 495nm excitation light, and calculate it as ΔFL 518 The linear equation was established by using the logarithm of bacterial concentration as the ordinate (FL-FL0, FL0 is the fluorescence intensity measured by this method when there is no Listeria monocytogenes). 1 CFU / mL-8×10 7 CFU / mL). As the concentration of Listeria monocytogenes increases, the fluorescence intensity increases. Figure 6 c is the relationship between the change in FAM-DNA fluorescence intensity and the logarithm of Listeria monocytogenes concentration. 518 ,FL-FL0, where FL0 is the fluorescence intensity at 518 nm in the absence of Listeria monocytogenes) and the logarithm of the concentration of Listeria monocytogenes in the range of 8×10 1 CFU / mL-8×10 7 The linear correlation is in the range of CFU / mL. The linear fit of the calibration is Y=6584.037722x+4050.04178, and the correlation coefficient is R 2 =0.99231, LOD is 4 CFU / mL.

[0100] 7. Specificity analysis

[0101] Example 7

[0102] In order to evaluate the recognition ability of the present invention for non-monocytogenes Listeria, the present invention uses different concentrations of Salmonella, Escherichia coli and Staphylococcus aureus for verification. According to the experimental steps of Example 6, the fluorescence and absorbance values ​​of these non-target substances are detected ( Figure 6 b,6d).

[0103] The results showed that only the presence of Listeria monocytogenes could induce the expected signal response, while the signal responses of other samples and blank samples were almost negligible, which verified that the method of the present invention had good selectivity.

[0104] 8. Actual sample analysis

[0105] Example 8

[0106] In order to evaluate the detection effect of the method described in the present invention on actual samples, milk, chicken and environmental water samples were processed according to the following method. Chicken: Weigh 25g of chicken sample and irradiate it under ultraviolet light for 30min; then mix a portion of it (5g) with sterilized PBS (5mL) and filter. Aspirate the filtrate and mix it with different concentrations of Listeria monocytogenes. Milk: Use a measuring cylinder to measure 25mL of milk purchased from the supermarket, add PBS (225mL), and homogenize the mixture for 3min. Then add different concentrations of Listeria monocytogenes solution. Pond water: Add different concentrations of Listeria solution to pond water. Finally, the samples prepared above were tested using the steps of Example 6, and the recovery rates of different samples were calculated.

[0107] The accuracy of the method of the present invention was investigated by performing a spike recovery test on negative samples. The standard solution was diluted into six concentration levels and added to actual samples (milk, chicken, pool water). Each addition level was repeated 3 times. The sample was pre-treated according to the above steps, and the detection and analysis were performed according to the steps of Example 6. The standard curve was substituted to calculate the content of Listeria monocytogenes in the sample, and the spike recovery rate and coefficient of variation were calculated. The results are shown in Table 1. The range of the spike recovery rate of the fluorescent method Listeria monocytogenes sample was 90.56-98.76%, and the coefficient of variation was 0.61-11.29%. The range of the spike recovery rate of the colorimetric method Listeria monocytogenes sample was 90.56-100%, and the coefficient of variation was 1.37-9.23%. It shows that the method of the present invention has good repeatability and stability.

[0108] Table 1 Recovery experiment and coefficient of variation of Listeria monocytogenes in actual samples (n=3)

[0109]

[0110]

[0111] As can be seen from Table 1, regardless of whether the colorimetric method or the fluorescence method is used, the nanoprobe and method of the present invention show a high recovery rate for the detection of Listeria monocytogenes in different matrices, indicating that the present invention has good sensitivity and accuracy; at the same time, it also has good reproducibility and wide adaptability, especially in three different matrices of chicken, pond water and milk, it shows good detection effect, all of which indicate that the present invention has good application prospects.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the technical solution. Those skilled in the art should understand that those modifications or equivalent substitutions of the technical solution of the present invention that do not depart from the purpose and scope of the technical solution should be included in the scope of the claims of the present invention.

Claims

1. A nanoprobe for identifying Listeria monocytogenes, characterized in that: The nanoprobe is composed of aptamer A8, HRP, and PAPP; wherein PAPP is obtained by loading Au, Pd, and Pt onto PCN-224 through an in-situ synthesis method; the sequence of the aptamer A8 is as follows: AT CCA TGG GGC GGA GAT GAG GGG GAG GAG GGC GGG TAC CCG GTT GAT; HRP is embedded horseradish peroxidase.

2. The nanoprobe according to claim 1, characterized in that: The nanoprobe is prepared by the following method: Step 1: Add HRP and PAPP into water and mix, and incubate at 30-40°C for 1-2 hours to embed HRP in the pores of PAPP to form a complex PAPPH; wherein the volume ratio of HRP to PAPP is 0.04-0.06; Step 2: resuspend the complex PAPPH in a buffer solution, add the aptamer A8 solution, and react at 4°C for 20 to 40 minutes; wherein the concentration of the aptamer A8 in the aptamer A8 solution is 1 μmoL / mL, and the amount added is 1 to 20 μL; Step 3: Centrifuge the product after the reaction in step 2 to obtain the nanoprobe PAPPH-Apt.

3. The nanoprobe according to claim 2, characterized in that: PAPP was prepared by the following steps: PCN-224 is dissolved in water to obtain a mixture; then, at 35-40° C., HAuCl4 aqueous solution, H2PtCl6 aqueous solution and PdCl4 aqueous solution are sequentially added to the mixture at intervals of 30 minutes to form a reaction system; then, a reducing agent NaBH4 is added to the reaction system, and the reaction is continued by stirring for 2-4 hours, and the product after the reaction is washed to obtain PAPP; Among them, the volume ratio of HAuCl4 aqueous solution, H2PtCl6 aqueous solution and PdCl4 aqueous solution is (500-1000):(50-100):1; calculated by mass percentage, the concentration of HAuCl4 in the HAuCl4 aqueous solution is 0.01%, the concentration of H2PtCl6 in the H2PtCl6 aqueous solution is 5%; the concentration of PdCl4 in the PdCl4 aqueous solution is 1 moL / mL; the amount of reducing agent NaBH4 added is 10 mg per 1 mL of the reaction system.

4. The nanoprobe according to claim 3, characterized in that: PCN-224 was prepared by the following steps: H2TCPP, ZrOCl2·8H2O and benzoic acid are added to DMF, stirred to make them uniformly dispersed, then stirred and reacted at 80-100°C for 4-6 hours, cooled and filtered after the reaction, and the collected precipitate is washed several times, and the residue is removed and dried to obtain PCN-224; Wherein, the mass ratio of H2TCPP, ZrOCl2·8H2O and benzoic acid is 1:(2-5):

28.

5. An application of a nanoprobe for identifying Listeria monocytogenes, characterized in that: The nanoprobe according to any one of claims 1 to 4 is used to detect Listeria monocytogenes in food or drinking water.

6. The use according to claim 5, characterized in that: The detection steps are as follows: S1: adding the sample to be tested to the detection platform and incubating it at 37°C for 20 to 40 minutes, then washing it with PBS buffer for multiple times, adding the nanoprobe and incubating it at 37°C for 10 to 20 minutes; wherein the amount of the nanoprobe added is 80 to 120 μL; S2: Collect the supernatant in the detection platform to read the fluorescence signal, so as to determine the concentration of Listeria monocytogenes in the sample to be tested.

7. The use according to claim 6, characterized in that: The detection platform is a container with a cell culture plate inside, and vancomycin is fixed on the cell culture plate.

8. The use according to claim 7, characterized in that: Vancomycin is immobilized on the cell culture plate by the following steps: (1) dissolving vancomycin in an EDC solution, and then adding an NHS solution and a BSA solution to obtain a mixed solution; wherein the amount of vancomycin added is 10 mg of vancomycin per 850 μL of the mixed solution, the concentration of the EDC solution is 20 mg / mL, the concentration of the NHS solution is 1.0 mg / mL, and the concentration of the BSA solution is 1.0 mg / mL; (2) incubating the mixed solution obtained in step (1) at 4° C. for at least 12 h, adding a Tris buffer solution to the mixed solution to terminate the chemical reaction, and obtaining a BSA-Van complex; (3) adding carbonate buffer to the BSA-Van complex in step (2) to dilute it to obtain a BSA-Van solution, adding the BSA-Van solution to a 96-well plate so that the BSA-Van solution covers the bottom of the 96-well plate, and standing at 4° C. for at least 12 h; (4) The 96-well plate treated in step (3) was washed multiple times with a PBST buffer solution composed of PBS and Tween 20, and then a blocking solution was added to the 96-well plate containing BSA-Van so that the blocking solution covered the 96-well plate, and the reaction was carried out at 37° C. for at least 1 hour, and then the 96-well plate was rinsed with a buffer solution to obtain a 96-well plate fixed with vancomycin; wherein, calculated by mass percentage, the blocking solution was a PBS buffer solution containing BSA, and the concentration of BSA in the fixing solution was 5 wt %.

9. The use according to claim 6, characterized in that: In step S2, the fluorescence signal is read using a colorimetric method, specifically: NaAc, TMB and H2O2 were added to the detection platform in sequence and incubated at 37°C for 15 min. Then, 40-60 μL of H2SO4 at a concentration of 2 moL / L was added to terminate the reaction. The absorbance value A of the solution after the reaction at 450 nm was collected using an ELISA reader, and a linear equation was established to calculate the concentration of Listeria monocytogenes in the sample to be tested.

10. The use according to claim 6, characterized in that: In step S2, the fluorescence signal is read using the fluorescence method, specifically: The supernatant was added to the fluorescence ELISA plate, followed by 100 μL FAM-DNA, mixed and incubated at 37°C; after the incubation, the fluorescence intensity FL of the solution at 518 nm under 495 nm excitation light was collected, and a linear equation was established to detect the concentration of Listeria monocytogenes in the sample to be tested.

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