Pt NPs@Mn3O4 composite material, and preparation method and application thereof

Abstract

The application belongs to the technical field of food safety detection, and particularly relates to a PtNPs@Mn3O4 composite material, a preparation method and application thereof. The composite material is obtained by reducing a platinum source into nano platinum PtNPs under the action of a reducing agent and dispersing and loading the PtNPs on Mn3O4 with a nanoflower surface structure, to obtain a PtNPs@Mn3O4 composite material with a hollow nano structure, and the particle size of the PtNPs@Mn3O4 composite material is 100nm-250nm. The application can provide more reactive sites due to the excellent catalytic performance of platinum nanoparticles on H2O2 and the hollow nanoflower structure of Mn3O4, can effectively catalyze the luminescence system of a luminescent reagent Luminol and a co-reactant H2O2 by taking the PtNPs@Mn3O4 as a co-reaction catalyst, can enhance the electrochemiluminescence signal, and thus can improve the sensitivity of a sensor.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food safety detection, and particularly relates to a PtNPs@Mn3O4 composite material and a preparation method and application thereof. BACKGROUND

[0002] Foodborne diseases caused by pathogens can cause fever, diarrhea and even death within 12-72 hours, and have become a focus of global public health concern. Among them, S. typhimurium is one of the most common foodborne pathogenic bacteria. In addition, S. typhimurium can invade the food production chain through various routes, and has a lot of opportunities for reproduction and growth at each link from the farm to the kitchen, causing various health problems. Therefore, early identification and timely diagnosis are crucial to prevent large-scale contamination and spread of S. typhimurium.

[0003] Currently, the main strategies for bacterial detection include microbial culture, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Microbial culture as a gold standard detection method, although accurate but need long at least 24-36 hours, is not suitable for rapid screening or on-site detection. Nucleic acid analysis based on PCR has high sensitivity and specificity, but the operation is complex, and requires professional personnel or expensive equipment. ELISA is widely used due to its high specificity, low cost and operation automation, but its sensitivity is relatively low.

[0004] Electrochemiluminescence (ECL) as a product of the combination of electrochemistry and chemiluminescence has the characteristics of high sensitivity and high efficiency, and can meet the needs of actual sample monitoring, so it is favored by many scholars in the field of food safety analysis and detection. Therefore, a kind of high-efficiency detection method based on electrochemiluminescence sensor is developed for S. typhimurium to prevent foodborne disease outbreaks caused by it and to ensure food safety. SUMMARY

[0005] The purpose of the present application is to meet the needs of instant detection of S. typhimurium and ensure the sensitivity of detection, and to provide a PtNPs@Mn3O4 composite material and a preparation method and application thereof. The PtNPs@Mn3O4 composite material is modified as a sensing substrate, based on a specific nucleic acid aptamer regulation strategy, and then designed into a sensor.

[0006] The present application solves the above technical problems through the following technical solutions.

[0007] The first object of the present application is to provide a PtNPs@Mn3O4 composite material, which is prepared by reducing a platinum source into nano platinum PtNPs and dispersively loading the nano platinum PtNPs on Mn3O4 with a nanoflower surface structure under the action of a reducing agent, so as to obtain a PtNPs@Mn3O4 composite material with a hollow nano structure, wherein the particle size of the PtNPs@Mn3O4 composite material is 100nm-250nm, and the particle size of the nano platinum PtNPs is 2nm-5nm.

[0008] The second object of the present application is to provide a preparation method of the above-mentioned PtNPs@Mn3O4 composite material, which comprises the following steps:

[0009] mixing KMnO4 with oleic acid, and calcining the mixture under an air atmosphere at 80℃-300℃ to obtain Mn3O4;

[0010] dissolving the Mn3O4 in a solvent, sequentially adding a platinum source and a reducing agent, and performing impregnation reduction at room temperature to obtain the PtNPs@Mn3O4 composite material.

[0011] Further, the mass molar ratio of Mn3O4 and PtNPs in the PtNPs@Mn3O4 composite material is 20mg-50mg:1.2x10 -3 mmol-1.6x10 -3 mmol.

[0012] Further, the molar ratio of the reducing agent and the platinum source is 3:0.1-0.3, the platinum source is chloroplatinic acid, the reducing agent is sodium borohydride or ascorbic acid, and the impregnation reduction time is 10min-60min.

[0013] Further, the mass volume ratio of KMnO4 and oleic acid is 0.1g-0.3g:1mL, and the calcination time is 200min-400min.

[0014] The third object of the present application is to provide the use of the above-mentioned PtNPs@Mn3O4 composite material in the preparation of a sensor for detecting Salmonella typhimurium, wherein the sensor is an electrochemiluminescence sensor.

[0015] Further, the sensor comprises a substrate and an electrode layer attached to the substrate, wherein the electrode layer comprises a working electrode layer, and the surface of the working electrode layer is sequentially coated with the PtNPs@Mn3O4 composite material, cDNA and Apt-DA.

[0016] Further, the nucleotide sequence of the cDNA is: 5'-SH-(CH2)6-CTGTCATAATGTCAA-3'; the nucleotide sequence of the Apt-DA is: 5'-COOH-(CH2)6-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG-3'.

[0017] Further, the method for detecting Salmonella typhimurium by the sensor comprises the following steps:

[0018] The sample to be detected is dropped on the surface of the working electrode layer of the sensor, and the reaction is carried out at room temperature for 40min-60min, and the quantitative detection of Salmonella typhimurium is realized by measuring the ECL response.

[0019] Further, the detection range of the sensor is 10 1 CFU / mL-10 5 CFU / mL.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] (1) The PtNCs@Mn3O4 composite material is prepared by high-temperature carbonization and immersion reduction, and due to the excellent catalytic performance of platinum nanoparticles on H2O2 and the hollow nanoflower structure of Mn3O4 which can provide more reaction active sites, the PtNPs@Mn3O4 as a co-reaction catalyst can effectively catalyze the luminescence system of the luminescent reagent Luminol and the co-reaction H2O2, enhance the electrochemiluminescence signal, modify the sensing interface, improve the electron transfer rate on the electrode surface and catalyze the electrochemiluminescence system, thereby improving the sensitivity of the sensor and enhancing the ECL response.

[0022] (2) The PtNPs@Mn3O4 composite material prepared in the present application exhibits stable behavior in an electrochemical environment, and can maintain its chemical and physical properties during a long operation process, thereby ensuring the stability and reliability of the sensor; the specific recognition of the target and the aptamer causes the distance between the electrically active molecules on the sensing interface to change, thereby causing the electrochemiluminescence signal to change significantly; at the same time, the sensor provided in the present application using PtNPs@Mn3O4 as a co-reaction catalyst also exhibits good selectivity and stability, and has wide application value.

[0023] (3) The electrochemiluminescence sensor prepared in the present application is used for the detection of Salmonella typhimurium, and has good stability, high sensitivity and wide linear range, and can realize simple, rapid, high-sensitivity and specific detection. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The schematic diagram of the principle of the electrochemiluminescence sensor provided by the application for detecting S. typhimurium.

[0025] Figure 2 The transmission electron microscope image of Mn3O4 prepared for the embodiment 1 of the application.

[0026] Figure 3 The transmission electron microscope image of PtNPs@Mn3O4 composite material prepared for the embodiment 1 of the application.

[0027] Figure 4 The X-ray photoelectron spectrogram of PtNPs@Mn3O4 composite material prepared for the embodiment 1 of the application.

[0028] Figure 5 The ECL test diagram of the catalytic performance of PtNPs@Mn3O4 composite material prepared for the embodiment 1 of the application.

[0029] Figure 6 The alternating current impedance spectrogram of the sensor construction process of the application.

[0030] Figure 7 The time-current curve diagram of the sensor corresponding to different concentrations of S. typhimurium of the application.

[0031] Figure 8 The linear fitting curve diagram of the logarithmic value and the current of S. typhimurium of different concentrations of the application.

[0032] Figure 9 The selectivity test result diagram of the sensor provided by the application.

[0033] Figure 10 The stability test result diagram of the sensor provided by the application. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.

[0035] It should be noted that the professional terms used in the application are only for the purpose of describing the specific embodiments, and are not intended to limit the protection scope of the application. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the following embodiments of the application can be purchased from the market or prepared by the existing method.

[0036] In one aspect, the present application provides a PtNPs@Mn3O4 composite material, which is prepared by reducing a platinum source into nano platinum (Pt NPs) and dispersively loading the Pt NPs on Mn3O4 with a nanoflower surface structure under the action of a reducing agent, to obtain a PtNPs@Mn3O4 composite material with a hollow nanostructure, wherein the particle size of the PtNPs@Mn3O4 composite material is 100 nm to 250 nm, and the particle size of the nano platinum (Pt NPs) is 2 nm to 5 nm.

[0037] In some specific embodiments, the mass molar ratio of Mn3O4 and Pt NPs in the PtNPs@Mn3O4 composite material is 20 mg to 50 mg: 1.2 x 10 -3 mmol to 1.6 x 10 -3 mmol, and the average particle size of the obtained composite material is about 150 nm. As a preferred scheme of the present application, the composite material is prepared by a reaction ratio of 40 mg: 1.5 x 10 -3 mmol, and the average particle size of the obtained composite material is about 150 nm.

[0038] It should be noted that the PtNPs@Mn3O4 in the present application is in the form of a solid powder, and the source of the Pt NPs is not limited, and an acid or salt solution of platinum can be used as a raw material for preparation. In some specific embodiments, the platinum source is chloroplatinic acid.

[0039] In the present application, the PtNPs@Mn3O4 composite material provided by the present application has the following advantages compared with other sensing substrates: (1) PtNPs@Mn3O4 has excellent catalytic performance, which can effectively catalyze the reaction of luminol (Luminol) and hydrogen peroxide (H2O2), and significantly enhance the electrochemiluminescence signal. This catalytic effect reduces the time required for detection and improves the efficiency of the detection process; (2) PtNPs@Mn3O4 has a hollow nanostructure, which provides a larger surface area and more active sites. The nano-sized platinum particles are uniformly distributed on the Mn3O4 nanoflower, which can significantly improve the electron transfer rate of the electrode interface; (3) PtNPs@Mn3O4 composite material exhibits stable behavior in an electrochemical environment, which can maintain its chemical and physical properties during a long operation process, thereby ensuring the stability and reliability of the sensor. Therefore, the novel co-reaction catalyst PtNPs@Mn3O4 provided by the present application as a sensing substrate can significantly improve the sensitivity of the sensor.

[0040] In another aspect, the present application provides a preparation method of the PtNPs@Mn3O4 composite material, which comprises the following steps:

[0041] Mixing KMnO4 with oleic acid, calcining at 80-300 DEG C for 200-400 min in air atmosphere to obtain Mn3O4;

[0042] Dissolving Mn3O4 in solvent, adding platinum source and reducing agent mixture in sequence, conducting impregnation reduction at room temperature, centrifuging to obtain PtNPs@Mn3O4 composite material.

[0043] The application prepares PtNCs@Mn3O4 composite material by high-temperature carbonization and impregnation reduction, which is used for modifying sensing interface, improving electrode surface electron transfer rate and catalyzing electrochemiluminescence system, and enhancing ECL response.

[0044] In some specific embodiments, the molar ratio of reducing agent and platinum source is 3:0.1-0.3, the platinum source is chloroplatinic acid, the reducing agent is sodium borohydride or ascorbic acid, the platinum source is a 20 mM chloroplatinic acid solution, the reducing agent is a 0.1 M sodium borohydride solution, and the solvent is water, preferably ultrapure water.

[0045] In some specific embodiments, the mass-volume ratio of KMnO4 and oleic acid is 0.1-0.3 g:1 mL.

[0046] In addition, the application provides a use of the above PtNPs@Mn3O4 composite material in preparing a sensor for detecting Salmonella typhimurium, wherein the sensor is an electrochemiluminescence sensor.

[0047] In some specific embodiments, the sensor comprises a substrate and an electrode layer attached to the substrate, wherein the electrode layer comprises a working electrode layer, and the surface of the working electrode layer is coated with PtNPs@Mn3O4 composite material, cDNA and Apt-DA in sequence.

[0048] In some specific embodiments, the nucleotide sequence of the cDNA is 5'-SH-(CH2)6-CTGTCATAATGTCAA-3', and the nucleotide sequence of the Apt-DA is 5'-COOH-(CH2)6-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG-3'.

[0049] In addition, the application also provides a method for quantitatively detecting Salmonella typhimurium, and the sensor is used for detection, and in some specific embodiments, the method for detecting Salmonella typhimurium by using the sensor comprises the following steps:

[0050] The sample to be detected is dropped on the surface of the working electrode layer of the sensor, and reaction is carried out at room temperature for 40-60 min, and the quantitative detection of Salmonella typhimurium is realized by measuring the ECL response. 1 ~10 5 CFU / mL.

[0051] The mechanism for detecting S.typhimurium by using the electrochemiluminescence sensor provided by the application is as shown in Figure 1 The specific mechanism is as follows:

[0052] When S.typhimurium does not exist, Apt-DA is combined with cDNA fixed on the surface of the electrode to form a double-stranded structure through DNA hybridization, and the ECL response is quenched due to the inhibition of the DA molecule on the luminescence system, and a "signal-off" state is presented. When S.typhimurium exists, the DA molecule is separated from the electrode interface based on the specific binding between the target and the aptamer, and the ECL response is significantly increased, and a "signal-on" state is presented. Therefore, the quantitative determination of S.typhimurium can be realized by the difference of the ECL signal, and the reliability of the detection is improved based on the recognition of the specific aptamer.

[0053] The PtNPs@Mn3O4 composite material prepared by the application can show good catalytic performance, and can be used as a sensing substrate of a sensor, which is conducive to improving the detection sensitivity. When the target S.typhimurium exists, S.typhimurium is combined with the aptamer modified with DA, so that DA is separated from the electrode surface, and the electrochemiluminescence signal is changed. Based on the specific recognition between the target and the nucleic acid aptamer and the quenching regulation strategy of dopamine, the quantitative detection of S.typhimurium is realized by comparing the relationship between the change of the electrochemiluminescence signal and the concentration of the target.

[0054] The sensor made of the PtNPs@Mn3O4 composite material has excellent catalytic performance for the electrochemiluminescence system, can effectively enhance the ECL intensity, and shorten the detection time. The PtNPs@Mn3O4 electrochemiluminescence sensor for detecting S.typhimurium constructed by the application has a detection range of 10 1 ~10 5The detection limit of the method is CFU / mL, and the detection limit is low, which has good application prospect in food safety detection.

[0055] The following is further illustrated by specific examples.

[0056] Example 1

[0057] A preparation method of a PtNPs@Mn3O4 composite material, comprising the following steps:

[0058] S1, preparation of Mn3O4: 0.5 g of KMnO4 is dissolved in 250 mL of ultrapure water, and stirred vigorously for 30 min to form a reaction mixture, 5 mL of oleic acid is added to the reaction mixture, and then stirring is continued at 28°C for 5 h to obtain a brown-black precipitate, which is washed with ultrapure water and ethanol in sequence to remove unreacted starting materials, and dried at 80°C for 10 h to obtain precursor particles, which are calcined at 200°C in an air atmosphere for 200 min to obtain monodisperse Mn3O4 nanoparticles;

[0059] S2, preparation of Pt NPs@Mn3O4: a simple immersion chemical reduction method is used for preparation, 100 mg of Mn3O4 sample is dispersed in 30 mL of deionized water, then 0.5 mL of 20 mM concentration of HPtCl4 solution is added and ultrasonic treatment is carried out for 30 min, after ultrasonic treatment, 3 mL of mixed solution containing 0.1 M concentration of NaBH4 and 0.2 M concentration of NaOH is quickly injected into the above solution, while stirring for 10 min, finally, the precipitate is separated by centrifugation and washed with deionized water and ethanol, and vacuum dried at 60°C for 24 h to obtain Pt NPs@Mn3O4.

[0060] Example 2

[0061] A preparation method of a PtNPs@Mn3O4 composite material, comprising the following steps:

[0062] S1, preparation of Mn3O4: 0.5 g of KMnO4 is dissolved in 250 mL of ultrapure water, and stirred vigorously for 30 min to form a reaction mixture, 5 mL of oleic acid is added to the reaction mixture, and then stirring is continued at 28°C for 5 h to obtain a brown-black precipitate, which is washed with ultrapure water and ethanol in sequence to remove unreacted starting materials, and dried at 80°C for 10 h to obtain precursor particles, which are calcined at 200°C in an air atmosphere for 200 min to obtain monodisperse Mn3O4 nanoparticles;

[0063] S2, Preparation of Pt NPs@Mn304: A simple impregnation chemical reduction method was used to prepare, 100 mg of Mn304 sample was dispersed in 30 mL of deionized water, then 0.5 mL of HPtCl4 solution with a concentration of 40 mM was added and ultrasonic treatment was performed for 30 min, after the ultrasonic treatment was completed, 3 mL of mixed solution containing 0.1 M of NaBH4 and 0.2 M of NaOH was quickly injected into the above solution while stirring for 40 min, finally, the precipitate was separated by centrifugation and washed with deionized water and ethanol, and vacuum dried at 60°C for 24 h to obtain Pt NPs@Mn304.

[0064] Example 3

[0065] A method for preparing a PtNPs@Mn304 composite material, comprising the following steps:

[0066] S1, Preparation of Mn304: 1.5 g of KMnO4 was dissolved in 250 mL of ultrapure water and stirred vigorously for 30 min to form a reaction mixture, 5 mL of oleic acid was added to the reaction mixture, then stirring was continued at 28°C for 5 h to obtain a brown-black precipitate, which was washed with ultrapure water and ethanol in sequence to remove unreacted starting materials, and dried at 80°C for 10 h to obtain precursor particles, which were calcined at 200°C in an air atmosphere for 400 min to obtain monodisperse Mn304 nanoparticles;

[0067] S2, Preparation of Pt NPs@Mn304: A simple impregnation chemical reduction method was used to prepare, 100 mg of Mn304 sample was dispersed in 30 mL of deionized water, then 0.5 mL of HPtCl4 solution with a concentration of 40 mM was added and ultrasonic treatment was performed for 30 min, after the ultrasonic treatment was completed, 3 mL of mixed solution containing 0.1 M of NaBH4 and 0.2 M of NaOH was quickly injected into the above solution while stirring for 40 min, finally, the precipitate was separated by centrifugation and washed with deionized water and ethanol, and vacuum dried at 60°C for 24 h to obtain Pt NPs@Mn304.

[0068] The PtNPs@Mn304 composite material prepared in Examples 1-3 has similar structure and effect, and the structure and performance of the PtNPs@Mn304 composite material prepared in Example 1 are described as follows:

[0069] The Mn304 nanoflower and PtNPs@Mn304 composite material prepared in Example 1 were observed by transmission electron microscopy, and the results are shown in Figure 2 and Figure 3 .

[0070] Figure 2 Transmission electron microscope image of Mn304 prepared in Example 1 of the present application. As shown in Figure 2 Mn304 nanoparticles are uniform in size, with a particle size of about 150 nm. There is a clear contrast difference between the inside and outside of the nanoparticles, which can be used to determine that the Mn304 particles are hollow structures, and also exhibit good dispersibility.

[0071] Figure 3 Transmission electron microscope image of PtNPs@Mn304 composite material prepared in Example 1 of the present application. As shown in Figure 3 The particle size of PtNPs is about 2 nm, and the PtNPs are uniformly distributed on the surface of the Mn304 nanoflower without obvious agglomeration. The prepared Mn304 nanoflower structure provides a good support for the Pt nanoparticles, which can effectively improve the overall catalytic performance of the composite material.

[0072] Further, X-ray photoelectron spectroscopy was used to explore whether the Pt NPs@Mn304 composite material was successfully constructed, and the results are shown in Figure 4

[0073] Figure 4 X-ray photoelectron spectrogram of Pt NPs@Mn304 composite material prepared in Example 1 of the present application. As shown in Figure 4 The full spectrum of XPS shows several characteristic peaks: 71.55, 284.85, 531.45, and 641.85 eV, respectively, corresponding to Pt4f, C1s, O1s, and Mn2p, respectively, indicating the presence of Pt, C, O, and Mn, thereby proving the successful synthesis of the PtNPs@Mn304 composite material.

[0074] Figure 5 ECL test chart of the catalytic performance of the PtNPs@Mn304 composite material prepared in Example 1 of the present application. As shown in Figure 5 The catalytic performance of the composite material prepared in Example 1 on the Luminol-H2O2 luminescence system under ECL test conditions was studied. First, the good hollow structure of Mn304 can expose more active sites for the catalysis of H2O2, thereby improving the ECL response. The ECL response of the PtNPs@Mn304 composite material is significantly enhanced relative to the bare electrode, indicating that the PtNPs@Mn304 composite material modified electrode can effectively promote the generation efficiency of hydroxyl radicals, and has good ECL performance in the luminol system. This is consistent with the predicted mechanism and qualitative analysis results.

[0075] Application Example

[0076] Example 1 was prepared from Mn304 and PtNPs at a ratio of 40 mg:1.5 x 10 -3 ​The Pt NPs@Mn3O4 composite material obtained in the preparation of the above-mentioned reaction ratio of 0.5 mmol is used to prepare an electrochemiluminescence sensor, and the preparation method of the sensor is as follows:

[0077] Construction of the electrode:

[0078] Step 1: polish the bare electrode to a mirror surface using 0.3 nm and 0.05 nm Al2O3 polishing powder in sequence, and clean the electrode surface with ultrapure water and dry it;

[0079] Step 2: drop 6 μL of 2.0 mg / mL Pt NPs@Mn3O4 nanoparticle solution on the surface of a glassy carbon electrode (GCE) to form a uniform nanofilm;

[0080] Step 3: then, 6 μL of 1.0 μg / mL cDNA sequence is fixed on the electrode interface through Pt-S bond;

[0081] Step 4: then, 6 μL of 6-mercaptohexanol (MCH) solution is added and incubated for 40 min to block the unreacted active sites;

[0082] Step 5: 6 μL of 1.0 μg / mL Apt-DA is added to the electrode surface on which the cDNA is fixed, and incubated at 37°C for 1 h to form double-stranded DNA;

[0083] Step 6: finally, different concentrations of Salmonella typhimurium solution are modified on the electrode surface, and incubated for 40 min to obtain an electrochemiluminescence sensor for detecting S. typhimurium.

[0084] The detection effect of the electrochemiluminescence sensor prepared by the above-mentioned method on S. typhimurium is tested, and the method is as follows:

[0085] The electrode assembly process is studied by electrochemical impedance spectroscopy (EIS), Figure 6 The electrochemical impedance spectrogram of the sensor construction process of the present application is as shown in Figure 6 The semicircle diameter represents the charge transfer resistance (Ret), which is related to the modification of the electrode surface. As shown in Figure 6 After the deposition of Pt NPs@Mn3O4 (curve b), the charge transfer resistance increases (curve b) because the poor conductivity of Pt NPs@Mn3O4 hinders the electron transfer. The cDNA is modified on the electrode surface through Pt-S bond, and Ret increases (curve c). Then MCH is added to block the non-specific active sites on the sensor interface, and because MCH forms an insulating layer to increase the steric hindrance, Ret further increases (curve d). After the addition of Apt-DA, the non-conductive material will seriously hinder the electron transfer of [Fe(CN)6] 3- / [Fe(CN)6]4- The Ret value of the modified electrode is much larger than that of the unmodified electrode. EIS results show that the electrochemiluminescence sensor is successfully constructed.

[0086] Take 1 mL of S. typhimurium in the late logarithmic phase in a 2 mL sterilized centrifuge tube, centrifuge at 8000 rpm for 5 minutes to collect the bacterial cells. Wash with an equal volume of 2x PBS and serially dilute the bacterial cells by ten times, and determine the bacterial number range of 10 1 CFU / mL ~ 10 5 CFU / mL. The test conditions of the ECL sensor for the ECL response curve of the target with different bacterial numbers are that the signal response of the experiment is measured in 15 mL of PBS (pH = 7.0) solution containing 1 mM luminol and 2 mM H2O2 by measuring the signal response of the experiment and recording the electrochemiluminescence curve under the condition of a potential range of 0 V ~ 0.8 V and a scanning rate of 50 mV / s, and the results are shown in Figure 7 and Figure 8 .

[0087] Figure 7 The time-current curve diagram of the sensor corresponding to different concentrations of S. typhimurium of the present application is shown in Figure 8 The linear fitting curve diagram of the logarithmic value and the current of different concentrations of S. typhimurium of the present application is shown in Figure 7 and Figure 8 , the concentration of S. typhimurium in the range of 10 1 CFU / mL ~ 10 5 CFU / mL shows a good linear correlation, and the ECL signal continuously increases with the increase of the concentration of S. typhimurium. The linear regression equation is ECL = 2279.16 LogC - 736.64, and the correlation coefficient R 2 is 0.997; in the equation, ECL is the change of ECL intensity, C is the target concentration, and the LOD is calculated to be 6 CFU / mL (S / N = 3).

[0088] Figure 9 The selectivity test results of the sensor provided by the present application are shown in Figure 9 From the results in , the results of the four interference strains and the negative control group are similar, because the non-target bacteria cannot be recognized by Apt, and no obvious signal change is generated. Only when the sample contains S. typhimurium, the target bacteria can be recognized by Apt, the Apt-DA falls off from the electrode surface, the ECL signal is restored, and the target sample and the mixed sample both generate obvious ECL signal changes. These phenomena show that Apt can only specifically bind to the target and is not affected by interference, and can well distinguish the mixed strain sample containing S. typhimurium.

[0089] In addition, the ECL sensor was continuously scanned for 10 cycles under the same conditions to test the stability of the sensor, and the results are shown in Figure 10

[0090] Figure 10 The stability test results of the sensor provided by the present application are shown in the figure. Figure 10 As can be seen from the figure, the relative standard deviation of the ECL signal response is 3.30%, which indicates that the sensor has good stability.

[0091] It should be noted that when the numerical range is involved in the present application, both endpoints of each numerical range and any number between the two endpoints can be selected. Since the same steps and examples are used, the preferred embodiments are described in the present application to prevent redundancy. Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to the embodiments once they know the basic creative concept. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0092] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application also intends to include these modifications and variations.​

Claims

1. The application of a Pt NPs@Mn3O4 composite material in the preparation of a sensor for detecting Salmonella typhimurium, characterized in that, The sensor is an electrochemiluminescence sensor; The preparation method of the Pt NPs@Mn3O4 composite material includes the following steps: Preparation of S1 and Mn3O4: 0.5 g of KMnO4 was dissolved in 250 mL of ultrapure water and stirred vigorously for 30 min to form a reaction mixture. 5 mL of oleic acid was added to the reaction mixture, and stirring was continued at 28 °C for 5 h to obtain a brownish-black precipitate. The precipitate was washed with ultrapure water and ethanol in turn to remove unreacted starting materials and dried at 80 °C for 10 h to obtain precursor particles. The precursor particles were calcined in air at 200 °C for 200 min to obtain monodisperse Mn3O4 nanoparticles. Preparation of S2 and Pt NPs@Mn3O4: A simple impregnation chemical reduction method was used. 100 mg of Mn3O4 sample was dispersed in 30 mL of deionized water, and then 0.5 mL of 20 mM HPtCl4 solution was added and sonicated for 30 min. After sonication, 3 mL of a mixed solution containing 0.1 M NaBH4 and 0.2 M NaOH was rapidly injected into the above solution while stirring for 10 min. Finally, the precipitate was separated by centrifugation and washed with deionized water and ethanol. The precipitate was then vacuum dried at 60 °C for 24 h to obtain Pt NPs@Mn3O4. The sensor includes a substrate and an electrode layer attached to the substrate. The electrode layer includes a working electrode layer, on which Pt NPs@Mn3O4 composite material, cDNA, and Apt-DA are sequentially coated.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the cDNA is: 5'-SH-(CH2)6-CTGTCATAATGTCAA-3'; the nucleotide sequence of the Apt-DA is: 5'-COOH-(CH2)6-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG-3'.

3. The application according to claim 1, characterized in that, The method for using the sensor to detect Salmonella typhimurium includes the following steps: The sample to be tested is dropped onto the surface of the working electrode layer of the sensor and reacted at room temperature for 40 to 60 minutes. The detection of Salmonella typhimurium is achieved by measuring the ECL response.

4. The application according to claim 3, characterized in that, The sensor has a detection range of 10. 1 CFU / mL ~10 5 CFU / mL.

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