Escherichia coli detection reagent, detection method and kit based on Raman enhanced spectroscopy

Abstract

The present invention discloses an Escherichia coli detection reagent, a detection method and a kit based on Raman enhanced spectroscopy. The detection reagent is prepared by the following method: S1, preparing Fe3O4 magnetic microspheres; S2, preparing silica-coated magnetic microspheres; S3, grafting copper-doped reduced carbon dots on the silica-coated magnetic microspheres; S4, in-situ depositing gold nanoparticles to prepare gold nanoparticle-modified magnetic microspheres; S5, grafting Escherichia coli aptamers on the gold nanoparticle-modified magnetic microspheres to obtain the Escherichia coli detection reagent. The present invention detects Escherichia coli by combining the prepared detection reagent with Raman detection means. The detection reagent can realize a significant enhancement of Raman signals. The detection method based on the detection reagent has the advantages of high sensitivity, short time consumption, simple operation, etc., and can realize on-site detection, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to an Escherichia coli detection reagent, a detection method and a kit based on Raman enhanced spectroscopy. Background Art

[0002] Pathogenic microbial contamination, especially foodborne pathogenic bacteria contamination, is a public health issue of worldwide concern, seriously affecting the health and life safety of the people. Escherichia coli is an important foodborne pathogen that can cause serious foodborne diseases. It is of great significance to develop a rapid and sensitive new foodborne pathogen detection method using Escherichia coli as a model.

[0003] The detection methods of Escherichia coli mainly include conventional culture detection, molecular biology detection, immunological detection, etc. For example, CN118362721A discloses a rapid detection method of Escherichia coli O157:H7 based on aptamers, CN114563568A discloses an Escherichia coli-Staphylococcus aureus biosensor and its application, and CN114836360B discloses an Escherichia coli genetic engineering bacterium for detecting the adaptive cost of drug-resistant genes and its construction method.

[0004] Conventional culture detection technology has simple requirements, but first the pathogenic bacteria must be cultured, a process that takes at least 48-72 hours to complete, and the detection is time-consuming and laborious. Molecular biology detection often extracts E. coli DNA or RNA, uses PCR technology to specifically amplify the extracted nucleic acid, and then detects E. coli by detecting the nucleic acid. This method has high detection sensitivity and strong specificity, but it cannot achieve on-site analysis and detection, and the operation is cumbersome and time-consuming. Although immunodetection of E. coli has strong detection specificity, its detection sensitivity is low.

[0005] Raman spectroscopy has been applied to the detection of Escherichia coli, but conventional Raman detection usually encounters problems such as Raman signals affecting detection sensitivity and accuracy.

[0006] Therefore, it is necessary to improve the existing technology to provide a more reliable solution. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide an Escherichia coli detection reagent, detection method and kit based on Raman enhanced spectroscopy in view of the deficiencies in the above-mentioned prior art.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is: In the first aspect of the present invention, there is provided an Escherichia coli detection reagent based on Raman enhanced spectroscopy, which is prepared by the following method:

[0009] S1. Preparation of Fe3O4 magnetic microspheres:

[0010] S2, preparing silica-coated magnetic microspheres;

[0011] S3, grafting copper-doped reduced carbon dots onto silica-coated magnetic microspheres;

[0012] S4, in situ deposition of gold nanoparticles to prepare gold nanoparticle-modified magnetic microspheres;

[0013] S5, grafting the E. coli aptamer onto the magnetic microspheres modified with gold nanoparticles to obtain the E. coli detection reagent;

[0014] During the detection, the detection reagent is mixed with the sample to be tested and incubated, and then Raman detection is performed. According to the presence and intensity of the Raman characteristic peak corresponding to Escherichia coli, qualitative and quantitative detection of Escherichia coli in the sample to be tested is achieved.

[0015] Preferably, step S1 specifically comprises:

[0016] Take 5.0-21.6g of ferric chloride hexahydrate and 6-24g of ferric chloride tetrahydrate, add them into 200-500mL of deionized water, add 6.5-18g of polyethylene glycol, ultrasonically disperse for 15-60min, then add 50-200mL of 2-6mol / L sodium hydroxide solution, stir and react at 60℃ under nitrogen protection for 0.5-3h, separate the magnetic product with a magnet, wash with deionized water until neutral, and vacuum dry at 60-80℃ to constant weight to obtain Fe3O4 magnetic microspheres.

[0017] Preferably, step S2 specifically comprises:

[0018] S2-1, take 1-4g Fe3O4 magnetic microspheres and add them to 100-400mL ethanol, ultrasonically disperse for 5-30min, then add 4-16mL ammonia water with a concentration of 10-20wt%, then add 1.75-7g vinyltriethoxysilane, stir and reflux at 70-80°C for 4-16h, separate by magnetic absorption, wash the magnetic product with ethanol, and obtain pretreated magnetic microspheres;

[0019] S2-2, take 1-4g of pretreated magnetic microspheres and add them to a mixed solution consisting of 75-300mL of ethanol and 50-200mL of deionized water, ultrasonically disperse for 5-30min, then add 3.5-13mL of 10-20wt% ammonia water, and then add 1.1-4.4ml of tetraethyl silicate dropwise under stirring, stir and react at 30-45℃ and 200-800rpm for 2.5-10h, separate by magnetic absorption, wash the magnetic product with ethanol, and obtain silica-coated magnetic microspheres: SiO2@Fe3O4.

[0020] Preferably, step S3 specifically comprises:

[0021] S3-1, take 1g of silica-coated magnetic microspheres and 0.6g of copper acetate, add them into 100mL of deionized water, and disperse them by ultrasonic for 20min to obtain a mixed solution A;

[0022] S3-2, add 960 mg of citric acid, 316 mg of pyridine, 264 mg of vitamin C and 105 mg of cysteine ​​to a solution consisting of 80 mL of deionized water and 60 mL of ethanol, and disperse by ultrasonic for 10 min to obtain a mixed solution B;

[0023] S3-3. Add mixed solution B to mixed solution A, ultrasonically disperse for 45 minutes, transfer the obtained precursor solution to a polytetrafluoroethylene-lined reactor, react at 190°C for 8 hours, cool to room temperature after the reaction, centrifuge and filter, wash the solid product with deionized water, and vacuum dry at 75°C for 12 hours to obtain grafted modified magnetic microspheres: CDs-Cu@SiO2@Fe3O4.

[0024] Preferably, step S4 is specifically:

[0025] Take 0.5-2g of grafted modified magnetic microspheres and add them into 50-200mL of deionized water, ultrasonically disperse for 15-60min to obtain a microsphere dispersion liquid; add 5-20mL of 0.075g / mL HAuCl4 solution to the microsphere dispersion liquid, stir and react at 300-800rpm and 60-80℃ for 1-4h, filter, wash the solid product with ethanol, and vacuum dry at 50-70℃ for 4-12h to obtain gold nanoparticle-modified magnetic microspheres: AuNPS-CDs-Cu@SiO2@Fe3O4.

[0026] Preferably, step S5 is specifically:

[0027] S5-1, adding 0.1 g of magnetic microspheres modified with gold nanoparticles into 20-80 mL of deionized water, and ultrasonically dispersing for 10-30 min to obtain a carrier solution;

[0028] S5-2. Take 10-40 μL of 12-50 μM E. coli aptamer solution and 50-200 μL of carrier solution and add them to 4-16 mL of 2-10 mM PBS solution with a pH of 7.0. Ultrasonic dispersion is performed for 5-20 minutes. The solution is incubated at room temperature for 30-90 minutes. The solution is separated by magnetic absorption. The magnetic product is washed with deionized water and freeze-dried to obtain the E. coli detection reagent: APT-AuNPS-CDs-Cu@SiO2@Fe3O4.

[0029] Preferably, the 5-end of the E. coli aptamer is modified with a thiol group, and the specific sequence is shown in SEQ ID NO: 1:

[0030] 5'-SH-AAAAAATCCGTCACACCTGCTCTATCAAATGTGCAGATATCAAGACGATTTGTACAAGATGG-3'.

[0031] A second aspect of the present invention provides a method for detecting Escherichia coli based on Raman enhanced spectroscopy, comprising the following steps:

[0032] The detection reagent as described above is prepared into a dispersion with a concentration of 0.1-5 mg / mL with deionized water, and then mixed with the sample to be tested, incubated at room temperature for 5-15 minutes, the magnetic substance is separated with a magnet, washed with deionized water, and then re-dispersed in deionized water to obtain a test sample, and the test sample is aspirated and dripped on a clean silicon wafer for Raman detection; qualitative and quantitative analysis of Escherichia coli is performed based on the obtained Raman spectrum;

[0033] The qualitative analysis method is: when there is a Raman characteristic peak corresponding to the target fungus in the Raman spectrum, it is judged that the sample to be tested contains Escherichia coli;

[0034] The quantitative analysis method is: according to the intensity of the Raman characteristic peak corresponding to Escherichia coli, the concentration of Escherichia coli in the sample to be tested is calculated in combination with a pre-constructed standard curve characterizing the relationship between the intensity of the Raman characteristic peak and the concentration of Escherichia coli.

[0035] Preferably, the Raman characteristic peak positions are selected from 718, 965, 1138, 1266, 1455 and 1585 cm -1 at least one of .

[0036] The detection reagent prepared by the present invention is used for Escherichia coli detection in combination with Raman detection means. The detection reagent can achieve a significant enhancement of the Raman signal. The detection method based on the detection reagent has the advantages of high sensitivity, short time consumption, simple operation, etc., and can realize on-site detection, and has broad application prospects.

[0037] The principle of the detection reagent is described in detail below in conjunction with the preparation process thereof to facilitate understanding of the present invention.

[0038] The invention firstly prepares Fe3O4 magnetic microspheres by a coprecipitation method, and then in-situ coats the surface of the Fe3O4 magnetic microspheres with nano-silicon dioxide. The coating of nano-silicon dioxide can form a barrier protection for the magnetic microspheres on the one hand, and can also prevent the Fe3O4 magnetic microspheres from causing oxidation to the synthesis of nano-gold when nano-gold is subsequently deposited on the other hand.

[0039] Then, the reductive carbon dots are grafted in situ on the nano-silica. First, a large amount of copper ions are bound to the surface of the nano-silica through the action of copper ions and the silanol groups on the nano-silica. Then, the affinity of copper ions for the carbon dot raw materials is used to synthesize carbon dots in situ on the surface of the nano-silica by hydrothermal method. In this process, copper ions play an intermediate bridging role and are subsequently converted into nano-particle-level elemental copper and copper oxide. The carbon dots not only have abundant functional groups such as carboxyl, hydroxyl, amino, and thiol groups on the surface, but also inherit the reducibility of pyridine and L-ascorbic acid, showing excellent antioxidant properties.

[0040] Then, the carboxyl, hydroxyl, amino and thiol groups on the surface of the carbon dots were combined with gold ions through coordination, electrostatic adsorption and other effects, and then with the help of the reducing property of the carbon dots, gold nanoparticles were deposited in situ on the surface of the carbon dots. Finally, the Escherichia coli aptamer modified with thiol groups was combined with gold nanoparticles to prepare the Escherichia coli detection reagent: APT-AuNPS-CDs-Cu@SiO2@Fe3O4.

[0041] During detection, the E. coli aptamer can specifically bind to E. coli, and after magnetic separation, it can be enriched for Raman detection, while AuNPS-CDs-Cu@SiO2@Fe3O4 can significantly enhance the Raman signal during Raman detection, thereby achieving high-sensitivity detection.

[0042] In AuNPS-CDs-Cu@SiO2@Fe3O4, Fe3O4 is used as the basic carrier and gives the whole system magnetic properties; carbon dots can achieve in-situ synthesis of gold nanoparticles by providing reducing ability without the need to add additional reducing agents, and can still provide antioxidant properties during storage and detection to prevent the system from being oxidized; on the other hand, the reducing properties of carbon dots can eliminate solvent oxygen and reduce carbonization induction during detection, thereby reducing interference with Raman enhanced signals (Chu Youping, Chen Shuo, Zheng Jufang, et al. Stannous chloride improves carbonization phenomenon of pyridine surface enhanced Raman spectroscopy [J]. 2008.). Furthermore, the nano-size effect of carbon dots, in combination with gold nanoparticles, can enhance the plasma resonance effect generated by gold nanoparticles, thereby forming a stronger Raman signal. Gold nanoparticles are in-situ grafted onto the surface of spherical carbon dots, and the carbon dots are evenly grafted onto the surface of spherical coated silica. Such a structural system can gradually increase the active surface area, allowing gold nanoparticles to be more evenly and stably dispersed, forming more "hot spots" and further improving the Raman enhancement effect.

[0043] The third aspect of the present invention provides an Escherichia coli detection kit, characterized in that it comprises the detection reagent as described above.

[0044] The beneficial effects of the present invention are:

[0045] The present invention provides an Escherichia coli detection reagent, a detection method and a kit based on Raman enhanced spectroscopy. The detection reagent prepared by the present invention is used for Escherichia coli detection in combination with Raman detection means. The detection reagent can realize a significant enhancement of Raman signals. The detection method based on the detection reagent has the advantages of high sensitivity, short time consumption, simple operation, etc., and can realize on-site detection, and has broad application prospects.

[0046] When the detection reagent of the present invention: APT-AuNPS-CDs-Cu@SiO2@Fe3O4 is used for detection, the Escherichia coli aptamer can specifically bind to Escherichia coli, and enrichment can be achieved after magnetic separation for Raman detection, while AuNPS-CDs-Cu@SiO2@Fe3O4 can greatly enhance the Raman signal during Raman detection, thereby achieving high-sensitivity detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is a flow chart of the preparation of the Escherichia coli detection reagent of the present invention;

[0048] Figure 2 The infrared absorption spectrum of the magnetic microspheres (AuNPS-CDs-Cu@SiO2@Fe3O4) deposited with gold nanoparticles prepared in Example 1;

[0049] Figure 3 The reducibility test results of the magnetic microspheres (AuNPS-CDs-Cu@SiO2@Fe3O4) deposited with gold nanoparticles prepared in Example 1;

[0050] Figure 4 The application examples and comparative examples of the present invention and the obtained Raman spectra;

[0051] Figure 5 This is the standard curve for Escherichia coli detection obtained in the present invention. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0053] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0054] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0055] Example 1

[0056] An Escherichia coli detection reagent based on Raman enhanced spectroscopy is prepared by the following method:

[0057] S1. Preparation of Fe3O4 magnetic microspheres:

[0058] Take 10.8g of ferric chloride hexahydrate and 12g of ferric chloride tetrahydrate and add them to 300mL of deionized water, add 12g of polyethylene glycol, and ultrasonically disperse for 30min, then add 100mL of 4mol / L sodium hydroxide solution, stir and react for 90min at 70℃ and 800rpm under nitrogen protection, separate the magnetic product with a magnet, wash with deionized water until neutral, and vacuum dry at 70℃ to constant weight to obtain Fe3O4 magnetic microspheres;

[0059] S2. Preparation of silica-coated magnetic microspheres:

[0060] S2-1, take 2g of Fe3O4 magnetic microspheres and add them to 200mL of ethanol, ultrasonically disperse them for 15min, then add 8mL of 15wt% ammonia water, then add 3.5g of vinyltriethoxysilane, stir and reflux at 75°C for 8h, separate by magnetic absorption, wash the magnetic product with ethanol, and obtain pretreated magnetic microspheres;

[0061] S2-2, take 2g of pretreated magnetic microspheres and add them to a mixed solution consisting of 150mL of ethanol and 100mL of deionized water, ultrasonically disperse for 15min, then add 6.5mL of 15wt% ammonia water, and then add 2.2ml of tetraethyl silicate dropwise under stirring, stir and react at 40°C and 400rpm for 5h, separate by magnetic absorption, and wash the magnetic product with ethanol to obtain silica-coated magnetic microspheres;

[0062] S3. Grafting copper-doped reduced carbon dots onto silica-coated magnetic microspheres:

[0063] S3-1, take 1g of silica-coated magnetic microspheres and 0.6g of copper acetate, add them into 100mL of deionized water, and disperse them by ultrasonic for 20min to obtain a mixed solution A;

[0064] S3-2, add 960 mg of citric acid, 316 mg of pyridine, 264 mg of vitamin C and 105 mg of cysteine ​​to a solution consisting of 80 mL of deionized water and 60 mL of ethanol, and disperse by ultrasonic for 10 min to obtain a mixed solution B;

[0065] S3-3, adding mixed solution B to mixed solution A, ultrasonically dispersing for 45 minutes, transferring the obtained precursor solution to a polytetrafluoroethylene-lined reactor, reacting at 190°C for 8 hours, cooling to room temperature after the reaction, centrifuging and filtering, washing the solid product with deionized water, and vacuum drying at 75°C for 12 hours to obtain grafted modified magnetic microspheres: CDs-Cu@SiO2@Fe3O4;

[0066] S4. In situ deposition of gold nanoparticles to prepare gold nanoparticle-modified magnetic microspheres:

[0067] 1 g of grafted modified magnetic microspheres was added to 100 mL of deionized water and ultrasonically dispersed for 30 min to obtain a microsphere dispersion liquid; 10 mL of 0.075 g / mL HAuCl4 solution was added to the microsphere dispersion liquid, stirred at 500 rpm and 70 ° C for 2 h, filtered, the solid product was washed with ethanol, and vacuum dried at 60 ° C for 8 h to obtain magnetic microspheres with deposited gold nanoparticles: AuNPS-CDs-Cu@SiO2@Fe3O4;

[0068] S5, grafted E. coli aptamer;

[0069] S5-1, adding 0.2 g of magnetic microspheres deposited with gold nanoparticles into 40 mL of deionized water, and ultrasonically dispersing for 20 min to obtain a carrier solution;

[0070] S5-2. Take 20 μL of 25 μM Escherichia coli aptamer solution (pre-prepared with Tris-HCl buffer) and 100 μL of carrier solution, add them to 8 mL of 5 mM PBS solution, ultrasonically disperse for 10 minutes, incubate at room temperature for 45 minutes, separate by magnetic absorption, wash the magnetic product with deionized water, and freeze-dry to obtain the detection reagent APT-AuNPS-CDs-Cu@SiO2@Fe3O4.

[0071] The 5-end of the E. coli aptamer is modified with a thiol group. The sequence of the E. coli aptamer in this example is designed according to the patent CN114563568A E. coli-Staphylococcus aureus biosensor and its application, which can specifically bind to E. coli. The only difference is that the amino group modified at the 5-end is replaced by a thiol group. The specific sequence of the E. coli aptamer is shown in SEQ ID NO: 1:

[0072] 5'-SH-AAAAAATCCGTCACACCTGCTCTATCAAATGTGCAGATATCAAGACGATTTGTACAAGATGG-3'.

[0073] Reference Figure 2 , is the infrared absorption spectrum of the magnetic microspheres deposited with gold nanoparticles (AuNPS-CDs-Cu@SiO2@Fe3O4) prepared in Example 1; it can be explained that the surface of the prepared carbon dots has abundant functional groups such as hydroxyl, carboxyl, amino, and thiol, and the appearance of characteristic peaks of bonds such as Cu-O and Si-O can explain the successful synthesis of AuNPS-CDs-Cu@SiO2@Fe3O4.

[0074] Reference Figure 3 , is the reducibility test result of AuNPS-CDs-Cu@SiO2@Fe3O4 prepared in Example 1. The present invention adopts DPPH free radical scavenging ability test kit (Hefei Lair Biotechnology Co., Ltd.) for testing. The AuNPS-CDs-Cu@SiO2@Fe3O4 prepared in Example 1 is prepared into a dispersion with a concentration of 0.3 mg / mL by using anhydrous ethanol. Then, the test is carried out according to the instructions of the kit, and the absorbance at 515 nm is detected at different times.

[0075] DPPH free radical is a very stable nitrogen-centered free radical with strong absorption near 515nm. When antioxidants are present, DPPH free radicals are scavenged and the absorbance at 515nm decreases. The absorbance at 515nm can reflect the reducing ability of the test substance.

[0076] according to Figure 3 The test results show that the AuNPS-CDs-Cu@SiO2@Fe3O4 prepared in Example 1 has good reducibility.

[0077] Example 2

[0078] A method for detecting Escherichia coli based on Raman enhanced spectroscopy comprises the following steps:

[0079] The detection reagent prepared in Example 1 was prepared into a dispersion with a concentration of 1 mg / mL with deionized water, and then mixed with the sample to be tested, incubated at room temperature for 10 minutes, the magnetic substance was separated with a magnet, washed with deionized water, and then re-dispersed in deionized water to obtain a test sample, and the test sample was aspirated and dripped on a clean silicon wafer for Raman detection; qualitative and quantitative analysis of Escherichia coli was performed based on the obtained Raman spectrum;

[0080] The qualitative analysis method is: when there is a Raman characteristic peak corresponding to the target fungus in the Raman spectrum, it is judged that the sample to be tested contains Escherichia coli;

[0081] The quantitative analysis method is: according to the intensity of the Raman characteristic peak corresponding to Escherichia coli, the concentration of Escherichia coli in the sample to be tested is calculated in combination with a pre-constructed standard curve characterizing the relationship between the intensity of the Raman characteristic peak and the concentration of Escherichia coli.

[0082] In this example, the Raman characteristic peak position is selected from 1455cm -1 .

[0083] Example 3

[0084] A kit for detecting Escherichia coli comprises the detection reagent of embodiment 1.

[0085] 1. Application examples

[0086] E. coli was inoculated and cultured in conventional culture medium and then diluted to different concentrations (10 2 -10 9 CFU mL -1 )spare.

[0087] The detection reagent prepared in Example 1 was prepared into a dispersion with a concentration of 1 mg / mL using deionized water, and then 5 mL of the dispersion was mixed with 1 mL of Escherichia coli liquid (10 7 CFU mL -1 ) and incubate at room temperature for 10 min. Use a magnet to separate the magnetic material, wash with deionized water, and then redisperse in 5 mL of deionized water to obtain a test sample. Pipette the test sample and drop it on a clean silicon wafer for Raman detection. ATR3110-27 Raman spectrometer, excitation wavelength 725 nm, integration time 3 s, laser power 200 mW.

[0088] 2. Comparative Example

[0089] Synthetic silver nanoparticles glue:

[0090] 0.35 g of citric acid and 0.2 g of PVP were added into 30 mL of deionized water to obtain a mixed solution 1;

[0091] 0.1 g of silver nitrate was added to 200 mL of deionized water, heated to 100°C, and then the mixed solution 1 was added, stirred and reacted at 90°C for 45 min, and cooled to obtain a silver nanoparticle colloid;

[0092] Take 1 mL of silver nanoparticle colloid and 0.2 mL of Escherichia coli solution (1×10 7 CFU mL -1 ) and incubate for 10 minutes to obtain a test solution. 5 μL of the test solution is pipetted and dropped onto a clean silicon wafer for Raman detection. The detection method is the same as that in the application example.

[0093] Reference Figure 4 , are the Raman spectra obtained for the control example and the application example. It can be seen that the Raman signal intensity of the application example has been significantly improved; among them, the Raman characteristic peak of Escherichia coli obtained in the application example is consistent with the report in the literature "Wang Y, Ni H, Li H. Plasmonic microneedle arrays for rapid extraction, SERS detection, and inactivation of bacteria[J]. Chemical engineering journal, 2022: 442P1. DOI: 10.1016 / j.cej.2022.136140."

[0094] 3. Constructing a standard curve for Raman detection of Escherichia coli

[0095] The Escherichia coli solutions of different concentrations (10 2 -10 9 CFU mL -1 ), perform Raman detection according to the method of the application example, and select 1455cm -1 The peak at the position was taken as the characteristic peak, the intensity of the characteristic peak was taken as the ordinate, and the logarithm (log C) value of the concentration of Escherichia coli liquid (concentration unit CFU / mL) was taken as the abscissa, and curve fitting was performed. Figure 5 , is the standard curve obtained by fitting, and its linear equation is: y = 747.81x + 940.67, and the correlation coefficient R 2 It is 0.998, indicating a good linear relationship.

[0096] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A reagent for detecting Escherichia coli based on Raman enhanced spectroscopy, characterized in that: It is prepared by the following method: S1, preparation of Fe3O4 magnetic microspheres; S2, preparing silica-coated magnetic microspheres; S3, grafting copper-doped reduced carbon dots onto silica-coated magnetic microspheres; S4, in situ deposition of gold nanoparticles to prepare gold nanoparticle-modified magnetic microspheres; S5, grafting the E. coli aptamer onto the magnetic microspheres modified with gold nanoparticles to obtain the E. coli detection reagent; When performing the test, the test reagent is mixed with the sample to be tested and incubated, and then Raman detection is performed to achieve qualitative and quantitative detection of Escherichia coli in the sample to be tested based on the presence and intensity of the Raman characteristic peak corresponding to Escherichia coli; Step S1 is specifically as follows: Take 5.0-21.6g of ferric chloride hexahydrate and 6-24g of ferric chloride tetrahydrate, add them into 200-500mL of deionized water, add 6.5-18g of polyethylene glycol, ultrasonically disperse for 15-60min, then add 50-200mL of 2-6mol / L sodium hydroxide solution, stir and react at 60°C for 0.5-3h under nitrogen protection, separate the magnetic product with a magnet, wash with deionized water until neutral, and vacuum dry at 60-80°C until constant weight to obtain Fe3O4 magnetic microspheres; Step S2 is specifically as follows: S2-1, take 1-4g Fe3O4 magnetic microspheres and add them to 100-400mL ethanol, ultrasonically disperse for 5-30min, then add 4-16mL ammonia water with a concentration of 10-20wt%, then add 1.75-7g vinyltriethoxysilane, stir and reflux at 70-80°C for 4-16h, separate by magnetic absorption, wash the magnetic product with ethanol, and obtain pretreated magnetic microspheres; S2-2, 1-4 g of pretreated magnetic microspheres were added to a mixed solution consisting of 75-300 mL of ethanol and 50-200 mL of deionized water, and ultrasonically dispersed for 5-30 min, and then 3.5-13 mL of 10-20 wt% ammonia water was added, and then 1.1-4.4 ml of tetraethyl silicate was added dropwise under stirring, and the mixture was stirred at 30-45 ° C and 200-800 rpm for 2.5-10 h, and magnetic separation was performed, and the magnetic product was washed with ethanol to obtain silica-coated magnetic microspheres: SiO2@Fe3O4; Step S3 is specifically as follows: S3-1, take 1g of silica-coated magnetic microspheres and 0.6g of copper acetate, add them into 100mL of deionized water, and disperse them by ultrasonic for 20min to obtain a mixed solution A; S3-2, add 960 mg of citric acid, 316 mg of pyridine, 264 mg of vitamin C and 105 mg of cysteine ​​to a solution consisting of 80 mL of deionized water and 60 mL of ethanol, and disperse by ultrasonic for 10 min to obtain a mixed solution B; S3-3, adding mixed solution B to mixed solution A, ultrasonically dispersing for 45 minutes, transferring the obtained precursor solution to a polytetrafluoroethylene-lined reactor, reacting at 190°C for 8 hours, cooling to room temperature after the reaction, centrifuging and filtering, washing the solid product with deionized water, and vacuum drying at 75°C for 12 hours to obtain grafted modified magnetic microspheres: CDs-Cu@SiO2@Fe3O4; Step S4 is specifically as follows: Take 0.5-2g of grafted modified magnetic microspheres and add them into 50-200mL of deionized water, and ultrasonically disperse them for 15-60min to obtain a microsphere dispersion; add 5-20mL of 0.075g / mL HAuCl4 solution to the microsphere dispersion, stir and react at 300-800rpm and 60-80℃ for 1-4h, filter, wash the solid product with ethanol, and vacuum dry at 50-70℃ for 4-12h to obtain gold nanoparticle-modified magnetic microspheres: AuNPS-CDs-Cu@SiO2@Fe3O4.

2. The Escherichia coli detection reagent based on Raman enhanced spectroscopy according to claim 1, characterized in that: Step S5 is specifically as follows: S5-1, adding 0.1 g of magnetic microspheres modified with gold nanoparticles into 20-80 mL of deionized water, and ultrasonically dispersing for 10-30 min to obtain a carrier solution; S5-2. Take 10-40 μL of 12-50 μM E. coli aptamer solution and 50-200 μL of carrier solution and add them to 4-16 mL of 2-10 mM PBS solution with a pH of 7.

0. Ultrasonic dispersion is performed for 5-20 minutes. The solution is incubated at room temperature for 30-90 minutes. The solution is separated by magnetic absorption. The magnetic product is washed with deionized water and freeze-dried to obtain the E. coli detection reagent: APT-AuNPS-CDs-Cu@SiO2@Fe3O4.

3. The Escherichia coli detection reagent based on Raman enhanced spectroscopy according to claim 2, characterized in that: The 5' end of the E. coli aptamer is modified with a thiol group, and the specific sequence is shown in SEQ ID NO: 1: 5'-SH-AAAAAATCCGTCACACCTGCTCTATCAAATGTGCAGATATCA AGACGATTTGTACAAGATGG-3'.

4. A method for detecting Escherichia coli based on Raman enhanced spectroscopy, characterized in that: The following steps are involved: The detection reagent described in any one of claims 1 to 3 is prepared into a dispersion with a concentration of 0.1-5 mg / mL with deionized water, and then mixed with the sample to be tested, incubated at room temperature for 5-15 minutes, separated by a magnet, washed with deionized water, and then re-dispersed in deionized water to obtain a test sample, and the test sample is aspirated and dripped on a clean silicon wafer for Raman detection; qualitative and quantitative analysis of Escherichia coli is performed according to the obtained Raman spectrum; The qualitative analysis method is: when there is a Raman characteristic peak corresponding to the target fungus in the Raman spectrum, it is judged that the sample to be tested contains Escherichia coli; The quantitative analysis method is: according to the intensity of the Raman characteristic peak corresponding to Escherichia coli, the concentration of Escherichia coli in the sample to be tested is calculated in combination with a pre-constructed standard curve characterizing the relationship between the intensity of the Raman characteristic peak and the concentration of Escherichia coli.

5. The method for detecting Escherichia coli based on Raman enhanced spectroscopy according to claim 4, characterized in that: The Raman characteristic peak positions are selected from 718, 965, 1138, 1266, 1455 and 1585 cm -1 at least one of .

6. An Escherichia coli detection kit, characterized in that: It comprises the detection reagent as described in any one of claims 1-4.

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