A dual-probe composition for detecting Listeria monocytogenes and preparation method thereof

Abstract

The present invention discloses a dual-probe composition for detecting Listeria monocytogenes and a preparation method thereof, and belongs to the technical field of food safety. The dual-probe composition is a composition of a mutually independent magnetic separation probe and a silicon-based nanozyme probe, the concentration of the magnetic separation probe is 1 mg / mL, and the concentration of the silicon-based nanozyme probe is 40 µg / mL. The magnetic separation probe is used to specifically bind to Listeria monocytogenes and separate and enrich the test sample; the silicon-based nanozyme probe is used to specifically bind to Listeria monocytogenes after separation and enrichment and generate Listeria monocytogenes concentration-dependent fluorescence signals, colorimetric signals, and photothermal signals. Compared with traditional methods, it also has lower detection time and detection sensitivity; in addition, under the interference of sea bass sashimi samples, the established linear regression equation still shows excellent applicability.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and particularly relates to a double-probe composition for detecting Listeria monocytogenes and a preparation method thereof. Background Art

[0002] Food safety is a livelihood issue that concerns the whole nation the most, so it is subject to key monitoring by the country. Data from the World Health Organization in 2022 showed that all foodborne pathogens are a major cause of foodborne diseases. Therefore, a variety of different methods are needed to detect pathogens in food, which is very important for food safety.

[0003] Listeria monocytogenes, as a typical foodborne pathogen, is widely present in the entire industry chain such as meat products, dairy products, instant foods, and frozen and refrigerated foods. It can survive under extreme conditions and cause food contamination. The mortality rate after eating contaminated food is as high as 20%-30%, which poses a great threat to public health and safety. Therefore, it is very important to establish a method for rapid, sensitive and reliable detection of Listeria monocytogenes.

[0004] For the detection of common pathogenic Listeria monocytogenes, traditional detection methods, such as microbial culture, immunoassay based on antigen-antibody specific recognition, and nucleic acid amplification based on the principle of base complementary pairing, are still recognized and reliable methods, but they require multiple steps such as bacterial isolation and culture, morphological observation, and biochemical identification, which are cumbersome and time-consuming and thus limited. The ELISA kit based on immunological technology is expensive and difficult to promote on an industrial scale.

[0005] Therefore, it is urgent to develop a simple, rapid, highly sensitive and accurate detection method for real-time detection of Listeria monocytogenes. In recent years, researchers at home and abroad have been committed to exploring foodborne pathogen detection methods suitable for on-site analysis, but they are generally single-mode detection, and existing methods have problems such as large matrix interference, poor signal accuracy, and single detection signal. Summary of the invention

[0006] In view of the problems that the existing detection technology of Listeria monocytogenes in food is time-consuming and easily affected by the sample matrix, the present invention provides a dual-probe composition for detecting Listeria monocytogenes, and at the same time, provides a preparation method of the dual-probe composition.

[0007] A dual-probe composition for detecting Listeria monocytogenes is a composition of a mutually independent magnetic separation probe and a silicon-based nanozyme probe, wherein the concentration of the magnetic separation probe is 1 mg / mL, and the concentration of the silicon-based nanozyme probe is 40 µg / mL;

[0008] The magnetic separation probe is prepared by amidation and covalent coupling of an amino-containing ferroferric oxide nanosphere solution with a mass concentration of 1 mg / mL and a carboxyl-containing daptomycin solution with a molar mass concentration of 50 µmol / L at a volume ratio of 250:1 to obtain a daptomycin-functionalized ferroferric oxide nanosphere solution, wherein the daptomycin is used to specifically identify Listeria monocytogenes; the particle size of the magnetic separation probe is 100 nm-200 nm, and there is an ultraviolet absorption peak at 235 nm;

[0009] The magnetic separation probe is used to specifically bind to Listeria monocytogenes and separate and enrich the bacteria from the test sample;

[0010] The silicon-based nanozyme probe is prepared by amidation covalently coupling an amino-containing silicon-based nanozyme microsphere solution with a mass concentration of 40ug / mL and a carboxyl-containing nucleic acid aptamer solution with a molar mass concentration of 10µmol / L at a volume ratio of 20:1 to obtain an aptamer-functionalized silicon-based nanozyme microsphere solution, the DNA sequence of the nucleic acid aptamer is shown in SEQ ID No: 1, the 5' end of the nucleic acid aptamer is modified with a carboxyl group, and the nucleic acid aptamer is used to specifically identify Listeria monocytogenes; the particle size of the silicon-based nanozyme probe is 10nm-30nm, and there is an ultraviolet absorption peak at 251nm;

[0011] The silicon-based nanozyme probe is used to specifically bind to Listeria monocytogenes after separation and enrichment treatment, and generate fluorescence signals, colorimetric signals and photothermal signals that are dependent on the concentration of Listeria monocytogenes.

[0012] The preparation steps of a dual-probe composition for detecting Listeria monocytogenes are as follows:

[0013] (1) Preparation of activation mixture

[0014] 10 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mmol / L N-hydroxysuccinimide were mixed in a volume ratio of 1:1 to obtain an activated mixed solution;

[0015] (2) Preparation of magnetic separation probe

[0016] (2.1) vortexing the carboxyl group-containing daptomycin solution and the activated mixed solution at a volume ratio of 0.012:1, and incubating on a shaker at room temperature to obtain an activated daptomycin solution;

[0017] (2.2) adding an amino-containing ferroferric oxide nanosphere solution to the activated daptomycin solution and vortex mixing, wherein the amount of the amino-containing ferroferric oxide nanosphere solution added is 250 times the volume of the carboxyl-containing daptomycin solution, incubating on a shaking table, and removing the supernatant by magnetic separation to obtain a daptomycin-functionalized ferroferric oxide nanosphere solution;

[0018] (2.3) adding a 1% by volume bovine serum albumin solution to the daptomycin-functionalized ferroferric oxide nanosphere solution to block the residual sites of ferroferric oxide, and removing the supernatant by magnetic separation again to obtain a blocked ferroferric oxide nanosphere solution;

[0019] (2.4) Washing the sealed ferroferric oxide nanosphere solution with sterile Tris-HCl buffer for three times, and resuspending to obtain a magnetic separation probe solution; the mass concentration of the magnetic separation probe solution is 1 mg / mL;

[0020] When the ZETA potential value of the amino-containing ferroferric oxide nanoparticle solution increased from +25.37 mV to +33.13 mV, it indicated that the carboxyl-containing daptomycin in the magnetic separation probe was successfully modified onto the amino-containing ferroferric oxide nanoparticles;

[0021] (3) Preparation of silicon-based nanozyme probes

[0022] (3.1) Vortex-mix the carboxyl-containing aptamer solution and the activation mixture at a volume ratio of 0.125:1, and incubate on a shaker at room temperature to obtain an activated aptamer solution;

[0023] (3.2) Adding an amino-containing silicon-based nanozyme microsphere solution to the activated nucleic acid aptamer solution and vortex mixing, wherein the amount of the amino-containing silicon-based nanozyme microsphere solution added is 20 times the volume of the carboxyl-containing nucleic acid aptamer solution, and incubating on a shaker at room temperature to obtain an aptamer-functionalized silicon-based nanozyme microsphere solution, i.e., a silicon-based nanozyme probe; the mass concentration of the silicon nanozyme probe is 40 ug / mL;

[0024] When the ZETA potential value of the amino-containing silicon-based nanozyme microsphere solution dropped from + 18.84 mV to + 8.27 mV, it indicated that the carboxyl-containing nucleic acid aptamer in the silicon-based nanozyme probe was successfully modified onto the amino-containing silicon-based nanozyme microsphere;

[0025] (4) Preparation of dual probe composition

[0026] The magnetic separation probe and the silicon-based nanozyme probe were stored at 4° C. in a volume ratio of 1:2 to prepare a dual-probe composition;

[0027] When used for detecting Listeria monocytogenes, the magnetic separation probe is first combined with Listeria monocytogenes in a detection sample solution, and the added amount of the detection sample solution is twice the volume of the magnetic separation probe to obtain a complex of the magnetic separation probe and Listeria monocytogenes;

[0028] A silicon-based nanozyme probe is added to the magnetic separation probe and Listeria monocytogenes complex to obtain a magnetic separation probe, Listeria monocytogenes and silicon-based nanozyme probe complex, and then the detection of the sample solution is completed through three signal channels: fluorescence, colorimetry and photothermal.

[0029] The further defined preparation operation technical scheme is as follows:

[0030] The specific operations of step (2) are as follows:

[0031] (2.1) 2.4 uL of 50 μmol / L carboxyl-containing daptomycin solution was vortexed with 200 uL of the activated mixture, and incubated at room temperature at 200 rpm on a shaker for 30 min to obtain an activated daptomycin solution with a molar concentration of 0.6 μmol / L;

[0032] (2.2) Add 600 uL of 3 mg / mL amino-containing ferroferric oxide nanosphere solution to 200 uL of 0.6 μmol / L activated daptomycin solution, vortex mix, incubate on a shaker at 180 rpm and 37 °C for 3 h, remove the supernatant, and obtain daptomycin-functionalized ferroferric oxide nanosphere precipitate;

[0033] (2.3) adding 600 uL of 1% bovine serum albumin solution to the daptomycin-functionalized ferroferric oxide nanosphere precipitate to block the residual sites of ferroferric oxide, performing magnetic separation again using a magnet, removing the supernatant, and obtaining a blocked ferroferric oxide nanosphere precipitate;

[0034] (2.4) Wash the blocked FeO nanosphere precipitate with sterile Tris-HCl buffer for three times, and resuspend the precipitate in 1 mL sterile Tris-HCl buffer to prepare a magnetic separation probe.

[0035] The sterile Tris-HCl buffer has a molar concentration of 25 mmol / L, a pH value of 7.4, and contains calcium chloride with a molar concentration of 5 mmol / L.

[0036] In step (3), the preparation operation of the amino-containing silicon-based nanozyme microsphere solution is as follows:

[0037] Add 12 mL of deionized water to 1 mL of N-(β-aminoethyl-γ-aminopropyl)methyldimethoxysilane; then add 10 mg of amikacin and 1 mL of 200 mmol / L ferrous sulfate solution, and stir continuously for 10 min; heat at 180°C for 8 h, cool to room temperature, and dilute 30 times to obtain an amino-containing silicon-based nanozyme microsphere solution.

[0038] The specific operations of step (3) are as follows:

[0039] (3.1) Vortex mix 25 µL of 10 µmol / L carboxyl-containing aptamer solution and 200 µL of activation mixture, and incubate on a shaker at 200 rpm for 30 min at room temperature to obtain an activated aptamer solution;

[0040] (3.2) Add 500 µL of amino-containing silicon-based nanozyme microsphere solution to 200 uL of activated nucleic acid aptamer solution with a molar concentration of 1.25 µmol / L, vortex mix, and incubate on a shaker at 80 rpm at room temperature for 2.5 h to obtain an aptamer-functionalized silicon-based nanozyme microsphere solution, i.e., a silicon-based nanozyme probe.

[0041] The detection steps of a dual-probe composition for detecting Listeria monocytogenes are as follows:

[0042] (1) Preparation of sample test solution

[0043] The food to be tested was cut into 25 g pieces; mixed with 25 mL of Tris-HCl buffer in a sterilization bag, homogenized for 2 minutes to obtain a Tris-HCl buffer mixture, the mixture was allowed to stand for 5 minutes, and the supernatant was taken to obtain the test solution.

[0044] (2) Detection

[0045] Next, the magnetic separation probe was mixed with the test solution obtained above at a volume ratio of 1:2, incubated at room temperature for 30 minutes, washed 3 times with a magnet for magnetic separation, and resuspended in a sterile Tris-HCl buffer with an equal volume as the blank control or the bacteria-containing test solution. Next, a silicon-based nanozyme probe with a volume twice that of the magnetic separation probe was added and incubated at room temperature for 20 minutes to obtain the test solution.

[0046] (2) Detection of fluorescence signals

[0047] Next, for the test liquid obtained above, the supernatant obtained by magnetic separation with a magnet was obtained to obtain a fluorescent signal detection solution, and the fluorescence intensity value generated at an excitation wavelength of 410 nm was detected using a fluorescence spectrophotometer. The linear regression equation was y=-0.11267x+1.12571, and the logarithmic value of Listeria monocytogenes was calculated.

[0048] (3) Detection of colorimetric signals

[0049] At the same time, in order to obtain, the precipitate obtained by magnetic separation using a magnet was added with an acetic acid-sodium acetate buffer of an equal volume to the test liquid, wherein the acetic acid-sodium acetate buffer was as follows: an acetic acid-sodium acetate buffer with a pH value of 4.5, purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a product number of R27435-500ml, and 3,3',5,5'-tetramethylbenzidine with a final concentration of 1mmol / L and a hydrogen peroxide solution with a final concentration of 1mmol / L were added, and the mixture was reacted at 35°C for 10min to obtain a colorimetric signal detection solution, and an ultraviolet spectrophotometer was used to detect the ultraviolet absorption intensity value generated at 653nm, and the value was substituted into the linear regression equation as y=0.13367x+0.05318, and the logarithmic value of Listeria monocytogenes was calculated.

[0050] (4) Detection of photothermal signals

[0051] In addition, the above colorimetric signal detection solution was directly used as a photothermal signal detection solution at 1.5 W / cm 2 The solution was irradiated with a 660 nm laser for 120 s, and an infrared thermal imager was used to take a thermal image of the photothermal detection solution temperature. The photothermal temperature value was read and substituted into the linear regression equation of y = 5.80693x + 17.9997, and the logarithmic value of Listeria monocytogenes was calculated.

[0052] The beneficial technical effects of the present invention are embodied in the following aspects:

[0053] 1. Creativity of the “Dual Probe Combination”

[0054] The present invention applies daptomycin as a recognition element to the identification and detection of Listeria monocytogenes for the first time, and assembles a magnetic separation probe into daptomycin and amino-containing ferric oxide nano-microspheres by covalent coupling, which is used to screen and separate Gram-positive bacteria, solving the problem that the existing Listeria monocytogenes lacks a suitable specific recognition element, thereby realizing the first recognition of the double probe composition, that is, when Listeria monocytogenes exists in the sample solution, the magnetic separation probe will be bound to the surface of Listeria monocytogenes, and the magnet will be used to separate and enrich Listeria monocytogenes from the sample solution, reduce the matrix interference of the sample solution, improve the accuracy and sensitivity of the detection, and list the related linear coefficients and detection limits of the three-modal detection. At the same time, a silicon-based nanozyme probe with multiple properties such as generating fluorescence signals, colorimetric signals and photothermal signals is synthesized by combining a silicon coupling agent, amphenol and ferrous sulfate at high temperature for the first time, and coupled with a specific aptamer as a recognition element to perform secondary screening and separation of strains. In the above process, the interference of various impurities such as proteins and fats in the sample is effectively removed, thereby improving the sensitivity of detection; at the same time, it has been verified that the correlation coefficient of the linear fitting degree of the ratio fluorescent probe prepared by the present invention is as high as 0.99337, while the linear fitting degree of the current prior art generally ranges from 0.90 to 0.98, and the ratio fluorescent probe prepared by the present invention has a high linear fitting degree; the ratio fluorescent probe prepared by the present invention has an extremely low detection limit, which is as low as 2.06 CFU / mL.

[0055] 2. Creativity of the preparation method of the present invention

[0056] Compared with traditional technologies (strand displacement reaction method, PCR amplification method, ELISA kit method), the detection time of traditional technologies is as long as 12h-3d, and the specificity of some common model strains is cross-linked. For example, the traditional chain displacement reaction uses the free energy difference of DNA molecular hybridization to replace the target single strand in the hybrid DNA double helix structure with a single-stranded DNA. Since the negatively charged phosphate group on the single-stranded DNA has an intramolecular electrostatic repulsion effect on the double-stranded DNA, the chain displacement reaction efficiency is relatively low; while the present invention adopts the three modes of fluorescence signal-colorimetric signal-photothermal signal for comparison detection, especially, through secondary screening and separation, the supernatant is subjected to fluorescence signal detection, the precipitate is resuspended for colorimetric signal detection, and then the resuspended precipitate after colorimetric signal detection is subjected to photothermal signal detection. The fluorescence-colorimetric-photothermal three-modal signals can be obtained simultaneously in one test, which not only enhances the sensitivity of detection, but also verifies each other between the three-modal signals, plays an internal proofreading role, improves the accuracy of the detection results, and avoids misjudgment caused by operational errors. The detection time of the present invention is within 1 hour, has a relatively low detection time, and has good specificity for common model strains such as Staphylococcus aureus and Escherichia coli.

[0057] 3. The creativity of the present invention's "dual probe combination" for detection

[0058] The linear regression equation established in the standard environment of the present invention can be directly applied to the detection of sea bass sashimi samples. There is no need to re-cultivate bacteria to establish a standard curve. It is only necessary to directly apply the established linear regression equation, which saves time and is suitable for quickly estimating the initial concentration of Listeria monocytogenes in food samples under emergency conditions. For example, the test results show that, after calculation, in the environment of artificial inoculation of Listeria monocytogenes in sea bass sashimi, the concentrations of Listeria monocytogenes measured by the three signals of fluorescence, colorimetry and photothermal are 1.03*10 6 CFU / mL, 1.06*10 6 CFU / mL, 1.05*10 6 CFU / mL, almost the same as its actual concentration 10 6 The CFU / mL are close. It can be seen that even if the corresponding standard curve and linear regression equation are not established in the environment of sea bass sashimi samples, the standard curve and linear regression equation established under Tris-HCl buffer can still have good versatility. It can be seen that the silicon-based nanozyme probe prepared by the present invention has good versatility and can be quickly applied to different sample detections. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is the infrared spectrum of the silicon-based nanozyme prepared in the present invention.

[0060] Figure 2 It is the fluorescence spectra of Listeria monocytogenes at different concentrations and the corresponding standard curve diagram in the present invention.

[0061] Figure 3 It is the ultraviolet absorption diagram of Listeria monocytogenes at different concentrations and the corresponding standard curve diagram in the present invention.

[0062] Figure 4 It is a standard curve diagram of the photothermal temperature values ​​of Listeria monocytogenes at different concentrations in the present invention.

[0063] Figure 5 This is a specific detection diagram of Listeria monocytogenes in the present invention. DETAILED DESCRIPTION

[0064] The present invention is further described below in conjunction with specific embodiments.

[0065] Unless otherwise defined, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0066] The raw materials used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified; the quantitative tests in the following examples are all repeated three times, and the results are averaged; the % in the following examples are all mass percentages unless otherwise specified.

[0067] In the following examples, the nucleic acid aptamers used were purchased from Shanghai Biotech Co., Ltd.; 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, N-(β-aminoethyl-γ-aminopropyl) methyldimethoxysilane, amphenol, and ferrous sulfate used were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; unless otherwise specified, other raw materials used were purchased from Sinopharm Chemical Reagent Co., Ltd. The sterile Tris-HCl buffer used had a molar concentration of 25 mmol / L and a pH value of 7.4, and contained calcium chloride at a molar concentration of 5 mmol / L.

[0068] All bacterial species used in the present invention are from Guangdong Institute of Microbiology, wherein the number of Listeria monocytogenes is ATCC 43251, the number of Staphylococcus aureus is ATCC 29213, the number of Salmonella is ATCC 14028, the number of Escherichia coli is ATCC 25922, the number of Cronobacter sakazakii is ATCC29544, and the number of Pseudomonas aeruginosa is ATCC 15442.

[0069] The instruments, equipment, raw materials, reagents or method steps used in the present invention are all ensured to be processed under sterile conditions.

[0070] The instruments, equipment, raw materials, reagents or method steps not mentioned in the present invention are conventional or well-known technical methods for those skilled in the art and will not be described in detail in the present invention.

[0071] Example 1

[0072] The preparation steps of a dual-probe composition for detecting Listeria monocytogenes are as follows:

[0073] (1) Preparation of activation mixture

[0074] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride at a concentration of 10 mmol / L and N-hydroxysuccinimide at a concentration of 5 mmol / L were mixed in a volume ratio of 1:1 to obtain an activated mixed solution.

[0075] (2) Preparation of magnetic separation probe

[0076] (2.1) Vortex mix 2.4 uL of 50 μmol / L carboxyl-containing daptomycin solution with 200 uL of activated mixture, and incubate at room temperature at 200 rpm on a shaker for 30 min to obtain an activated daptomycin solution with a molar concentration of 0.6 μmol / L.

[0077] (2.2) Add 600 uL of 3 mg / mL amino-containing ferroferric oxide nanosphere solution to 200 uL of 0.6 μmol / L activated daptomycin solution, vortex mix, and incubate on a shaker at 180 rpm and 37 °C for 3 h. Remove the supernatant to obtain daptomycin-functionalized ferroferric oxide nanosphere precipitate.

[0078] (2.3) Add 600uL of 1% bovine serum albumin solution to the daptomycin-functionalized ferroferric oxide nanosphere precipitate to block the residual sites of ferroferric oxide. Use a magnet for magnetic separation again, remove the supernatant, and obtain the blocked ferroferric oxide nanosphere precipitate.

[0079] (2.4) Wash the blocked FeO nanosphere precipitate with sterile Tris-HCl buffer for three times, and resuspend the precipitate in 1 mL sterile Tris-HCl buffer to prepare a magnetic separation probe.

[0080] The mass concentration of the magnetic separation probe is 1 mg / mL; the particle size of the magnetic separation probe is 100 nm-200 nm, and the magnetic separation probe has an ultraviolet absorption peak at 235 nm.

[0081] When the ZETA potential value of the amino-containing ferroferric oxide nanoparticle solution increased from +25.37 mV to +33.13 mV, it indicated that the carboxyl-containing daptomycin in the magnetic separation probe was successfully modified onto the amino-containing ferroferric oxide nanoparticle.

[0082] The magnetic separation probe is used to specifically bind to Listeria monocytogenes and separate and enrich the bacteria from the test sample.

[0083] (3) Preparation of silicon-based nanozyme probes

[0084] (3.1) Vortex mix 25 µL of 10 µmol / L carboxyl-containing aptamer solution and 200 µL of activation mixture, and incubate on a shaker at 200 rpm for 30 min at room temperature to obtain an activated aptamer solution.

[0085] (3.2) Add 500 µL of amino-containing silicon-based nanozyme microsphere solution to 200 uL of activated nucleic acid aptamer solution with a molar concentration of 1.25 µmol / L, vortex mix, and incubate on a shaker at 80 rpm at room temperature for 2.5 h to obtain an aptamer-functionalized silicon-based nanozyme microsphere solution, i.e., a silicon-based nanozyme probe.

[0086] The mass concentration of the silicon-based nanozyme probe is 40µg / mL; the particle size of the silicon-based nanozyme probe is 10nm-30nm, and the silicon-based nanozyme probe has an ultraviolet absorption peak at 251nm.

[0087] The DNA sequence of the nucleic acid aptamer in the silicon-based nanozyme probe is shown in SEQ ID No: 1. The 5' end of the nucleic acid aptamer is modified with a carboxyl group. The nucleic acid aptamer is used to specifically identify Listeria monocytogenes.

[0088] The preparation operation of the amino-containing silicon-based nanozyme microsphere solution is as follows: add 12 mL of deionized water to 1 mL of N-(β-aminoethyl-γ-aminopropyl)methyldimethoxysilane; then add 10 mg of amphenol and 1 mL of 200 mmol / L ferrous sulfate solution, and stir continuously for 10 minutes; heat at 180° C. for 8 hours, cool to room temperature, and dilute 30 times to obtain the amino-containing silicon-based nanozyme microsphere solution.

[0089] See also Figure 1 The infrared spectrum information of the amino-containing silicon-based nanozyme microsphere solution is as follows: 2926cm -1 1600cm is the antisymmetric stretching vibration of CH2; -1 The carboxylate COO -1 Antisymmetric stretching vibration or H2O angular vibration or NH3 + Asymmetric angle vibration or aromatic ring skeleton vibration; 1460cm -1 It may be carboxylate COO -1 Symmetric stretching vibration or NH3 + Symmetrical angle vibration or CH2 angle vibration or aromatic ring skeleton vibration; 1257cm -1 The position may be the out-of-plane swing vibration of CH2 or the stretching vibration of carboxylic acid C-OH; 1020cm -1 The position may be the C-OH stretching vibration of alcohol or the CC stretching vibration of straight chain or the CO stretching vibration of ester group or the CH in-plane bending vibration of aromatic ring; 909cm -1 The position may be the out-of-plane bending vibration of carboxylic acid COH or the twisting vibration of NH2; 791cm -1 The carboxylate COO -1 Angular vibration or out-of-plane bending vibration of aromatic ring CH.

[0090] When the ZETA potential value of the amino-containing silicon-based nanozyme microsphere solution dropped from + 18.84 mV to + 8.27 mV, it indicated that the carboxyl-containing nucleic acid aptamer in the silicon-based nanozyme probe was successfully modified onto the amino-containing silicon-based nanozyme microsphere.

[0091] The silicon-based nanozyme probe is used to specifically bind to Listeria monocytogenes after separation and enrichment, and produce Listeria monocytogenes concentration-dependent fluorescence signals, colorimetric signals and photothermal signals.

[0092] (4) Preparation of dual probe composition

[0093] The magnetic separation probe and the silicon-based nanozyme probe were stored separately in a refrigerator at 4° C. in a volume ratio of 1:2 to prepare a dual-probe composition.

[0094] During detection, the magnetic separation probe is first used to combine with Listeria monocytogenes in the detection sample solution, and the amount of the detection sample solution added is twice the volume of the magnetic separation probe to obtain a complex of the magnetic separation probe and Listeria monocytogenes; the silicon-based nanozyme probe is added to the complex of the magnetic separation probe and Listeria monocytogenes to obtain a complex of the magnetic separation probe, Listeria monocytogenes and the silicon-based nanozyme probe, and then the detection of the detection sample solution is completed through the three signal channels of fluorescence-colorimetry-photothermal.

[0095] Example 2

[0096] Construction of linear regression equation for detection of Listeria monocytogenes using fluorescence-colorimetry-photothermal trimodal signals

[0097] The dual-probe composition prepared in Example 1 is used to establish a linear regression equation for detecting Listeria monocytogenes. The operating steps are as follows:

[0098] (1) Preprocessing

[0099] First, a test solution containing no bacteria of the test sample is prepared, which is set as a blank control test solution, and the blank control test solution is artificially contaminated with Listeria monocytogenes, and a test solution containing bacteria is set to be diluted in a linear gradient; in this embodiment, during the experiment, a sterile Tris-HCl buffer is selected as the blank control test solution, and at the same time, the bacterial solution of Listeria monocytogenes is centrifuged and washed three times with a sterile Tris-HCl buffer, and then resuspended and gradiently diluted with a sterile Tris-HCl buffer to prepare concentrations of 10 1 CFU / mL, 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6CFU / mL, 10 7 CFU / mL of bacteria-containing test solution;

[0100] Next, the magnetic separation probe was mixed with the blank control or bacteria-containing test solution obtained above in a volume ratio of 1:2, incubated at room temperature for 30 minutes, washed 3 times with a magnet for magnetic separation, and resuspended in a sterile Tris-HCl buffer with an equal volume as the blank control or bacteria-containing test solution. Next, a silicon-based nanozyme probe with a volume twice that of the magnetic separation probe was added and incubated at room temperature for 20 minutes to obtain a test solution.

[0101] (2) Detection of fluorescence signals

[0102] Next, for the above-obtained test liquid, the supernatant obtained by magnetic separation with a magnet is obtained to obtain a fluorescent signal detection solution, and the fluorescence intensity value generated at an excitation wavelength of 410 nm is detected by a fluorescence spectrophotometer. The read fluorescence intensity value is selected as the ordinate, and the logarithm of the bacterial concentration of the test liquid is used as the abscissa to draw a standard curve, and the correlation coefficient and the detection limit are obtained according to the linear regression equation; Figure 2 As shown, see Figure 2 A, from top to bottom: Curve 0 represents the fluorescence spectrum curve with a bacterial concentration of 0 CFU / mL (blank control test solution), Curve 1 represents the fluorescence spectrum curve with a bacterial concentration of 10 1 The fluorescence spectrum curve of CFU / mL, curve 2 indicates that the bacterial concentration is 10 2 The fluorescence spectrum curve of CFU / mL, curve 3 indicates that the bacterial concentration is 10 3 The fluorescence spectrum curve of CFU / mL, curve 4 indicates that the bacterial concentration is 10 4 The fluorescence spectrum curve of CFU / mL, curve 5 indicates that the bacterial concentration is 10 5 The fluorescence spectrum curve of CFU / mL, curve 6 indicates that the bacterial concentration is 10 6 The fluorescence spectrum curve of CFU / mL, curve 7 indicates that the bacterial concentration is 10 7 The fluorescence spectrum curve of CFU / mL, curve 8 indicates that the bacterial concentration is 10 8 CFU / mL fluorescence spectrum curve; at the same time, combined with Figure 2 B, take the above Figure 2 The fluorescence intensity value at 528 nm on each fluorescence spectrum curve in A is taken as the ordinate, and the logarithmic value of the bacterial concentration corresponding to each fluorescence spectrum curve is taken as the abscissa. The linear regression equation is y=-0.11267x+1.12571, and the detection limit is 3.62 CFU / mL. 2 The value is 0.98534.

[0103] (3) Detection of colorimetric signals

[0104] At the same time, in order to obtain, the precipitate obtained by magnetic separation using a magnet is added with an acetic acid-sodium acetate buffer of an equal volume to the test liquid, wherein the acetic acid-sodium acetate buffer is as follows: an acetic acid-sodium acetate buffer with a pH value of 4.5 is purchased from Shanghai Yuanye Biotechnology Co., Ltd., and its product number is R27435-500ml, and 3,3',5,5'-tetramethylbenzidine with a final concentration of 1mmol / L and a hydrogen peroxide solution with a final concentration of 1mmol / L are added, and the reaction is carried out at 35°C for 10min to obtain a colorimetric signal detection solution, and an ultraviolet spectrophotometer is used to detect the ultraviolet absorption intensity value generated at 653nm, and the read ultraviolet absorption intensity value is selected as the ordinate, and the logarithmic value of the bacterial concentration of the test liquid is used as the abscissa to draw a standard curve, and the correlation coefficient and the detection limit are obtained according to the linear regression equation; Figure 3 As shown, see Figure 3 A, from top to bottom: Curve 0 represents the UV absorption curve with a bacterial concentration of 0 CFU / mL (blank control test solution), Curve 1 represents the UV absorption curve with a bacterial concentration of 10 1 The UV absorption curve of CFU / mL, curve 2 indicates that the bacterial concentration is 10 2 The UV absorption curve of CFU / mL, curve 3 indicates that the bacterial concentration is 10 3 The UV absorption curve of CFU / mL, curve 4 indicates that the bacterial concentration is 10 4 The UV absorption curve of CFU / mL, curve 5 indicates that the bacterial concentration is 10 5 The UV absorption curve of CFU / mL, curve 6 indicates that the bacterial concentration is 10 6 The UV absorption curve of CFU / mL, curve 7 indicates that the bacterial concentration is 10 7 The UV absorption curve of CFU / mL, curve 8 indicates that the bacterial concentration is 10 8 CFU / mL UV absorption curve; at the same time, combined with Figure 3 B, take the above Figure 3 The ultraviolet absorption intensity value of 653 on each ultraviolet absorption curve in A is taken as the ordinate, and the logarithmic value of the bacterial concentration corresponding to each ultraviolet absorption curve is taken as the abscissa. The linear regression equation is y=0.13367x+0.05318, and the detection limit is 2.06CFU / mL. Its R 2 The value is 0.99116.

[0105] (4) Detection of photothermal signals

[0106] In addition, the above colorimetric signal detection solution was directly used as a photothermal signal detection solution at 1.5 W / cm 2The 660nm laser was irradiated for 120s, and the infrared thermal imager was used to take the thermal image of the photothermal detection solution temperature. The photothermal temperature value read was selected as the ordinate, and the logarithm of the bacterial concentration of the detected solution was used as the abscissa to draw the standard curve. The correlation coefficient and detection limit were obtained according to the linear regression equation. Figure 4 As shown in the figure, the temperature values ​​corresponding to different concentrations of Listeria monocytogenes are used as the ordinate, and the logarithmic value of the corresponding bacterial concentration is used as the abscissa. The linear regression equation is y=5.80693x+17.9997, and the detection limit is 3.7 CFU / mL. 2 The value is 0.99337.

[0107] Example 3

[0108] Testing of real samples

[0109] The dual-probe composition prepared in Example 1 and the linear regression equation constructed in Example 2 were used to detect real food samples, and sea bass sashimi samples were selected for testing:

[0110] In order to verify whether the linear regression equation constructed according to Example 2 can be effectively applied to the determination of interference environments such as seabass sashimi, this Example 3 is carried out by artificial quantitative bacterial infection to explore the difference between the number of bacteria detected by the dual-probe composition in seabass sashimi samples, so as to find out that even in real food samples, the dual-probe composition established in this application can effectively perform detection under the influence of high protein.

[0111] The sea bass sashimi samples involved in this Example 3 were purchased from HEMA Fresh. When purchased, the sea bass sashimi was stored in a refrigerator at 4°C. The nutritional components per 100g are as follows: energy 378kJ, protein 21.1g, fat 3.0g, carbohydrates 4.6g, and sodium 749mg.

[0112] The inoculation volume of Listeria monocytogenes in sea bass sashimi was 10 8 CFU / g, and let it stand for 2 hours at room temperature to obtain the inoculated sea bass sashimi, and cut the inoculated sea bass sashimi into pieces; in a sterilization bag, 25g of the inoculated sea bass sashimi cut into pieces was mixed with 25mL of sterile Tris-HCl buffer, and homogenized for 2 minutes to prepare a Tris-HCl buffer mixture, and the mixture was allowed to stand for 5 minutes, and the supernatant was taken; by adding sterile Tris-HCl buffer, the final concentration of Listeria monocytogenes in the supernatant was adjusted to 10 6 CFU / mL, as the test solution. Subsequently, in combination with Example 2, the fluorescence-colorimetry-photothermal trimodal signal detection was performed, and the obtained fluorescence intensity value-ultraviolet absorption intensity value-photothermal temperature value was substituted into the obtained linear regression equation to obtain the concentration of Listeria monocytogenes in the real sample.

[0113] The experimental results show that, according to calculations, the concentrations of Listeria monocytogenes measured in the artificial inoculation of sea bass sashimi were 1.03*10 6 CFU / mL, 1.06*10 6 CFU / mL, 1.05*10 6 CFU / mL corresponds to the detection values ​​of fluorescence, colorimetry, and photothermal signals, which are almost 10 times the actual concentration. 6 The CFU / mL are close. It can be seen that even if the corresponding standard curve and linear regression equation are not established in the environment of sea bass sashimi samples, the standard curve and linear regression equation established in pure sterile Tris-HCl buffer can still have good versatility. It can be seen that the dual-probe composition prepared by the present invention has good versatility and can be quickly applied to different sample detections.

[0114] Example 4

[0115] Basically the same as Example 2, except that:

[0116] Listeria monocytogenes was replaced with Staphylococcus aureus, Salmonella, Escherichia coli, Cronobacter sakazakii, and Pseudomonas aeruginosa; and the concentrations of 10 6 CFU / mL of the test solution containing bacteria.

[0117] See also Figure 5For A, 5B and 5C, after adding Staphylococcus aureus (serial number "1" in the figure), Salmonella (serial number "2" in the figure), Escherichia coli (serial number "3" in the figure), Cronobacter sakazakii (serial number "4" in the figure) and Pseudomonas aeruginosa (serial number "5" in the figure), the fluorescence intensity value, ultraviolet absorption intensity value and photothermal temperature value of (Staphylococcus aureus) were 0.99234, 0.15023 and 20.2, respectively; the fluorescence intensity value, ultraviolet absorption intensity value and photothermal temperature value of (Salmonella) were 0.99013, 0.14219 and 19.4, respectively; the fluorescence intensity value, ultraviolet absorption intensity value and photothermal temperature value of (Escherichia coli) were 0.9876, 0.12782 and 19.1, respectively; the fluorescence intensity value, ultraviolet absorption intensity value and photothermal temperature value of (Cronobacter sakazakii) were The fluorescence intensity value, ultraviolet absorption intensity value and photothermal temperature value of (Pseudomonas aeruginosa) were 0.98376, 0.13207 and 20.1, respectively. At the same time, the fluorescence intensity value, ultraviolet absorption intensity value and photothermal temperature value of the bacteria-containing test solution after adding Listeria monocytogenes under the same conditions (serial number "6" in the figure) were 0.43889, 0.85848 and 52.5, respectively. In addition, the fluorescence intensity value, ultraviolet absorption intensity value and photothermal temperature value of the blank control test solution under the same conditions (serial number "0" in the figure) were 0.98221, 0.11985 and 18.7, respectively. This shows that the dual-probe composition prepared by the present invention has good specificity for Listeria monocytogenes.

[0118] It will be easily understood by those skilled in the art that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A dual-probe composition for detecting Listeria monocytogenes, characterized in that: The dual-probe composition is a composition of a mutually independent magnetic separation probe and a silicon-based nanozyme probe, wherein the concentration of the magnetic separation probe is 1 mg / mL, and the concentration of the silicon-based nanozyme probe is 40 µg / mL; The magnetic separation probe is prepared by amidation and covalent coupling of an amino-containing ferroferric oxide nanosphere solution with a mass concentration of 1 mg / mL and a carboxyl-containing daptomycin solution with a molar mass concentration of 50 µmol / L at a volume ratio of 250:1 to obtain a daptomycin-functionalized ferroferric oxide nanosphere solution, wherein the daptomycin is used to specifically identify Listeria monocytogenes; the particle size of the magnetic separation probe is 100 nm-200 nm, and there is an ultraviolet absorption peak at 235 nm; The magnetic separation probe is used to specifically bind to Listeria monocytogenes and separate and enrich the bacteria from the test sample; The silicon-based nanozyme probe is prepared by amidation covalently coupling an amino-containing silicon-based nanozyme microsphere solution with a mass concentration of 40ug / mL and a carboxyl-containing nucleic acid aptamer solution with a molar mass concentration of 10µmol / L at a volume ratio of 20:1 to obtain an aptamer-functionalized silicon-based nanozyme microsphere solution, the DNA sequence of the nucleic acid aptamer is shown in SEQ ID No: 1, the 5' end of the nucleic acid aptamer is modified with a carboxyl group, and the nucleic acid aptamer is used to specifically identify Listeria monocytogenes; the particle size of the silicon-based nanozyme probe is 10nm-30nm, and there is an ultraviolet absorption peak at 251nm; The silicon-based nanozyme probe is used to specifically bind to Listeria monocytogenes after separation and enrichment treatment, and generate fluorescence signals, colorimetric signals and photothermal signals that are dependent on the concentration of Listeria monocytogenes; The preparation operation of the amino-containing silicon-based nanozyme microsphere solution is as follows: Add 12 mL of deionized water to 1 mL of N-(β-aminoethyl-γ-aminopropyl)methyldimethoxysilane; then add 10 mg of amikacin and 1 mL of 200 mmol / L ferrous sulfate solution, and stir continuously for 10 min; heat at 180 ° C for 8 h, cool to room temperature, and dilute 30 times to obtain an amino-containing silicon-based nanozyme microsphere solution; The preparation operation of the silicon-based nanozyme probe is as follows: (1) Vortex mix 25 µL of 10 µmol / L carboxyl-containing aptamer solution and 200 µL of activation mixture, and incubate on a shaker at 200 rpm for 30 min at room temperature to obtain an activated aptamer solution; (2) Add 500 µL of amino-containing silicon-based nanozyme microsphere solution to 200 uL of activated nucleic acid aptamer solution with a molar concentration of 1.25 µmol / L, vortex mix, and incubate on a shaker at 80 rpm at room temperature for 2.5 h to obtain an aptamer-functionalized silicon-based nanozyme microsphere solution, which is a silicon-based nanozyme probe.

2. The method for preparing a dual-probe composition for detecting Listeria monocytogenes according to claim 1, characterized in that: The steps are as follows: (1) Preparation of activation mixture Mix 10 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 5 mmol / L N-hydroxysuccinimide in a volume ratio of 1:1 to obtain an activated mixed solution; (2) Preparation of magnetic separation probe (2.1) vortexing the carboxyl group-containing daptomycin solution and the activated mixed solution at a volume ratio of 0.012:1, and incubating on a shaker at room temperature to obtain an activated daptomycin solution; (2.2) adding an amino-containing ferroferric oxide nanosphere solution to the activated daptomycin solution and vortex mixing, wherein the amount of the amino-containing ferroferric oxide nanosphere solution added is 250 times the volume of the carboxyl-containing daptomycin solution, incubating on a shaking table, and removing the supernatant by magnetic separation to obtain a daptomycin-functionalized ferroferric oxide nanosphere solution; (2.3) adding a 1% by volume bovine serum albumin solution to the daptomycin-functionalized ferroferric oxide nanosphere solution to block the residual sites of ferroferric oxide, and removing the supernatant by magnetic separation again to obtain a blocked ferroferric oxide nanosphere solution; (2.4) Washing the sealed ferroferric oxide nanosphere solution with sterile Tris-HCl buffer for three times, and resuspending to obtain a magnetic separation probe solution; the mass concentration of the magnetic separation probe solution is 1 mg / mL; When the ZETA potential value of the amino-containing ferroferric oxide nanoparticle solution increased from +25.37 mV to +33.13 mV, it indicated that the carboxyl-containing daptomycin in the magnetic separation probe was successfully modified onto the amino-containing ferroferric oxide nanoparticles; (3) Preparation of silicon-based nanozyme probes (3.1) Vortex-mix the carboxyl-containing aptamer solution and the activation mixture at a volume ratio of 0.125:1, and incubate on a shaker at room temperature to obtain an activated aptamer solution; (3.2) Adding an amino-containing silicon-based nanozyme microsphere solution to the activated nucleic acid aptamer solution and vortex mixing, wherein the amount of the amino-containing silicon-based nanozyme microsphere solution added is 20 times the volume of the carboxyl-containing nucleic acid aptamer solution, and incubating on a shaker at room temperature to obtain an aptamer-functionalized silicon-based nanozyme microsphere solution, i.e., a silicon-based nanozyme probe; the mass concentration of the silicon-based nanozyme probe is 40 ug / mL; When the ZETA potential value of the amino-containing silicon-based nanozyme microsphere solution dropped from + 18.84 mV to + 8.27 mV, it indicated that the carboxyl-containing nucleic acid aptamer in the silicon-based nanozyme probe was successfully modified onto the amino-containing silicon-based nanozyme microsphere; (4) Preparation of dual probe composition The magnetic separation probe and the silicon-based nanozyme probe were stored at 4° C. in a volume ratio of 1:2 to prepare a dual-probe composition; When used for detecting Listeria monocytogenes, the magnetic separation probe is first combined with Listeria monocytogenes in a detection sample solution, and the added amount of the detection sample solution is twice the volume of the magnetic separation probe to obtain a complex of the magnetic separation probe and Listeria monocytogenes; A silicon-based nanozyme probe is added to the magnetic separation probe and Listeria monocytogenes complex to obtain a magnetic separation probe, Listeria monocytogenes and silicon-based nanozyme probe complex, and then the detection of the sample solution is completed through three signal channels: fluorescence, colorimetry and photothermal.

3. The method for preparing the dual-probe composition according to claim 2, characterized in that: In step (2), (2.1) 2.4 uL of 50 μmol / L carboxyl-containing daptomycin solution was vortexed with 200 uL of the activated mixture, and incubated at room temperature at 200 rpm on a shaker for 30 min to obtain an activated daptomycin solution with a molar concentration of 0.6 μmol / L; (2.2) Add 600 uL of 3 mg / mL amino-containing ferroferric oxide nanosphere solution to 200 uL of 0.6 μmol / L activated daptomycin solution, vortex mix, incubate on a shaker at 180 rpm and 37 °C for 3 h, remove the supernatant, and obtain daptomycin-functionalized ferroferric oxide nanosphere precipitate; (2.3) adding 600 uL of 1% bovine serum albumin solution to the daptomycin-functionalized ferroferric oxide nanosphere precipitate to block the residual sites of ferroferric oxide, performing magnetic separation again using a magnet, removing the supernatant, and obtaining a blocked ferroferric oxide nanosphere precipitate; (2.4) Washing the sealed ferroferric oxide nanosphere precipitate with sterile Tris-HCl buffer for three times, and resuspending the precipitate in 1 mL of sterile Tris-HCl buffer to prepare a magnetic separation probe; the sterile Tris-HCl buffer has a molar concentration of 25 mmol / L, a pH value of 7.4, and contains a molar concentration of 5 mmol / L of calcium chloride.

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