Based on Cu + A method for homogeneous immunodetection of foodborne pathogens using low-field nuclear magnetic resonance mediated click chemistry gel system

Abstract

The present invention discloses a Cu-based + The invention relates to a method for homogeneous immunodetection of foodborne pathogens by low-field nuclear magnetic resonance mediated click chemistry gel system, which is characterized by comprising the following steps: 1) binding a foodborne pathogen aptamer to the surface of HMSMs@SAA to obtain a signal unit HMSMs@SAA&Apt; 2) coupling an alkynyl group and an azide group to PEG and PAA to obtain gel precursors Azide-PEG-Azide and PAA-Alkyne; 3) mixing a PAA-Alkyne solution, an Azide-PEG-Azide solution, an HMSMs@SAA&Apt dispersion, and a CuSO4 solution in a volume ratio of 10:10:1:1 to obtain an LF-NMR test solution, which is then mixed with a foodborne pathogen sample to determine the concentration of the foodborne pathogen in an unknown sample. The method has the advantages of high sensitivity and accuracy, strong specificity, and simple and rapid operation.

Description

Technical Field

[0001] The present invention relates to a method for detecting foodborne pathogens, in particular to a method based on Cu + A method for homogeneous immunodetection of foodborne pathogens using low-field nuclear magnetic resonance mediated click chemistry gel system. Background Art

[0002] Foodborne pathogens refer to pathogenic bacteria that can cause food poisoning or spread through food. Common foodborne pathogens include: Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli, Salmonella, etc. Current detection technologies mainly include: (1) Traditional microbial enumeration methods, such as agar plate and broth dilution assay, inhibition zone method, membrane filtration method, etc. (2) Molecular biology detection, including polymerase chain reaction (PCR), polymerase spiral reaction (PSR), loop-mediated isothermal amplification (LAMP), nucleic acid sequence amplification (NASBA), gene chip, etc. (3) Immunological detection, including electrochemiluminescence analysis (ECL), enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FIA), electrochemical impedance spectroscopy (EIS), surface enhanced Raman scattering (SERS), colorimetry, etc. These methods offer advantages such as high specificity and sensitivity, but they typically require skilled technicians, complex sample pretreatment steps, and expensive instrumentation. This makes them difficult to meet the requirements for rapid, real-time, and portable on-site testing in economically underdeveloped regions and areas experiencing sudden, large-scale epidemics. Therefore, developing a method that can achieve highly sensitive, on-site detection of common pathogens is crucial.

[0003] Nuclear magnetic resonance (NMR) relaxometry utilizes time-domain NMR at low magnetic fields (<1.0T) to measure the relaxation process of water and food samples. Water molecules are the primary signal source. The relaxation time is related to the mobility and dynamics of water molecules, which in turn are closely related to their surroundings. Therefore, NMR relaxometry using water molecules as the signal source primarily modulates the surrounding environment to affect the state of water molecules, thereby detecting the target. The most commonly used method utilizes a magnetic relaxation switch (MRS) sensor to achieve signal transitions. MRS offers advantages such as simple sample pretreatment, fast response, high sensitivity, and low background signal. It has been applied to the detection of biomacromolecules, pathogens, pesticide residues, and heavy metals. The detection principle relies on the target-mediated changes in the aggregation and dispersion state or number of magnetic nanoparticles, which affect the transverse relaxation time (T2) of water molecules within the system, enabling qualitative and quantitative analysis of the target. However, this method has limitations, including instability and low sensitivity due to the wide particle size distribution of magnetic labels, uncontrollable aggregation, and the low saturation magnetization of paramagnetic ions. Therefore, it is imperative to develop a non-magnetic relaxation switch sensor that can analyze water molecules by directly changing their state and affecting their relaxation time. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a Cu-based + A method for homogeneous immunodetection of foodborne pathogens using low-field nuclear magnetic resonance mediated click chemistry gel system.

[0005] The technical solution adopted by the present invention to solve the above technical problems is: a Cu-based + A method for homogeneous immunodetection of foodborne pathogens using low-field nuclear magnetic resonance mediated click chemistry gel system, which is not intended for diagnosis or treatment, comprises the following steps:

[0006] (1) Synthesis of signaling unit HMSMs@SAA&Apt

[0007] A. Loading sodium ascorbate (SAA) into aminated mesoporous hollow silica spheres (HMSMs) to obtain HMSMs@SAA.

[0008] B. The foodborne pathogen aptamer (Apt) was bound to the surface of HMSMs@SAA through electrostatic attraction and then all mesopores were sealed to obtain the signaling unit HMSMs@SAA&Apt.

[0009] (2) Preparation of LF-NMR (low-field nuclear magnetic resonance) test solution

[0010] A. Dissolve 80-120 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 10-20 mg of N-hydroxysuccinimide (NHS), and 250-300 mg of dicarboxy polyethylene glycol (HOOC-PEG-COOH) in 10 mL of water, adjust the pH to 5.0, and stir at room temperature for 30 min. Add 0.5-1 mL of 0.5 mol / L 3-azidopropylamine solution and allow the EDC / NHS coupling reaction to proceed for 3 h. After the reaction, transfer the product to a dialysis membrane with a molecular weight cutoff of 1000 Da and dialyze for 12 h to remove unreacted 3-azidopropylamine, EDC, and NHS. Then, freeze-dry the product to obtain azido-modified polyethylene glycol (Azide-PEG-Azide) powder.

[0011] B. Dissolve 500-550 mg of EDC, 300-350 mg of NHS, and 0.5-1 g of 35 wt% polyacrylic acid (PAA) in 10 mL of water, adjust the pH to 5.0, and stir at room temperature for 30 min. Add 100-200 mg of propargylamine and allow the EDC / NHS coupling reaction to proceed for 3 h. The product is dialyzed for 12 h using a 1000 Da molecular weight cutoff dialysis membrane to remove unreacted EDC, NHS, and propargylamine to obtain a propargylamine-modified polyacrylic acid (PAA-Alkyne) solution.

[0012] C. 2 wt% PAA-Alkyne solution, 1.4×10 -2 mol / L Azide-PEG-Azide solution, 0.5 mg / mL HMSMs@SAA&Apt dispersion, 1.0×10 -4 mol / L CuSO4 solution in a volume ratio of 10:10:1:1 to obtain the LF-NMR test solution;

[0013] (3) Low-field NMR homogeneous immunoassay

[0014] 1.0 mL of LF-NMR test solution and 100 μL of a foodborne pathogen sample solution were added to a 1.5 mL sample vial. After 15 minutes of reaction, T2 measurements were performed. The CPMG pulse sequence measurement method was used to determine the transverse relaxation time difference ΔT2 of water protons corresponding to a series of different foodborne pathogen concentrations. A quantitative relationship was established between the transverse relaxation time difference of water protons and the concentration of foodborne pathogens. Based on this quantitative relationship, the concentration of foodborne pathogens in unknown samples could be determined. ΔT2 was calculated using the following formula: ΔT2 = T2(negative) – T2(positive), where T2 is the transverse relaxation time of water protons, T2(negative) is the average T2 in the absence of foodborne pathogens, and T2(positive) is the average T2 in the presence of foodborne pathogens. A higher concentration of foodborne pathogens corresponds to a smaller reduction in T2.

[0015] Furthermore, the specific process of step (1) A is as follows: 550-650 mg of SAA is dissolved in 1 mL of 1 mg / mL HMSMs dispersion, the pH is adjusted to 5.0, and the mixture is stirred in the dark at room temperature for 24 h to allow SAA to fully enter the internal cavity of HMSMs to obtain a HMSMs@SAA dispersion.

[0016] Furthermore, the specific process of step (1) B is as follows: adjust the pH of HMSMs@SAA dispersion to 7.0, add optical density OD 600 The dispersion of foodborne pathogen aptamers (Apt) with a value of 1 to 2 was stirred at room temperature for 30 minutes. Apt bound to the surface of HMSMs@SAA through electrostatic attraction and sealed all mesopores. The suspension was centrifuged at 3405×g for 5 minutes, washed with water to remove free Apt, and then dispersed in 2 mL of water to obtain the signal unit HMSMs@SAA&Apt dispersion.

[0017] Furthermore, the CPMG pulse sequence measurement method is as follows: T2 is measured at 35°C, and the parameters are as follows: main frequency 19.00 MHz, sampling frequency 100 kHz, RF delay 0.5 ms, waiting time 5000 ms, echo time 1.2 ms, number of echoes 18000, accumulation times 2, digital gain 3, and analog gain 15.0 dB.

[0018] Furthermore, the foodborne pathogenic bacteria are selected from Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli and Salmonella.

[0019] Principle of the invention: The signal unit is HMSMs@SAA&Apt, which can quickly identify the target. When the target foodborne pathogens appear, Apt falls off the surface of HMSMs@SAA&Apt, releasing the SAA saturated solution inside the HMSMs@SAA. The release of SAA alone cannot be converted into a change in the nuclear magnetic resonance signal. Therefore, this work designed a gel relaxation switch sensor with amplified signal effect, which relies on Cu + The hydrogel system acts as a bridge for signal conversion. The release of SAA can convert Cu 2+ Reduction to Cu + The precursors Azide-PEG-Azide and PAA-Alkyne in the hydrogel system are + A click chemistry reaction occurs under the guidance of a sol-gel transition mechanism, forming a three-dimensional network gel structure. The sol-gel transition causes a change in the transverse relaxation time T2 of water molecules in the test solution, and the concentration of the target compound is estimated based on the change in T2, ΔT2. By incorporating click chemistry, this design successfully converts the SAA release signal into a sol-gel transition signal. Furthermore, the system contains no magnetic materials, avoiding their negative impact and simplifying the detection process, achieving a successful "one-step" detection method.

[0020] According to the immunological principle of the present invention, HMSMs@SAA and Apt are combined with each other through electrostatic adsorption, which makes the signal probe specific. When the target is present, HMSMs@SAA&Apt will specifically recognize the target, causing Apt to fall off and release SAA, which in turn leads to the occurrence of a copper-mediated click chemistry reaction, causing the solution to undergo a sol-gel transition. The test liquid is placed in a low-field nuclear magnetic resonance instrument for detection. Adjusting the concentration of the target can directly control the concentration of SAA and indirectly control the degree of cross-linking of the gel system, thereby controlling the strength of the effect on the water molecules T2 in the system. The reduction amount ΔT2 of the water molecules T2 in the system shows a certain relationship with the concentration of foodborne pathogens. Under a specific working curve, the unknown concentration of foodborne pathogens in the sample can be tested.

[0021] Compared with the prior art, the advantages of the present invention are:

[0022] 1. Simple sample pretreatment: Because LF-NMR signals originate from water molecules rather than photoelectric properties, and because magnetic materials are virtually absent in the detection environment, LF-NMR is virtually free of background interference and can even directly detect turbid samples. For individual pathogens, direct testing can be performed without DNA extraction and amplification.

[0023] 2. Avoiding the frontal zone effect caused by traditional MRS: Traditional MRS sensors derive their magnetic signal from the state of the magnetic probe, often resulting in low detection limits and a narrow linear range due to issues with the target-to-probe ratio. The sensor signal of the present invention derives from changes in the state of water molecules, thus avoiding these issues.

[0024] 3. Dual signal amplification improves detection sensitivity: (1) The hydrogel's three-dimensional network structure contains abundant hydrophilic groups, with a large amount of water combining with them to form bound water, achieving a "binding" effect. (2) The hydrogel's three-dimensional network structure forms a cavity-like structure, which can be seen as small pores with similar pore sizes hindering the free movement of water molecules, "binding" them. The gel system achieves dual signal amplification through these two "binding" effects. Dual signal amplification greatly improves detection sensitivity.

[0025] In summary, the present invention is based on Cu + A method for homogeneous immunodetection of foodborne pathogens using low-field nuclear magnetic resonance mediated click chemistry gel system was developed. A non-magnetic relaxation switch sensor was designed to realize a "one-step" T2 detection strategy. The click chemistry hydrogel system with controllable sol-gel transition process was combined with low-field nuclear magnetic resonance detection. Compared with the traditional method of affecting T2 by changing the state of magnetic labels, the detection method of affecting T2 by directly changing the state of water protons by using the binding effect of the gel structure on water molecules effectively avoids the instability caused by magnetic labels, and the detection limit of VP reaches 10 CFU / mL. This detection method can achieve sensitive and convenient detection of different pathogens by simply changing the corresponding target recognition elements. It has broad prospects in the field of rapid on-site detection of pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the principle of low-field nuclear magnetic resonance homogeneous immunoassay for foodborne pathogens of the present invention;

[0027] Figure 2 This is an electron microscope image of the sol state of the present invention;

[0028] Figure 3 This is an electron microscope image of the gel state of the present invention;

[0029] Figure 4 This is the linear relationship diagram of low-field nuclear magnetic resonance detection of different foodborne pathogens (VP) concentrations;

[0030] Figure 5 Figure 3. Sensor specificity diagram for low-field NMR detection of different bacterial species at the same concentration. DETAILED DESCRIPTION

[0031] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments. 1. Specific embodiments

[0033] Based on Cu + A method for homogeneous immunoassay of foodborne pathogens using low-field nuclear magnetic resonance mediated click chemistry gel system. This method is not intended for diagnosis or treatment. Figure 1 As shown, the following steps are included:

[0034] (1) Synthesis of signaling unit HMSMs@SAA&Apt

[0035] A. Sodium ascorbate (SAA) was loaded onto aminated mesoporous hollow silica spheres (HMSMs) to obtain HMSMs@SAA. The specific process was as follows: 600 mg of SAA was dissolved in 1 mL of a 1 mg / mL HMSMs dispersion, the pH was adjusted to 5.0, and the mixture was stirred in the dark at room temperature for 24 h to allow SAA to fully enter the HMSMs cavity, resulting in a HMSMs@SAA dispersion.

[0036] B. The foodborne pathogen aptamer (Apt) was bound to the surface of HMSMs@SAA through electrostatic attraction to seal all the mesopores, and the signal unit HMSMs@SAA&Apt was obtained. The specific process was as follows: the pH of the HMSMs@SAA dispersion was adjusted to 7.0, and OD 600 A dispersion of foodborne pathogen aptamers (Apt) with a pH value of 1 to 2 was stirred at room temperature for 30 min. Apt bound to the surface of HMSMs@SAA through electrostatic attraction and sealed all mesopores. The suspension was centrifuged at 3405 × g for 5 min, washed with water to remove free Apt, and then dispersed in 2 mL of water to obtain a dispersion of signaling unit HMSMs@SAA&Apt.

[0037] (2) Preparation of LF-NMR (low-field nuclear magnetic resonance) test solution

[0038] A. Dissolve 100 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 15 mg of N-hydroxysuccinimide (NHS), and 280 mg of HOOC-PEG-COOH in 10 mL of water, adjust the pH to 5.0, and stir at room temperature for 30 min. Add 0.8 mL of 0.5 mol / L 3-azidopropylamine solution and allow the EDC / NHS coupling reaction to proceed for 3 h. After the reaction, transfer the product to a dialysis membrane with a molecular weight cutoff of 1000 Da and dialyze for 12 h to remove unreacted 3-azidopropylamine, EDC, and NHS. Then, freeze-dry to obtain Azide-PEG-Azide powder.

[0039] B. Dissolve 520 mg of EDC, 320 mg of NHS, and 0.8 g of 35 wt% PAA in 10 mL of water, adjust the pH to 5.0, and stir at room temperature for 30 min. Add 150 mg of propargylamine and allow the EDC / NHS coupling reaction to proceed for 3 h. The product is then dialyzed using a 1000 Da dialysis membrane for 12 h to remove unreacted EDC, NHS, and propargylamine to obtain a PAA-Alkyne solution.

[0040] C. 2 wt% PAA-Alkyne solution, 1.4×10 -2 mol / L Azide-PEG-Azide solution, 0.5 mg / mL HMSMs@SAA&Apt dispersion, 1.0×10 -4 mol / L CuSO4 solution in a volume ratio of 10:10:1:1 to obtain the LF-NMR test solution;

[0041] (3) Low-field NMR homogeneous immunoassay

[0042] 1.0 mL of LF-NMR test solution and 100 μL of foodborne pathogen sample solution were added to a 1.5 mL sample vial. After 15 minutes of reaction, T2 measurement was performed. The CPMG pulse sequence measurement method was used to determine the magnitude of the transverse relaxation time difference ΔT2 of water protons corresponding to a series of different concentrations of foodborne pathogens. A quantitative relationship between the transverse relaxation time difference of water protons and the concentration of foodborne pathogens was established. Based on this quantitative relationship, the concentration of foodborne pathogens in unknown samples can be determined. The ΔT2 value is calculated by the following formula: ΔT2 = T2(negative) – T2(positive), where T2 is the transverse relaxation time of water protons, T2(negative) is the average T2 in the absence of foodborne pathogens, and T2(positive) is the average T2 in the presence of foodborne pathogens. The CPMG pulse sequence measurement method is as follows: T2 is measured at 35°C with the following parameters: main frequency 19.00 MHz, sampling frequency 100 kHz, RF delay 0.5 ms, waiting time 5000 ms, echo time 1.2 ms, number of echoes 18000, accumulation times 2, digital gain 3, and analog gain 15.0 dB.

[0043] The foodborne pathogenic bacteria are selected from Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli and Salmonella.

[0044] Example 2

[0045] The same as the above embodiment 1, the difference is:

[0046] Step (1) Synthesis of the signal unit HMSMs@SAA&Apt: 550-650 mg of SAA was dissolved in 1 mL of a 1 mg / mL HMSMs dispersion, the pH was adjusted to 5.0, and the mixture was stirred in the dark at room temperature for 24 h to obtain a HMSMs@SAA dispersion.

[0047] Step (2) Preparation of LF-NMR test solution:

[0048] A. Dissolve 80 mg of EDC, 10 mg of NHS, and 250-300 mg of HOOC-PEG-COOH in 10 mL of water, adjust the pH to 5.0, and stir at room temperature for 30 min. Add 0.5 mL of 0.5 mol / L 3-azidopropylamine solution and allow the EDC / NHS coupling reaction to proceed for 3 h. After the reaction, dialyze for 12 h to obtain Azide-PEG-Azide powder.

[0049] B. Dissolve 500 mg of EDC, 300 mg of NHS, and 0.5 g of 35 wt% PAA in 10 mL of water, adjust the pH to 5.0, and stir at room temperature for 30 min. Add 100 mg of propargylamine and allow the EDC / NHS coupling reaction to proceed for 3 h. The product is dialyzed using a dialysis membrane for 12 h to obtain a PAA-Alkyne solution.

[0050] C. 2 wt% PAA-Alkyne solution, 1.4×10 -2 mol / L Azide-PEG-Azide solution, 0.5 mg / mL HMSMs@SAA&Apt dispersion, 1.0×10 -4 mol / L CuSO4 solution in a volume ratio of 10:10:1:1 to obtain the LF-NMR test solution.

[0051] Example 3

[0052] The same as the above embodiment 1, the difference is:

[0053] In step (1) synthesis of the signal unit HMSMs@SAA&Apt: 650 mg of SAA was dissolved in 1 mL of a 1 mg / mL HMSMs dispersion, the pH was adjusted to 5.0, and the mixture was stirred in the dark at room temperature for 24 h to obtain a HMSMs@SAA dispersion; in step (2) preparation of the LF-NMR test solution:

[0054] A. Dissolve 120 mg of EDC, 20 mg of NHS, and 300 mg of HOOC-PEG-COOH in 10 mL of water, adjust the pH to 5.0, and stir at room temperature for 30 min. Add 1 mL of 0.5 mol / L 3-azidopropylamine solution. Incubate the EDC / NHS coupling reaction for 3 h. After the reaction, dialyze for 12 h to obtain Azide-PEG-Azide powder.

[0055] B. Dissolve 550 mg of EDC, 350 mg of NHS, and 1 g of 35 wt% PAA in 10 mL of water, adjust the pH to 5.0, and stir at room temperature for 30 min. Add 200 mg of propargylamine and allow the EDC / NHS coupling reaction to proceed for 3 h. The product is dialyzed using a dialysis membrane for 12 h to obtain a PAA-Alkyne solution.

[0056] C. 2 wt% PAA-Alkyne solution, 1.4×10 -2 mol / L Azide-PEG-Azide solution, 0.5 mg / mL HMSMs@SAA&Apt dispersion, 1.0×10 -4 mol / L CuSO4 solution in a volume ratio of 10:10:1:1 to obtain the LF-NMR test solution.

[0057] 2. Experimental Results Analysis

[0058] 1. Gel system analysis

[0059] The LF-NMR test solution is in a sol state without the target object to be detected. Figure 2 As shown in Figure 2, in the sol state, uneven micropores can be observed on the surface of the precursor, which is caused by the sublimation of water during the freeze-drying process. There is no obvious three-dimensional network structure. After adding VP to the LF-NMR test solution, the solution changes from a sol state to a gel state. Figure 3 As shown in the figure, the three-dimensional network structure of the hydrogel can be clearly observed in the gel state, which indicates that the hydrogel with PAA and PEG as precursors can fix water molecules very well, thereby directly affecting the T2 of the system and achieving effective sensing.

[0060] 2. Sensitivity analysis

[0061] The method of the first embodiment was used to study the Cu + The detection performance of the relaxation switch sensor mediated by click chemistry gel system for VP. As the VP concentration increases from 1 to 1.0×10 8 As CFU / mL increases, the transverse relaxation decay rate accelerates and T2(positive) shortens. Therefore, when T2(negative) remains unchanged, ΔT2 increases with the increase of VP concentration. Figure 4As shown, in the range of 10 CFU / mL to 1.0×10 8 Within the CFU / mL concentration range, the logarithm of ΔT2(y,ms) and VP concentration (x,CFU / mL) showed a good linear relationship, and the linear regression equation was y=7.65+102.214logx, R 2 =0.998, and the detection limit of VP by LF-NMR was 10 CFU / mL, demonstrating excellent sensitivity and a wide linear detection range. This is due to the abundant hydrophilic groups on the surface of the gel network structure and the dual "binding" of the network framework, which achieves dual signal amplification.

[0062] 3. Specificity analysis experimental method: In order to investigate the selectivity of the sensor, 1.0×10 4 CFU / mL of VP standard solution, Listeria monocytogenes (LM), Staphylococcus aureus (SA), Vibrio vulnificus (VV), Escherichia coli (EC), Salmonella (SM), blank samples and mixed samples (containing 1.0×10 4 The experiments were performed with SA, EC, VV, LM, SM and VP at a concentration of 100 CFU / mL.

[0063] like Figure 5 As shown, 1.0×10 4 The ΔT2 of LM, SA, VV, EC, SM and blank samples with CFU / mL were very small and had no significant difference. 4 The ΔT2 generated by the CFU / mL VP standard solution was almost the same, indicating that the sensor has good selectivity. This is attributed to the specific recognition ability of Apt on the signal unit, and other bacteria do not produce a response signal.

[0064] 4. Accuracy analysis

[0065] Real seawater and oyster samples were used for spike recovery testing. The seawater samples were collected from the East China Sea and filtered through a 0.45 μm filter membrane before use. Different concentrations of VP (1.0×10 2 ,1.0×10 3 ,1.0×10 4 ,1.0×10 5CFU / mL) was added to real seawater samples for experiments. Oyster samples were purchased from the local seafood market in Ningbo, placed in a fresh-keeping ice box and transported to the laboratory. 30g of oyster meat was taken, ground, spread on a culture dish, and irradiated with ultraviolet light for 30 minutes to remove the VP in the oyster sample. VP standard solutions with different concentration gradients were injected into the oyster sample and incubated for 30 minutes. The above-mentioned spiked oyster sample was added to 50mL of sterile PBS buffer, shaken for 5 minutes, and the upper homogenate was taken for experiment. The results were compared with the plate count method, and the recovery rate was calculated. The negative blank sample used sterile PBS buffer instead of oysters and seawater for experiments. The detection method was as described in Specific Example 1, and there were five parallel samples. The results are shown in Table 1. Table 1. Detection of VP content in seawater and oyster samples ( n=5)

[0066]

[0067]

[0068] As shown in Table 1, the recoveries of seawater samples ranged from 96.6% to 106.4%, with RSDs of 2.8% to 6.3%. The recoveries of oyster samples ranged from 97.6% to 105.3%, with RSDs of 2.5% to 6.3%. A t-test was performed between the groups and the plate count results. The calculated t values ​​for seawater and oyster samples were 1.19 and 0.11, respectively, both less than t 0.10,3 =2.35, indicating that there was no significant difference between the two methods. This method has good precision and accuracy and can be used to reliably detect real samples.

[0069] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. A Cu-based + A method for homogeneous immunodetection of foodborne pathogens by low-field nuclear magnetic resonance mediated click chemistry gel system, which is not intended for diagnosis or treatment, is characterized by The following steps are involved: (1) Synthesis of signaling unit HMSMs@SAA&Apt A. Loading sodium ascorbate SAA into aminated mesoporous hollow silica spheres HMSMs to obtain HMSMs@SAA; B. The foodborne pathogen aptamer Apt was bound to the surface of HMSMs@SAA through electrostatic attraction and then all mesopores were sealed to obtain the signaling unit HMSMs@SAA&Apt. (2) Preparation of low-field nuclear magnetic resonance (LF-NMR) test solution A. Dissolve 80-120 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 10-20 mg of N-hydroxysuccinimide (NHS), and 250-300 mg of dicarboxy polyethylene glycol (HOOC)-PEG-COOH in 10 mL of water, adjust the pH to 5.0, and stir at room temperature for 30 minutes. Add 0.5-1 mL of 0.5 mol / L 3-azidopropylamine solution and allow the coupling reaction to proceed for 3 hours. After the reaction, transfer the product to a dialysis membrane with a molecular weight cutoff of 1000 Da and dialyze it for 12 hours. Then, freeze-dry it to obtain azido-modified polyethylene glycol (Azide-PEG-Azide) powder. B. Dissolve 500-550 mg of EDC, 300-350 mg of NHS, and 0.5-1 g of 35 wt% polyacrylic acid (PAA) in 10 mL of water, adjust the pH to 5.0, and stir at room temperature for 30 min. Add 100-200 mg of propargylamine and allow the coupling reaction to proceed for 3 h. The product is dialyzed for 12 h using a 1000 Da molecular weight cutoff dialysis membrane to obtain a propargylamine-modified polyacrylic acid (PAA)-Alkyne solution. C. 2 wt% PAA-Alkyne solution, 1.4×10 -2 mol / L Azide-PEG-Azide solution, 0.5 mg / mL HMSMs@SAA&Apt dispersion, 1.0×10 -4 mol / L CuSO4 solution in a volume ratio of 10:10:1:1 to obtain the LF-NMR test solution; (3) Low-field NMR homogeneous immunoassay 1.0 mL of LF-NMR test solution and 100 μL of foodborne pathogen sample solution were added to a sample vial. After a 15-minute reaction, the CPMG pulse sequence measurement method was used to measure the transverse relaxation time difference ΔT2 of water protons corresponding to a series of different concentrations of foodborne pathogens. A quantitative relationship between the transverse relaxation time difference of water protons and the concentration of foodborne pathogens was established. Based on this quantitative relationship, the concentration of foodborne pathogens in unknown samples can be determined. The ΔT2 value is calculated by the following formula: ΔT2 = T2(negative) – T2(positive), where T2 is the transverse relaxation time of water protons, T2(negative) is the average T2 in the absence of foodborne pathogens, and T2(positive) is the average T2 in the presence of foodborne pathogens.

2. The Cu-based + A method for homogeneous immunodetection of foodborne pathogens by low-field nuclear magnetic resonance mediated click chemistry gel system, which is not intended for diagnosis or treatment, is characterized by The specific process of step (1) A is as follows: 550-650 mg of SAA is dissolved in 1 mL of 1 mg / mL HMSMs dispersion, the pH is adjusted to 5.0, and the mixture is stirred in the dark at room temperature for 24 h to obtain a HMSMs@SAA dispersion.

3. The Cu-based + A method for homogeneous immunodetection of foodborne pathogens by low-field nuclear magnetic resonance mediated click chemistry gel system, which is not intended for diagnosis or treatment, is characterized by The specific process of step (1) B is as follows: adjust the pH of HMSMs@SAA dispersion to 7.0, add OD 600 The foodborne pathogen aptamer dispersion with a value of 1 to 2 was stirred at room temperature for 30 minutes, and the suspension was centrifuged at 3405×g for 5 minutes, washed with water, and then dispersed in 2 mL of water to obtain the signal unit HMSMs@SAA&Apt dispersion.

4. The Cu-based + A method for homogeneous immunodetection of foodborne pathogens by low-field nuclear magnetic resonance mediated click chemistry gel system, which is not intended for diagnosis or treatment, is characterized by The CPMG pulse sequence measurement method described in step (3) is as follows: T2 is measured at 35°C, and the parameters are as follows: main frequency 19.00 MHz, sampling frequency 100 kHz, RF delay 0.5 ms, waiting time 5000 ms, echo time 1.2 ms, number of echoes 18000, accumulation times 2, digital gain 3, and analog gain 15.0 dB.

5. The Cu-based method according to any one of claims 1 to 4. + A method for homogeneous immunodetection of foodborne pathogens by low-field nuclear magnetic resonance mediated click chemistry gel system, which is not intended for diagnosis or treatment, is characterized by The foodborne pathogenic bacteria are selected from Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli and Salmonella.

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