A method for detecting foodborne pathogens using a dual-mode well-plate low-field nuclear magnetic resonance / colorimetry method

Abstract

The present invention discloses a method for detecting foodborne pathogens using a dual-mode low-field nuclear magnetic resonance (NMR) / colorimetry method in a well plate. The method comprises the following steps: 1) synthesizing a signal unit MNS@Ab2; 2) forming an MNS@Ab2-VP-Ab1 composite structure on the wall of a 96-well plate, adding sulfuric acid to each well, and then adding a potassium permanganate solution, and measuring the absorbance with a microplate reader for colorimetric analysis; 3) disassembling the 96-well plate into separate small cups and placing them in a NMR test tube, performing T1 measurement using an IR pulse sequence measurement method, and calculating the ΔT1 value using the following formula: ΔT1=T1(negative)–T1(positive). The concentration of the foodborne pathogens in the test solution is calculated based on the current signal value. The method has the advantages of high sensitivity, strong specificity, good accuracy, 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 for detecting foodborne pathogens using a well plate type low-field nuclear magnetic resonance / colorimetry dual mode. Background Art

[0002] Nuclear magnetic resonance switch (MRS) magnetic biosensors use magnetic nanoparticles as signal tags and low-field nuclear magnetic resonance as a method to detect targets such as chemical molecules, biological molecules, viruses, and pathogens. The detection principle is that the presence of the target causes the dispersion state or concentration of the magnetic nanoparticles to change, so the local magnetic field generated by them changes, affecting the relaxation process of the surrounding water molecules. By establishing a functional relationship between the change in the transverse relaxation time T2 or longitudinal relaxation time T1 of water molecules and the concentration of the target, quantitative detection of the target can be completed. Since biological or environmental samples generally do not contain magnetic substances, magnetic biosensors have unique advantages in anti-interference ability. However, MRS magnetic biosensors also have some technical obstacles: (1) Low sensitivity. The main reason is that the magnetic saturation intensity of the nanoscale signal tags is not high and the number is limited. (2) Poor stability. The composition and size of the magnetic nanoparticles themselves are not completely uniform, and they tend to settle under the action of gravity. Therefore, their distribution in the system is difficult to be homogeneous, resulting in limited stability of the analysis results. Colorimetry is a method of determining the content of a component to be tested by comparing or measuring the color depth of a colored substance solution.

[0003] The so-called dual-mode detection is to use two methods with different detection principles to detect the same target object. The interference factors of methods with different detection principles are different. For example, the magnetic method is easily affected by the surrounding electromagnetic environment and ambient temperature, the optical method is easily affected by ambient light, scattering, refraction, etc., and the electrical signal is easily affected by the surrounding electromagnetic environment, the circuit system itself, etc. By using dual-mode detection, the results obtained by the two methods can be cross-checked, effectively eliminating their respective system errors and improving the accuracy and reliability of the detection. At present, there are no public research reports at home and abroad on the method of detecting foodborne pathogens based on the dual-channel output of low-field nuclear magnetic resonance / colorimetry. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for detecting foodborne pathogens by a dual-mode well-plate low-field nuclear magnetic resonance / colorimetry method with high sensitivity, strong specificity, good accuracy, and simple and rapid operation.

[0005] The technical solution adopted by the present invention to solve the above technical problems is: a method for detecting foodborne pathogens using a dual-mode method of a well plate low-field nuclear magnetic resonance / colorimetry method. This method is not intended for diagnosis or treatment and comprises the following steps:

[0006] (1) Synthesis of signaling unit MNS@Ab2

[0007] 25 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 10 mg of N-hydroxysuccinimide (NHS) were dissolved in 10 mL of a 0.5 mg / mL two-dimensional magnetic nanosheet (MNS) dispersion. The pH was adjusted to 5.0 with 0.01 mol / L hydrochloric acid and the mixture was stirred at room temperature for 30 min. 100 μL of a 0.5 mg / mL polyclonal antibody against foodborne pathogens, Ab2, was added. After a coupling reaction at room temperature for 3 h, Ab2 was assembled onto the MNS. Subsequently, 200 μL of a 2 wt% bovine serum albumin (BSA) solution was added to block nonspecific binding sites. After magnetic washing, the resulting precipitate was dispersed in 10 mL of a 0.1 mol / L PBS solution (pH 7.4) to obtain a signal unit MNS@Ab2 dispersion.

[0008] (2) Colorimetric detection

[0009] Take a clean 96-well plate, add 100 μL of 2.0 μg / mL foodborne pathogen polyclonal antibody Ab1 to each well, incubate at 4°C for 10 hours, modify Ab1, and wash three times with 0.1 mol / L PBS to remove free Ab1; at room temperature, use 250 μL of 2wt% BSA to block nonspecific binding sites, and finally wash with water to remove excess BSA; add 200 μL of the test sample to each well, incubate at room temperature for 30 minutes, pour out the remaining liquid and add 200 μL of 0.5 mg / mL signal unit MNS@Ab2 dispersion; after incubation for 30 minutes, wash three times to remove unreacted MNS@Ab2; after immune reaction, MNS@Ab2-VP-Ab1 composite structure is formed on the wall of the 96-well plate, 100 μL of 3 mol / L sulfuric acid is added to each well to dissolve Fe3O4 nanoparticles, and then 100 μL 0.25mmol / L potassium permanganate solution, the absorbance is measured by microplate reader for colorimetric analysis, and the concentration of foodborne pathogens in unknown samples can be determined based on the quantitative relationship between the absorbance value and the concentration of foodborne pathogens;

[0010] (3) Low-field NMR homogeneous immunoassay

[0011] The 96-well plate was disassembled into separate small cups and placed in a nuclear magnetic resonance test tube. The longitudinal relaxation time T1 was measured using the IR pulse sequence measurement method at 35°C. The concentration of foodborne pathogens in unknown samples could be determined based on the quantitative relationship between the longitudinal relaxation time difference of water protons and the concentration of foodborne pathogens. The ΔT1 value was calculated by the following formula: ΔT1 = T1(negative) – T1(positive), where T1(negative) is the average T1 in the absence of foodborne pathogens, and T1(positive) is the average T1 in the presence of foodborne pathogens.

[0012] Furthermore, the preparation method of the two-dimensional magnetic nanosheets (MNS) described in step (1) is as follows: 540 mg FeCl3·6H2O, 200 mg trisodium citrate, 7 mL ethylene glycol and 13 mL diethylene glycol are mixed and stirred at room temperature for 30 min to dissolve; then 2 g sodium acetate and 25 mg single-layer graphene oxide GO powder are added and stirred at room temperature for 30 min. The mixture is transferred to a polytetrafluoroethylene reactor and reacted at 200°C for 8 h. The obtained black precipitate is washed with water and ethanol and placed in a vacuum drying oven at 60°C to obtain graphene oxide GO with Fe3O4 nanoparticles assembled on the surface, i.e., two-dimensional magnetic nanosheets.

[0013] Furthermore, the parameters of the IR pulse sequence measurement method described in step (3) are as follows: main frequency 19.00 MHz, sampling bandwidth 100 kHz, RF delay 0.08 ms, number of sampling points 2048, waiting time 8000 ms, number of inversion times 20, number of accumulation times 2 times, digital gain 3, and analog gain 15.0 dB.

[0014] Furthermore, the foodborne pathogenic bacteria are selected from Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli and Salmonella.

[0015] The detection principle of the present invention is as follows Figure 1 As shown in the figure, taking Vibrio parahaemolyticus VP as an example, the capture antibody Ab1 coated in the 96-well plate can specifically recognize and capture the target VP. The signal unit MNS@Ab2 binds to the captured VP through the antibody-antigen reaction to form the MNS@Ab2-VP-Ab1 immune complex structure. After adding sulfuric acid, the Fe3O4 nanoparticles loaded on the MNS surface are dissolved to obtain the strong paramagnetic Fe 3+ and weakly paramagnetic Fe 2+ After adding KMnO4 standard solution in excess and quantitatively, Fe 2+ With non-paramagnetic, purple-red MnO4 ￣ Fe 3+ and the strongly paramagnetic, colorless Mn 2+ .Fe 3+ and Mn2+ The longitudinal relaxation time (T1) of protons in water molecules in the system is reduced, which enables LF-NMR detection; purple-red MnO4 ￣ Reduced to form colorless Mn 2+ , based on which colorimetric analysis can be performed. In this way, dual-mode detection is achieved.

[0016] Compared with the existing technology, the advantages of the present invention are: the present invention discloses for the first time a method for detecting foodborne pathogens by dual-mode detection of foodborne pathogens using a well plate type low-field nuclear magnetic resonance / colorimetry method. Since a large amount of Fe3O4 is loaded on the surface of MNS to amplify the signal, and the addition of KMnO4 provides more Fe 3+ With Mn 2+ The longitudinal relaxation of water molecules is further shortened, achieving secondary amplification of the signal. The acid hydrolysis of multifunctional two-dimensional magnetic nanomaterials realizes the sensing strategy of transforming the signal unit from nanomaterials to magnetic ions and from heterogeneous analysis to homogeneous analysis. The introduction of the redox reaction system can not only assist in LF-NMR signal amplification, but also perform colorimetric analysis to achieve dual-mode detection. Of particular importance is that the entire detection process, including target capture, immune assembly, heterogeneous to homogeneous conversion, colorimetric analysis, and LF-NMR detection, is completed entirely within a 96-well plate, greatly improving the precision and accuracy of the detection, and providing a new path for achieving sensitive, accurate, and reliable rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the principle of the orifice plate type low-field nuclear magnetic resonance / colorimetry dual-mode detection method of the present invention;

[0018] Figure 2 Characterization of the signal unit MNS@Ab2, including A: XRD images of MNS and GO; B: infrared images of GO, MNS and MNS@Ab; C: SEM image of MNS; D: EDX element scanning surface scanning (mapping) image of MNS (distribution of O); E: mapping image of MNS (distribution of C); F: mapping image of MNS (distribution of Fe); G: EDX measurement element content chart of MNS;

[0019] Figure 3 This is the T1 signal attenuation diagram corresponding to different concentrations of VP;

[0020] Figure 4 is the detection sensitivity curve of LF-NMR sensor;

[0021] Figure 5 The absorbance graph corresponding to different concentrations of VP;

[0022] Figure 6is the detection sensitivity curve of the colorimetric sensor;

[0023] Figure 7 The specificity of the dual-signal sensor;

[0024] Figure 8 For the stability of the dual signal sensor. DETAILED DESCRIPTION

[0025] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments. 1. Specific embodiments

[0027] A method for detecting foodborne pathogens using a dual-mode, well-plate low-field nuclear magnetic resonance / colorimetry assay, not intended for diagnosis or treatment, comprises the following steps:

[0028] (1) Synthesis of signaling unit MNS@Ab2

[0029] 25 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 10 mg of N-hydroxysuccinimide (NHS) were dissolved in 10 mL of a 0.5 mg / mL two-dimensional magnetic nanosheet (MNS) dispersion, and the pH was adjusted to 5.0 with 0.01 mol / L hydrochloric acid. The mixture was stirred at room temperature for 30 min, and 100 μL of a 0.5 mg / mL polyclonal antibody against foodborne pathogens (Ab2) was added. After a coupling reaction at room temperature for 3 h, Ab2 was assembled onto the MNS. Subsequently, 200 μL of a 2 wt% bovine serum albumin (BSA) solution was added to block nonspecific binding sites. After magnetic cleaning, the resulting precipitate was dispersed in 10 mL of a 0.1 mol / L PBS solution (pH 7.4) to obtain a signal unit (MNS@Ab2) dispersion. The preparation method of the two-dimensional magnetic nanosheet (MNS) was as follows: 540 mg of FeCl3·6H2O, 200 mg of trisodium citrate, 7 mL of ethylene glycol, and 13 mL of diethylene glycol were mixed and stirred at room temperature for 30 min to dissolve. Then, 2 g of sodium acetate and 25 mg of single-layer graphene oxide (GO) powder were added and stirred at room temperature for 30 min. The mixture was transferred to a polytetrafluoroethylene reactor and reacted at 200°C for 8 h. The obtained black precipitate was washed with water and ethanol and dried in a vacuum drying oven at 60°C to obtain graphene oxide (GO) with Fe3O4 nanoparticles assembled on the surface, i.e., two-dimensional magnetic nanosheets.

[0030] (2) Colorimetric detection

[0031] Take a clean 96-well plate, add 100 μL of 2.0 μg / mL foodborne pathogen polyclonal antibody Ab1 to each well, incubate at 4°C for 10 hours, modify Ab1, and wash three times with 0.1 mol / L PBS to remove free Ab1; at room temperature, use 250 μL of 2wt% BSA to block nonspecific binding sites, and finally wash with water to remove excess BSA; add 200 μL of the test sample or blank water sample to each well, incubate at room temperature for 30 minutes, pour out the remaining liquid and add 200 μL of 0.5 mg / mL signal unit MNS@Ab2 dispersion; after incubation for 30 minutes, wash three times to remove unreacted MNS@Ab2; after immune reaction, MNS@Ab2-VP-Ab1 composite structure is formed on the wall of the 96-well plate, 100 μL of 3 mol / L sulfuric acid is added to each well to dissolve Fe3O4 nanoparticles, and then 100 μL 0.25mmol / L potassium permanganate solution, the absorbance is measured by microplate reader for colorimetric analysis, and the concentration of foodborne pathogens in unknown samples can be determined based on the quantitative relationship between the absorbance value and the concentration of foodborne pathogens;

[0032] (3) Low-field NMR homogeneous immunoassay

[0033] The 96-well plate was disassembled into individual cuvettes and placed in an NMR test tube. T1 measurements were performed at 35°C using the IR pulse sequence method. The concentration of foodborne pathogens in unknown samples can be determined based on the quantitative relationship between the difference in the longitudinal relaxation time of water protons and the concentration of foodborne pathogens. ΔT1 was calculated using the following formula: ΔT1 = T1(negative) – T1(positive), where T1(negative) is the average T1 in the absence of foodborne pathogens and T1(positive) is the average T1 in the presence of foodborne pathogens. The IR pulse sequence measurement parameters were as follows: main frequency 19.00 MHz, sampling bandwidth 100 kHz, RF delay 0.08 ms, number of sampling points 2048, wait time 8000 ms, number of inversion times 20, number of accumulations 2, digital gain 3, and analog gain 15.0 dB.

[0034] The foodborne pathogenic bacteria are selected from Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli and Salmonella.

[0035] 2. Experimental Results and Analysis Experimental Instruments: A Nova NanoSEM 450 scanning electron microscope (FEI, USA) was used for morphological characterization. A D8 Focus X-ray diffractometer (Bruker, Germany) was used to characterize the phase properties of the magnetic two-dimensional materials and GO. A Nicolet FTIR 6700 infrared spectrometer (Thermo Fisher, USA) was used for functional group characterization. A UV-1800 UV-visible spectrophotometer (Shimadzu, Japan) was used to quantify the VP concentration. A PQ001-20-015V NMR contrast agent relaxivity analyzer (Newmai, Suzhou, China) was used to determine the longitudinal relaxation time T1. A TECAN spark multifunctional microplate reader (Austria) was used for colorimetric analysis of the samples.

[0036] Cultivation of experimental strains: Prepare a liquid culture by dissolving 0.9 g of nutrient broth in 50 mL of water and autoclaving at 121°C for 15 min. Add the VP strain to the liquid culture and shake at 37°C for 24 h. Then, centrifuge the mixture at 1737 × g for 5 min to isolate the fresh VP strain. The UV-Vis optical density (OD) at 600 nm was used to determine the strain's activity. 600 =1.000), and the concentration of VP strain was adjusted to 1.0×10 8 CFU / mL, and serially diluted to obtain a series of concentrations of 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 , 1.0×10 3 , 1.0×10 2 , 1.0×10 1 CFU / mL VP standard solution. Seawater samples were collected from the East China Sea and directly used for spike recovery testing of real samples.

[0037] 1. Characterization of the signaling unit MNS@Ab2

[0038] The XRD pattern of MNS at room temperature is as follows Figure 2 As shown in Figure A, compared with GO (curve a), the XRD pattern of MNS (curve b) contains six main diffraction peaks: (220), (311), (400), (422), (511), and (440), which are consistent with the diffraction peaks of Fe3O4 (JCPDS card number: 880315) and the diffraction peaks of Fe3O4 of the Joint Committee on Powder Diffraction Standards of JCPDS (JCPDS card number: 880315), indicating that the surface of MNS is covered with Fe3O4 with a cubic spinel structure.

[0039] The successful assembly of the signaling unit MNS@Ab2 was characterized by FT-IR. Figure 2As shown in B, compared with GO (curve a), MNS (curve b) has a -1 The absorption peak at 3390 cm corresponds to the Fe-O stretching vibration, indicating that the MNS surface contains Fe3O4 nanoparticles. -1 、1633cm -1 、1351cm -1 The absorption peaks at 3428 cm correspond to the stretching vibration peaks of OH, C=O, and CO in the carboxyl group, respectively. The above dark peaks show that MNS has abundant carboxyl groups. -1 、1633cm -1 , 1400cm -1 The absorption peaks correspond to the stretching vibration peaks of NH, C=O, and N-HC-N of the amide bond, which indicates that Ab2 is successfully assembled on the MNS surface through the EDC / NHS reaction to obtain the signal unit MNS@Ab2.

[0040] like Figure 2 C SEM images also show that MNS is a sheet structure of GO surface covered with Fe3O4 nanoparticles. In order to verify the distribution of each element, Figure 2 D, E, and F mapping images show that the elements are evenly distributed, indicating that Fe3O4 nanoparticles are evenly distributed on the GO surface. Figure 2 As shown in Figure 1, EDX elemental analysis and spectrum show that the elemental composition of MNS is mainly composed of three elements: C, Fe, and O, with contents of 35.61%, 9.89%, and 54.51%, respectively. The following specific embodiment method is used to detect Vibrio parahaemolyticus VP as the detection target.

[0041] 2. Sensitivity analysis

[0042] like Figure 3 As shown in the figure, the low-field nuclear magnetic resonance analysis results using the specific embodiment method show that as the VP concentration increases from 1 to 1.0×10 8 As the CFU / mL range increases, the longitudinal relaxation decay rate accelerates and T1(positive) shortens. Therefore, when T1(negative) remains unchanged, ΔT1 increases with increasing VP concentration.

[0043] like Figure 4 As shown, in 60~1.0×10 8 Within the CFU / mL concentration range, the logarithm of ΔT1 (y, ms) and VP concentration (x, CFU / mL) showed a good linear relationship, and the linear regression equation was y = 238.47logx–469.74, R 2 = 0.996. Based on S / N = 3, the detection limit was estimated to be 60 CFU / mL.

[0044] The colorimetric detection results showed that as the VP concentration increased from 1 to 1.0×10 8 As the CFU / mL increases, the color of the solution gradually changes from purple-red to colorless and transparent. The sample concentrations were scanned using an enzyme-labeled instrument. The results are as follows: Figure 5 As shown in the figure, the absorbance of KMnO4 spectrum curve decreases with the increase of pathogen concentration in the sample. The reason is that the concentration of pathogens is closely related to Fe 2+ The absorbance at 524 nm of samples containing pathogenic bacteria with a concentration gradient decreased with the increase of bacterial concentration.

[0045] like Figure 6 As shown, in 10 3 CFU / mL~1.0×10 8 Within the CFU / mL concentration range, the absorbance at 524 nm (y) and the logarithm of the VP concentration (x, CFU / mL) showed a good linear relationship, and the linear regression equation was y = 0.025logx–0.243, R 2 =0.988. The detection limit of colorimetric VP detection was 100 CFU / mL.

[0046] 3. Selectivity analysis

[0047] like Figure 7 As shown, 1.0×10 5 There was no significant difference in ΔT1 and absorbance between the CFU / mL of Staphylococcus aureus (SA), Escherichia coli (EC), Vibrio vulnificus (VV), Listeria monocytogenes (LM), and Salmonella (SM) and the blank sample. 5 CFU / mL VP standard solution and 1×10 5 The ΔT1 and absorbance of the mixed samples of SA, EC, VV, LM, and SM with CFU / mL VP were almost the same, indicating that the dual-mode sensor had good selectivity.

[0048] 4. Reproducibility and stability analysis

[0049] Measure 1.0×10 5 T1 and absorbance of VP standard solution of CFU / mL. Figure 8 As shown in the figure, in the LF-NMR measurement, the seven-day average value of ΔT1 was 804.93ms, and the relative standard deviation (RSD) was 0.98%; in the colorimetric measurement, the seven-day average value of absorbance was 0.1513, and the relative standard deviation (RSD) was 0.31%, both showing good reproducibility and stability. This is attributed to the fact that the LF-NMR signal depends on the Fe 3+ With Mn2+ Solution concentration, colorimetric signal depends on MnO4 ￣ Concentration, these ions are uniformly distributed in water, avoiding the problem of signal instability caused by agglomeration or sedimentation of magnetic nanoparticle signal units.

[0050] 5. Precision and accuracy

[0051] The standard spike recovery experiment of VP was conducted using seawater samples from the East China Sea. The results are shown in Table 1.

[0052] Table 1. Spike recovery test of VP in seawater ( n=5)

[0053]

[0054] As shown in Table 1, the LF-NMR recovery rates ranged from 98.6% to 109.4%, with RSDs of 1.5% to 8.9%. The colorimetric recovery rates ranged from 98.3% to 107.1%, with RSDs of 1.9% to 7.7%. The excellent precision and accuracy are attributed to the simplicity of this method and minimal background interference. Importantly, the entire assay process is performed entirely within the well plate, effectively controlling systematic instrument errors and eliminating accidental operational errors. Due to the simple sample pretreatment and minimal background interference, this method exhibits excellent recovery and reproducibility.

[0055] 6. Consistency of dual-mode detection

[0056] The real samples of VP were tested with East China Sea seawater and fish meat samples using the plate count method and the dual-mode detection method. The results are shown in Table 2.

[0057] Table 2. Testing of East China Sea seawater and fish samples ( n=5).

[0058]

[0059] As shown in Table 2, the data obtained by LF-NMR, colorimetry, and plate count were very similar, with no significant differences between any two groups, as shown by t-tests with P values ​​> 0.05. This demonstrates that the LF-NMR and colorimetry dual-mode sensor is accurate and reliable, and can be used to reliably detect real samples.

[0060] 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 method for detecting foodborne pathogens using a dual-mode, well-plate-type low-field nuclear magnetic resonance / colorimetry assay, not intended for diagnosis or treatment, characterized in that The following steps are involved: (1) Synthesis of signaling unit MNS@Ab2 25 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 10 mg of N-hydroxysuccinimide (NHS) were dissolved in 10 mL of 0.5 mg / mL two-dimensional magnetic nanosheet (MNS) dispersion, and the pH was adjusted to 5.0 with 0.01 mol / L hydrochloric acid. The mixture was stirred at room temperature for 30 min, and 100 μL of 0.5 mg / mL polyclonal antibody Ab2 against foodborne pathogens was added. After the coupling reaction was carried out at room temperature for 3 h, 200 μL of 2 wt% bovine serum albumin (BSA) solution was added to block nonspecific binding sites. After magnetic washing, the resulting precipitate was dispersed in 10 mL of PBS solution (pH = 7.4, 0.1 mol / L) to obtain the signal unit MNS@Ab2 dispersion. The preparation method of the two-dimensional magnetic nanosheet is as follows: 540 mg of FeCl3·6H2O, 200 mg of trisodium citrate, 7 mL of ethylene glycol and 13 mL diethylene glycol was mixed and stirred at room temperature for 30 min to dissolve; then 2 g sodium acetate and 25 mg single-layer graphene oxide (GO) powder were added and stirred at room temperature for 30 min. The mixture was transferred to a polytetrafluoroethylene reactor and reacted at 200°C for 8 h. The obtained black precipitate was washed with water and ethanol and dried in a vacuum drying oven at 60°C to obtain graphene oxide (GO) with Fe3O4 nanoparticles assembled on the surface, i.e., two-dimensional magnetic nanosheets. (2) Colorimetric detection A clean 96-well plate was added, and 100 μL of 2.0 μg / mL foodborne pathogen polyclonal antibody Ab1 was added to each well. After incubation at 4 °C for 10 h, the plate was washed three times with 0.1 mol / L PBS to remove free Ab1. At room temperature, 250 μL of 2wt% BSA was used to block nonspecific binding sites, and finally, excess BSA was removed by washing with water. 200 μL of the test sample was added to each well and incubated at room temperature for 30 min. The remaining liquid was poured out and 200 μL of 0.5 mg / mL signal unit MNS@Ab2 dispersion was added. After incubation for 30 min, the plate was washed three times to remove unreacted MNS@Ab2. After immune reaction, a MNS@Ab2-VP-Ab1 composite structure was formed on the wall of the 96-well plate. 100 μL of 3 mol / L sulfuric acid was added to each well to dissolve Fe3O4 nanoparticles, and then 100 μL of 0.25 mmol / L potassium permanganate solution, the absorbance is measured by enzyme-labeled instrument for colorimetric analysis, and the concentration of foodborne pathogens in unknown samples can be determined based on the quantitative relationship between the absorbance value and the concentration of foodborne pathogens; (3) Low-field nuclear magnetic resonance homogeneous immunoassay The 96-well plate was disassembled into separate small cups and placed in a nuclear magnetic resonance test tube. The longitudinal relaxation time T1 was measured using the IR pulse sequence measurement method at 35°C. The concentration of foodborne pathogens in unknown samples could be determined based on the quantitative relationship between the longitudinal relaxation time difference of water protons and the concentration of foodborne pathogens. The ΔT1 value was calculated by the following formula: ΔT1 = T1(negative) – T1(positive), where T1(negative) is the average T1 in the absence of foodborne pathogens, and T1(positive) is the average T1 in the presence of foodborne pathogens.

2. The method of claim 1 for detecting foodborne pathogens using a dual-mode, low-field nuclear magnetic resonance (NMR) / colorimetric method, which is not intended for diagnosis or treatment, and is characterized in that The parameters of the IR pulse sequence measurement method described in step (3) are as follows: main frequency 19.00 MHz, sampling bandwidth 100 kHz, RF delay 0.08 ms, number of sampling points 2048, waiting time 8000 ms, number of inversion times 20, number of accumulation times 2 times, digital gain 3, and analog gain 15.0 dB.

3. The method of claim 1 for detecting foodborne pathogens using a dual-mode, low-field nuclear magnetic resonance (NMR) / colorimetry method, which is not intended for diagnosis or treatment, and is characterized by: The foodborne pathogenic bacteria are selected from Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli and Salmonella.

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