Method for detecting foodborne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous immunoassay based on gadolinium quantum dot explosive amplification strategy

Abstract

The application discloses a method for detecting foodborne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous immunoassay based on gadolinium quantum dot explosive amplification strategy, and the method is not for diagnosis or treatment, and is characterized by comprising the following steps: 1) coupling foodborne pathogenic bacteria polyclonal antibodies to the surface of magnetic graphene oxide by coupling reaction to obtain a capture unit MGO@Ab; 2) coupling foodborne pathogenic bacteria polyclonal antibodies to a spherical bush-like PS@Gd-CQDs compound by coupling reaction to obtain a signal unit PS@Gd-CQDs@Ab compound; 3) mixing the PS@Gd-CQDs@Ab, the MGO@Ab and a foodborne pathogenic bacteria sample to prepare a capture unit-foodborne pathogenic bacteria-signal unit sandwich structure, and determining the concentration of foodborne pathogenic bacteria in an unknown sample by low-field nuclear magnetic resonance homogeneous immunoassay; and the method has the advantages of high sensitivity and accuracy, strong specificity and simple and rapid operation.

Description

TECHNICAL FIELD

[0001] The present application relates to a detection method of foodborne pathogenic bacteria, and particularly relates to a method for detecting foodborne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous immunoassay based on gadolinium quantum dot explosion amplification strategy. BACKGROUND

[0002] Foodborne pathogenic bacteria refers to pathogenic bacteria that can cause food poisoning or food as a transmission medium. Food is easily contaminated by foodborne pathogenic bacteria during collection, processing, transportation and other links, and food poisoning and disease outbreak events occur frequently. The economic loss caused by foodborne pathogenic bacteria in China is as high as 17 billion US dollars per year. Common foodborne pathogenic bacteria include Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli and Salmonella, and the traditional method for detecting foodborne pathogenic bacteria is biochemical culture identification method. This method is tedious, time-consuming and laborious. With the rapid development of molecular biology technology, polymerase chain reaction (PCR), DNA hybridization, loop-mediated isothermal amplification (LAMP) and biochip methods have also been applied to detect foodborne pathogenic bacteria, and good accuracy and sensitivity have been achieved. However, there are still many problems in practical application: high false positive rate, expensive equipment, high detection cost, complex detection steps, long detection time, etc. Therefore, it is an urgent need to develop a sensitive, accurate, simple and rapid detection method for foodborne pathogenic bacteria.

[0003] In recent years, magnetic relaxation switch (MRS) sensors have been used to detect biomacromolecules, pathogenic bacteria, pesticide residues, and heavy metals. The detection principle of MRS is that the interaction between magnetic nanoprobes and targets in a homogeneous system changes the state (dispersion or aggregation) of the magnetic nanoprobes or the concentration of the magnetic probes, thereby generating a local magnetic field, affecting the precession frequency of water protons in the system, and producing a change in the relaxation time, i.e., a magnetic signal. The strength of the magnetic signal is determined by the degree of aggregation or the concentration of the magnetic probes, so MRS sensors have derived various sensing strategies. (1) Traditional MRS sensors obtain signals by changing the state of magnetic probes in the system. This sensor has two analysis modes, Type I and Type II, and many factors affect the selection of the analysis mode, such as the size of the magnetic probes and the detection target. Traditional MRS sensors are not sensitive enough to trace amounts of detection targets and are easily affected by non-specific aggregation to produce signal interference. In addition, the stability and reliability of traditional MRSw sensors are easily affected by the "front zone effect", i.e., the excessive presence of detection targets will saturate the binding sites on the surface of the detection targets, hindering the aggregation of magnetic nanoprobes, and reducing the sensitivity and signal-to-noise ratio. (2) Magnetic separation MRS sensors obtain signals by changing the number of magnetic probes in the detection system. The signal strength of such sensors is only related to the concentration of the magnetic probes, which can avoid the instability and front zone effect caused by non-specific aggregation of traditional MRS sensors. However, such sensors have the disadvantage of low sensitivity to trace amounts of detection targets. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a method for low-field nuclear magnetic resonance homogeneous immunoassay of foodborne pathogenic bacteria based on gadolinium quantum dot explosive amplification strategy, which has high sensitivity and accuracy, strong specificity, and simple and rapid operation.

[0005] The technical solution adopted by the present application to solve the above technical problem is: a method for low-field nuclear magnetic resonance homogeneous immunoassay of foodborne pathogenic bacteria based on gadolinium quantum dot explosive amplification strategy, which is not for diagnosis or treatment, comprising the following steps:

[0006] (1) Synthesis of capture unit MGO@Ab

[0007] A. Magnetic graphene oxide is prepared by a double solvent method;

[0008] B. Foodborne pathogenic bacteria polyclonal antibodies are coupled to the surface of magnetic graphene oxide (MGO) through EDC / NHS coupling reaction, and bovine serum albumin is used to block non-specific binding sites, thereby obtaining the capture unit MGO@Ab;

[0009] (2) Synthesis of signal unit PS@Gd-CQDs@Ab

[0010] A. Mix 0.8~1.5 g diethylenetriaminepentaacetic acid (DTPA), 0.1~0.3 g GdCl3·6H2O and 10 mL water, ultrasonic dispersion for 10 min, transfer to a polytetrafluoroethylene reaction kettle, heat at 200℃ for 6~8 h, then the product is dialyzed with water for 12 h, change water every 3 h, freeze-dried to obtain Gd-CQDs;

[0011] B. L-cysteine is coupled to Gd-CQDs by EDC / NHS coupling reaction to obtain thiol-modified Gd-CQDs;

[0012] C. Add 500~1000 µL 2.5 mg / mL carboxyl-modified polystyrene (PS) beads, 12.2 mg EDC and 7.2 mg NHS to 10 mL ethanol, stir at room temperature for 30 min, add 200 µL ethylenediamine-terminated polyethyleneimine, react at room temperature for 3 h by EDC / NHS reaction, centrifuge the mixture at 3405 ×g for 5~10 min, and then wash and disperse in 10 mL 10wt% N,N-dimethylformamide (DMF) in pH = 7.5, 0.1 M PBS solution, add 150~300 µL 0.01 M succinimidyl 3-(2-pyridyldithio)propionate (SPDP), react at room temperature for 1~3 h, centrifuge and wash, then re-disperse in 10 mL 10% DMF in pH = 8.0, 0.1 M PBS solution, add 1 mL thiol Gd-CQDs dispersion, and react at 4 ℃ for 8~10 h, centrifuge and wash, then disperse the product in 10~20 mL water to obtain a spherical bush-like PS@Gd-CQDs composite dispersion;

[0013] D. Foodborne pathogenic bacteria polyclonal antibody is coupled to the spherical bush-like PS@Gd-CQDs composite by EDC / NHS coupling reaction to obtain a biofunctionalized spherical bush-like PS@Gd-CQDs@Ab composite;

[0014] (3) Low-field nuclear magnetic resonance homogeneous immunoassay

[0015] Take 200 μL PS@Gd-CQDs@Ab dispersion liquid and 50~200 μL MGO@Ab dispersion liquid, 1 mL of the sample to be tested foodborne pathogenic bacteria sample, add the sample bottle to mix, shake incubation for 30~40 min, magnetic separation to obtain the capture unit-foodborne pathogenic bacteria-signal unit sandwich structure, the capture unit-foodborne pathogenic bacteria-signal unit sandwich structure is added to 800 μL 50 mM dithiothreitol (DTT) solution, reaction for 20 min, magnetic washing, 200 μL 0.01 M hydrochloric acid is added, reaction for 5 min, placed in low-field nuclear magnetic resonance contrast agent relaxation analyzer, T1 is collected at 35℃, using IR (inversion recovery) pulse sequence measurement (Carr-Purcell-Meiom-Gill), the size of the transverse relaxation time difference ΔT1 of water protons corresponding to a series of different concentrations of foodborne pathogenic bacteria is determined, and the quantitative relationship between the longitudinal relaxation time difference of water protons and the concentration of foodborne pathogenic bacteria is established, according to the quantitative relationship, the concentration of foodborne pathogenic bacteria in unknown sample can be determined.

[0016] Further, the specific process of step (1) A is as follows: 200~520 mg FeCl3·6H2O, 7 mL ethylene glycol (EG) and 13 mL diethylene glycol (DEG) are mixed, and after dissolution at room temperature for 10~30 min, 0.5~2.5 g polyvinylpyrrolidone (PVP) is added, and after stirring in an oil bath at 120℃ until the PVP is dissolved, 1~3 g sodium acetate and 5~30 mg graphene oxide powder are added, and stirred at room temperature for 30 min. The mixture is transferred to a polytetrafluoroethylene reaction kettle, and reacted at 200℃ for 8~10 h. The black precipitate obtained is washed with water and ethanol, and dried in a vacuum drying box at 60℃, to obtain magnetic graphene oxide.

[0017] Further, the specific process of step (1) B is as follows: 12.2 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 7.2 mg N-hydroxysuccinimide (NHS) are dissolved in 10 mL 0.5 mg / mL MGO dispersion (solvent water), and the pH is adjusted to 5.0 with 0.01 M hydrochloric acid, and stirred at room temperature for 30 min. 1.5 mg / mL foodborne pathogenic bacteria polyclonal antibody (Ab) solution 100 μL is added, and reacted at room temperature for 3 h. Through EDC / NHS coupling reaction, the foodborne pathogenic bacteria polyclonal antibody (Ab) is assembled on the MGO. Then 200 µL 2 wt% BSA solution is added to block the non-specific binding sites. After magnetic washing, the obtained precipitate is dispersed in pH = 7.4, 10 mL, 0.01 M PBS solution, to obtain the capture unit MGO@Ab dispersion.

[0018] Further, the step (2)B is specifically as follows: 1 mg of Gd-CQDs, 12.2 mg of EDC, and 7.2 mg of NHS are dissolved in 10 mL of water, the pH is adjusted to 5.0 by 0.01 M hydrochloric acid, and stirred at room temperature for 30 min, 1 mL of 0.1 M L-cysteine is added, and reacted at room temperature for 4 h, and then dialyzed for 12 h by using a dialysis membrane with a molecular weight cut-off of 1000 Da to remove excess L-cysteine, and the product is dispersed in 5 mL of 0.1 M PBS solution at pH = 7.5 to obtain a thiol-modified Gd-CQDs dispersion.

[0019] Further, the step (2)D is specifically as follows: 12.2 mg of EDC and 7.2 mg of NHS are added to the spherical bush-like PS@Gd-CQDs composite dispersion obtained in step (2)C, and reacted at room temperature for 30 min, 200 μL of 1.5 mg / mL foodborne pathogen polyclonal antibody solution is added, and reacted at room temperature for 1-3 h, and then 200 μL of 2 wt% BSA solution is added to block the non-specific binding sites, the mixture is centrifuged at 3405 ×g for 7 min, and then washed to remove the unbound foodborne pathogen polyclonal antibody, and then dispersed in 10 mL of 0.01 M PBS solution at pH = 7.4 to obtain a biologically functionalized spherical bush-like PS@Gd-CQDs@Ab composite.

[0020] Further, the value of ΔT1 in step (3) is calculated by the following formula: ΔT1 = T1(negative) - T1(positive), wherein T1 is the longitudinal relaxation time of water protons, T1(negative) is the average T1 without foodborne pathogens, and T1(positive) is the average T1 with foodborne pathogens. The greater the concentration of foodborne pathogens, the smaller the corresponding T1 shortening.

[0021] Further, T1 is measured at 35 ℃ by using an IR pulse sequence measurement with the following parameters: a main frequency of 19.00 MHz, a sampling frequency of 100 kHz, a sampling delay of 0.08 ms, a sampling point number of 2048, a waiting time of 20000 ms, an inversion number of 20, an accumulation number of 2 times, a digital gain of 3, and an analog gain of 15.0 dB.

[0022] Further, the foodborne pathogens include Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli, and Salmonella.

[0023] Invention principle: Based on the principle of magnetic separation-magnetic relaxation switch sensor (MS-MRSw) sensor, combined with PS@Gd-CQDs signal probe, a "sub-munition" type signal amplification strategy MS-MRS detection VP is constructed. When detecting, through DTT (dithiothreitol) cutting disulfide bond, Gd-CQDs is separated from the surface of PS@Gd-CQDs and diffused into the solution, and the state is changed (from aggregation to dispersion) to realize the second signal amplification. In this work, in order to avoid the front zone effect of the traditional MRS sensor, further enhance the detection sensitivity and reliability, we propose a magnetic and fluorescent dual-mode sensor relying on the concentration change of magnetic probes, and a signal amplification strategy after pre-assembly and dispersion. We choose magnetic graphene (MGO) material as the capture unit, and the two-dimensional nanomaterial with large specific surface area can provide a large number of binding sites, and the functionalized magnetic material can capture and enrich the target in the complex sample, thereby improving the capture efficiency. With PS@Gd-CQDs as the signal unit, the surface of PS ball is modified with dendrimer (ethylenediamine-terminated polyethyleneimine PEI), and a large number of amino groups on the surface of PEI can connect a large number of Gd-CQDs. The dendritic structure of PEI can greatly increase the number of Gd-CQDs in the signal unit, realizing the first signal enhancement. When detecting, through DTT (dithiothreitol) cutting the disulfide bond contained in SPDP, the aggregated Gd-CQDs is separated from the surface of PS@Gd-CQDs and released and dispersed into the homogeneous solution, after the Gd-CQDs is released into the solution, hydrochloric acid is added, H + compete with Gd3+ in the Gd-CQDs, Gd3+ is released into the solution, the target of homogeneous solution is realized, and the second signal amplification is generated. After magnetic washing to remove MGO, dilute hydrochloric acid is added to release Gd 3+ in the Gd-CQDs, so that the detection solution changes from Gd-CQDs dispersion to Gd 3+ solution, the signal molecules are more dispersed, and the detection solution is more homogeneous, realizing the third signal amplification. The concentration change of Gd 3+ changes the T1 time of the surrounding water molecules, and the change of T1 time can quantitatively detect the concentration of the target.

[0024] The immunological principle of the application, MGO is connected to the surface of GO through EDC / NHS to form a capture probe with magnetic property and specificity. PS@Gd-CQDs is connected to Ab through EDC / NHS to make the signal probe have specificity. When VP exists, MGO and PS@Gd-CQDs can specifically recognize VP to form a MGO-VP-PS@Gd-CQDs sandwich structure. After magnetic washing, DTT cutting and hydrochloric acid release, the detection solution is placed in a low-field nuclear magnetic resonance instrument for detection, and the concentration of VP can be directly controlled to control the Gd 3+The concentration of VP is controlled, so that the influence on the water molecules T1 in the system is controlled. The reduction amount of the water molecules T1 in the system and the concentration of VP have a certain relationship, and under a specific working curve, the unknown concentration of VP in the sample can be tested.

[0025] Compared with the prior art, the application has the advantages that:

[0026] 1. Simple sample pretreatment: the signal of LF-NMR is derived from magnetism rather than photoelectricity, and considering that there is almost no magnetic substance in the detection environment, LF-NMR has almost no background interference, and even turbid samples can be directly detected. The detection object is pathogenic bacteria individuals, and DNA extraction and amplification are not required, so the sample can be directly detected.

[0027] 2. Avoiding the front effect caused by traditional MRS: the magnetic signal of the traditional MRS sensor is derived from the state of the magnetic probe, and the detection limit is low and the linear range is narrow due to the problem of the target and the probe ratio. The signal of the sensor of the application is derived from the concentration of Gd 3+ , and the above problems are avoided.

[0028] 3. Three amplification signals improve the detection sensitivity: (1) Signal molecule quantity amplification. The dendritic macromolecule modified on the surface of PS can combine a large number of signal molecules Gd-CQDs, and realize the first-order enhancement of the signal. (2) State change of the magnetic label Gd-CQDs. The Gd-CQDs are aggregated and attached to the surface of the PS ball, and are dispersed into the solution by DTT cutting, contact more water molecules, and realize the second-order enhancement of the signal. (3) Dispersity reaches a homogeneous solution. H + In the solution will attack Gd 3+ In Gd-CQDs, so that Gd 3+ From the coordination state to the free state, the detection liquid becomes a homogeneous Gd 3+ Solution, realizing the third-order enhancement of the signal. The three amplification signals greatly improve the detection sensitivity.

[0029] In summary, the application first prepares a low-field nuclear magnetic resonance homogeneous immune detection method of foodborne pathogenic bacteria based on the explosive amplification strategy of gadolinium quantum dots. MGO is used as a capture probe, and PS@Gd-CQDs is used as a signal probe. After meeting the target pathogenic bacteria antigen, a sandwich structure is formed. The change of the T1 signal only depends on Gd 3+The concentration is improved, and the detection stability is improved. By using the technical framework, the triple signal amplification can be realized by the following paths: (1) signal molecule quantity amplification; (2) state of magnetic label Gd-CQDs; (3) dispersion reaches homogeneous solution, the MS-MRSw of the sensing strategy is three times of the signal amplification compared with the traditional MS-MRSw, and the detection sensitivity is improved. The novel double-signal magnetic separation MRS sensor has the advantages of high signal-to-noise ratio, simple operation, high accuracy and efficiency, and has great potential in the field of on-site detection of foodborne pathogens. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The electron micrograph of the capture unit MGO@Ab of the application;

[0031] Figure 2 The electron micrograph of the signal unit PS@Gd-CQDs@Ab of the application;

[0032] Figure 3 The sensor principle and triple-amplified signal diagram of the application;

[0033] Figure 4 The linear relationship diagram of low-field nuclear magnetic resonance detection under different VP concentrations;

[0034] Figure 5 The sensor specificity diagram of low-field nuclear magnetic resonance detection of different types of bacteria under the same concentration. DETAILED DESCRIPTION

[0035] The application will be further described in detail below in combination with the embodiments of the drawings. Specific embodiment one

[0037] Example 1

[0038] A method for detecting foodborne pathogens by low-field nuclear magnetic resonance homogeneous immunoassay based on gadolinium quantum dot explosion amplification strategy, the method is not for diagnosis or treatment, comprising the following steps:

[0039] (1) Synthesis of capture unit MGO@Ab

[0040] A. The magnetic graphene oxide was prepared by a double solvent method. The specific process was as follows: 300 mg FeCl3·6H2O, 7 mL ethylene glycol (EG) and 13 mL diethylene glycol (DEG) were mixed, and after stirring at room temperature for 20 min to dissolve, 1.5 g polyvinylpyrrolidone (PVP) was added, and after stirring in an oil bath at 120°C until the PVP was dissolved, 2 g sodium acetate and 15 mg graphene oxide powder were added, and stirred at room temperature for 30 min. The mixture was transferred to a polytetrafluoroethylene reaction kettle, and reacted at 200°C for 9 h. The obtained black precipitate was washed with water and ethanol, and dried in a vacuum drying oven at 60°C. Thus, the magnetic graphene oxide was obtained;

[0041] B. The polyclonal antibody of Vibrio parahaemolyticus (VP) was coupled to the surface of the magnetic graphene oxide (MGO) by EDC / NHS coupling reaction, and the non-specific binding sites were blocked with bovine serum albumin. Thus, the capture unit MGO@Ab was obtained. The specific process was as follows: 12.2 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 7.2 mg N-hydroxysuccinimide (NHS) were dissolved in 10 mL 0.5 mg / mL MGO dispersion (solvent water), and the pH was adjusted to 5.0 with 0.01 M hydrochloric acid. After stirring at room temperature for 30 min, 100 μL of 1.5 mg / mL Vibrio parahaemolyticus polyclonal antibody (Ab) solution was added, and reacted at room temperature for 3 h. Then, the polyclonal antibody of Vibrio parahaemolyticus (Ab) was assembled on the MGO by EDC / NHS coupling reaction. Then, 200 μL of 2 wt% BSA solution was added to block the non-specific binding sites. After magnetic cleaning, the obtained precipitate was dispersed in 10 mL of pH = 7.4, 0.01 M PBS solution. Thus, the capture unit MGO@Ab dispersion was obtained. The electron microscope image of the prepared capture unit MGO@Ab is shown in FIG. 2B, and it can be seen from the image that the MGO is a few-layer sheet-shaped GO uniformly covered with Fe3O4 nanoparticles with a diameter of about 120 nm on the surface, which is easy to be attracted by a magnet. Figure 1 Figure 1 It can be seen from the image that the MGO is a few-layer sheet-shaped GO uniformly covered with Fe3O4 nanoparticles with a diameter of about 120 nm on the surface, which is easy to be attracted by a magnet.

[0042] (2) Synthesis of signal unit PS@Gd-CQDs@Ab

[0043] A. 1.2 g diethylenetriamine pentaacetic acid (DTPA), 0.2 g GdCl3·6H2O and 10 mL water were mixed and ultrasonically dispersed for 10 min, and then transferred to a polytetrafluoroethylene reaction kettle. After heating at 200°C for 7 h, the product was dialyzed with water for 12 h, and the water was changed every 3 h. After freeze-drying, Gd-CQDs were obtained;

[0044] ​B. L-cysteine was coupled to Gd-CQDs by EDC / NHS coupling reaction, i.e. to obtain thiol-modified Gd-CQDs, the specific process is as follows: 1 mg of Gd-CQDs, 12.2 mg of EDC, 7.2 mg of NHS were dissolved in 10 mL of water, the pH was adjusted to 5.0 with 0.01 M hydrochloric acid, stirred at room temperature for 30 min, 1 mL of 0.1 M L-cysteine was added, and the reaction was carried out at room temperature for 4 h, then the product was dialyzed against a dialysis membrane with a molecular weight cut-off of 1000 Da for 12 h to remove excess L-cysteine, and the product was dispersed in 5 mL of 0.1 M PBS solution at pH = 7.5 to obtain a thiol-modified Gd-CQDs dispersion;

[0045] C. 800 μL of 2.5 mg / mL carboxyl-modified polystyrene (PS) microspheres were added to 10 mL of ethanol with 12.2 mg of EDC, 7.2 mg of NHS, stirred at room temperature for 30 min, 200 μL of ethylenediamine-capped polyethyleneimine was added, and the mixture was reacted by EDC / NHS reaction at room temperature for 3 h, then the mixture was centrifuged at 3405 x g for 5~10 min, and after washing, it was dispersed in 10 mL of 0.1 M PBS solution containing 10wt% N,N-dimethylformamide (DMF) at pH = 7.5, 200 μL of 0.01 M succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was added, and the reaction was carried out at room temperature for 2 h, then the mixture was centrifuged and washed, and then it was redispersed in 10 mL of 0.1 M PBS solution containing 10wt% DMF at pH = 8.0, 1 mL of thiol Gd-CQDs dispersion was added, and the reaction was carried out at 4°C for 8~10 h, then the mixture was centrifuged and washed, and the product was dispersed in 10~20 mL of water to obtain a spherical bush-like PS@Gd-CQDs composite dispersion;

[0046] D. The polyclonal antibody of Vibrio parahaemolyticus was coupled to the spherical bush-like PS@Gd-CQDs complex by EDC / NHS coupling reaction to obtain a biofunctionalized spherical bush-like PS@Gd-CQDs@Ab complex, and the specific process was as follows: 12.2 mg of EDC and 7.2 mg of NHS were added to the spherical bush-like PS@Gd-CQDs complex dispersion obtained in step (2) C, and the reaction was carried out at room temperature for 30 min, 200 μL of 1.5 mg / mL polyclonal antibody solution of Vibrio parahaemolyticus was added, and the reaction was carried out at room temperature for 2 h, then 200 μL of 2 wt% BSA solution was added to block the non-specific binding sites, the mixture was centrifuged at 3405 ×g for 7 min, and then washed to remove the unbound polyclonal antibody of Vibrio parahaemolyticus, and then dispersed in 10 mL of 0.01 M PBS solution with pH = 7.4 to obtain the biofunctionalized spherical bush-like PS@Gd-CQDs@Ab complex. The electron microscope image of the prepared PS@Gd-CQDs@Ab complex is shown in FIG. 8, and it can be seen from the figure that the average particle size of the signal unit PS@Gd-CQDs@Ab is about 220 nm ~300 nm or so, which is mainly because the dendrimer PEI is coated on the surface of PS, and a large number of Gd-CQDs and Ab are further assembled. Figure 2 Figure 2

[0047] (3) Low-field nuclear magnetic resonance homogeneous immune detection

[0048] Take 200 μL of PS@Gd-CQDs@Ab dispersion and 300 μL of MGO@Ab dispersion, 1 mL of Vibrio parahaemolyticus sample to be detected, add to the sample bottle and mix, shake and incubate for 30~40 min, and then magnetically separate to obtain a capture unit-Vibrio parahaemolyticus-signal unit sandwich structure, and then add the capture unit-Vibrio parahaemolyticus-signal unit sandwich structure to 800 μL of 50 mM dithiothreitol (DTT) solution, react for 20 min, magnetically wash, add 200 μL of 0.01 M hydrochloric acid, react for 5 min, and then place in a low-field nuclear magnetic resonance contrast agent relaxation analyzer, collect T1 at 35°C, use an IR (inversion recovery) pulse sequence measurement (Carr-Purcell-Meiom-Gill), measure the size of the difference ΔT1 of the transverse relaxation time of water protons corresponding to a series of different concentrations of Vibrio parahaemolyticus, establish the quantitative relationship between the longitudinal relaxation time difference of water protons and the concentration of Vibrio parahaemolyticus, and according to the quantitative relationship, the concentration of Vibrio parahaemolyticus in the unknown sample can be determined.

[0049] ​​wherein the value of ΔΤ1 is calculated by the following equation: ΔΤ1 = T1 (negative) - T1 (positive), wherein T1 is the longitudinal relaxation time of water proton, T1 (negative) is the average T1 without V. parahaemolyticus, and T1 (positive) is the average T1 with V. parahaemolyticus. The greater the concentration of foodborne pathogenic bacteria, the smaller the corresponding T1 shortening.

[0050] T1 was measured at 35 °C using an IR pulse sequence measurement with the following parameters: main frequency 19.00 MHz, sampling frequency 100 kHz, sampling delay 0.08 ms, sampling points 2048, waiting time 20000 ms, number of inversions 20, number of accumulations 2 times, digital gain 3, analog gain 15.0 dB.

[0051] Example 2

[0052] The same as Example 1 above, except that:

[0053] In step (1), 200 mg of FeCl3·6H2O, 7 mL of ethylene glycol and 13 mL of diethylene glycol were mixed, stirred at room temperature for 10 min to dissolve, then 0.5 g of polyvinylpyrrolidone was added, and the mixture was stirred in an oil bath at 120 °C until the PVP was dissolved. Then 1 g of sodium acetate and 5 mg of graphene oxide powder were added, and the mixture was stirred at room temperature for 30 min. The mixture was transferred to a polytetrafluoroethylene reaction kettle and reacted at 200 °C for 8 h. The black precipitate obtained was washed with water and ethanol and dried in a vacuum drying oven at 60 °C to obtain magnetic graphene oxide.

[0054] In step (2) A, 0.8 g of diethylenetriamine pentaacetic acid (DTPA), 0.1 g of GdCl3·6H2O and 10 mL of water were mixed and ultrasonically dispersed for 10 min, then transferred to a polytetrafluoroethylene reaction kettle and heated at 200 °C for 6-8 h. The product was dialyzed with water for 12 h, changing the water every 3 h, and then freeze-dried to obtain Gd-CQDs.

[0055] In step (2)C, 500 μL of 2.5 mg / mL carboxyl-modified polystyrene (PS) beads were added to 12.2 mg of EDC and 7.2 mg of NHS in 10 mL of ethanol, stirred at room temperature for 30 min, 200 μL of ethylenediamine-capped polyethyleneimine was added, and the mixture was reacted through EDC / NHS at room temperature for 3 h. The mixture was centrifuged at 3405 x g for 5-10 min, washed, and then dispersed in 10 mL of a 10wt% N,N-dimethylformamide (DMF) solution in 0.1 M PBS with pH = 7.5. 150 μL of 0.01 M succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was added, and the reaction was carried out at room temperature for 1 h. After centrifugation and washing, the product was re-dispersed in 10 mL of a 10% DMF solution in 0.1 M PBS with pH = 8.0. 1 mL of a thiol Gd-CQDs dispersion was added, and the reaction was carried out at 4 ℃ for 8 h. After centrifugation and washing, the product was dispersed in 10 mL of water to obtain a spherical bush-like PS@Gd-CQDs composite dispersion;

[0056] In step (3), 200 μL of the PS@Gd-CQDs@Ab dispersion and 50 μL of the MGO@Ab dispersion, and 1 mL of the Vibrio parahaemolyticus sample to be tested were mixed in a sample bottle, shaken and incubated for 30 min, and the capture unit-Vibrio parahaemolyticus-signal unit sandwich structure was obtained by magnetic separation.

[0057] Example 3

[0058] In step (1), 520 mg of FeCl3·6H2O, 7 mL of ethylene glycol, and 13 mL of diethylene glycol were mixed, stirred at room temperature for 30 min to dissolve, 2.5 g of polyvinylpyrrolidone was added, and the mixture was stirred at 120 ℃ in an oil bath until the PVP was dissolved. Then, 3 g of sodium acetate and 30 mg of graphene oxide powder were added, and the mixture was stirred at room temperature for 30 min. The mixture was transferred to a polytetrafluoroethylene reaction kettle, and the reaction was carried out at 200 ℃ for 8-10 h. The obtained black precipitate was washed with water and ethanol, and dried in a vacuum drying oven at 60 ℃ to obtain magnetic graphene oxide.

[0059] In step (2)A, 1.5 g of diethylenetriamine pentaacetic acid (DTPA), 0.3 g of GdCl3·6H2O, and 10 mL of water were mixed and ultrasonically dispersed for 10 min. The mixture was transferred to a polytetrafluoroethylene reaction kettle and heated at 200 ℃ for 6-8 h. The product was dialyzed against water for 12 h, with water being changed every 3 h. After freeze-drying, Gd-CQDs were obtained.

[0060] In step (2)C, 1000 μL of 2.5 mg / mL carboxyl-modified polystyrene (PS) beads were added to 12.2 mg of EDC and 7.2 mg of NHS in 10 mL of ethanol, stirred at room temperature for 30 min, 200 μL of ethylenediamine-capped polyethyleneimine was added, and reacted through EDC / NHS reaction at room temperature for 3 h. The mixture was centrifuged at 3405 x g for 5-10 min, washed, and then dispersed in 10 mL of a pH = 7.5, 0.1 M PBS solution containing 10 wt% N,N-dimethylformamide (DMF). 300 μL of 0.01 M succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was added, and reacted at room temperature for 3 h. After centrifugation and washing, the product was redispersed in 10 mL of a pH = 8.0, 0.1 M PBS solution containing 10% DMF. 1 mL of a thiol Gd-CQDs dispersion was added, and reacted at 4 ℃ for 8-10 h. After centrifugation and washing, the product was dispersed in 10-20 mL of water to obtain a spherical bush-like PS@Gd-CQDs composite dispersion;

[0061] In step (3), 200 μL of the PS@Gd-CQDs@Ab dispersion and 200 μL of the MGO@Ab dispersion, and 1 mL of the Vibrio parahaemolyticus sample to be detected were mixed in a sample bottle, shaken and incubated for 40 min, and the capture unit-Vibrio parahaemolyticus-signal unit sandwich structure was obtained by magnetic separation.

[0062] As shown in Figure 3 , the present application is based on the principle of a magnetic separation-magnetic relaxation switch sensor (MS-MRSw) sensor, combined with a PS@Gd-CQDs signal probe, to construct a MS-MRS detection VP of a "cluster bomb" type signal amplification strategy. The surface of the PS beads is modified with dendrimer ethylenediamine-capped PEI, and a large number of amino groups on the surface of PEI can connect a large number of Gd-CQDs, realizing the amplification of the number of signal molecules. During detection, DTT (dithiothreitol) is used to cut the disulfide bond, Gd-CQDs is separated from the surface of PS@Gd-CQDs and diffuses into the solution, a change in state (from aggregation to dispersion) is achieved, and a second signal amplification is realized. After magnetic washing to remove MGO, dilute hydrochloric acid is added to release Gd 3+ from the Gd-CQDs dispersion to a Gd 3+ solution, the signal molecules are more dispersed, the detection solution is more homogeneous, and a third signal amplification is realized. The change in Gd 3+ concentration changes the T1 time of the surrounding water molecules, and the change in T1 time can quantitatively detect the concentration of the target.

[0063] In addition to Vibrio parahaemolyticus, this detection method can also be applied to Vibrio vulnificus, Staphylococcus aureus, Escherichia coli and Salmonella. Specific embodiment 2

[0065] Sensitivity testing

[0066] Figure 4 The linear relationship diagram of low-field nuclear magnetic resonance detection of different VP concentrations was investigated. The sensitivity of MRSw in detecting VP of Vibrio parahaemolyticus was investigated with 30 min as the optimal incubation time. The quantitative detection range of 0.5 CFU / mL~1.0×10 6 CFU / mL of pathogenic bacteria at different concentrations. Figure 4 A, among different samples, △T2 increases with the increase of VP concentration. Figure 4 As shown in B, at a concentration of 1.0×10 2 CFU / mL~1.0×10 5 Within the CFU / mL concentration range, there is a good linear relationship between △T2(y) and the logarithm of VP concentration (x), and the linear regression equation is: y =347.47 logx – 100.32, R 2 = 0.995. Figure 4 A, The detection limit of MRSw for VP was 10 CFU / mL, thus demonstrating that MRSw is sensitive for detecting VP. Specific embodiment three

[0068] Specificity of low-field nuclear magnetic resonance in detecting different bacterial species at the same concentration

[0069] The specificity experiment of MRSw sensor was conducted to simulate the complex bacterial environment of seawater under the optimal experimental conditions by detecting the concentration of 1.0×10 4 CFU / mL of Staphylococcus aureus ( Staphylococcus aureus, SA ), Escherichia coli ( Escherichia Coli, E. coli ), Vibrio vulnificus ( Vibrio Vulnificus, VV ), Listeria monocytogenes ( Listeria monocytogenes, LM ),salmonella( Salmonella, SM ), Vibrio parahaemolyticus ( Vibrio Parahemolyticus, VP ), and contains 1.0×10 4 Mixed samples of VP with different CFU / mL concentrations and blank samples were added to PBS to verify the specificity of the MRSw sensor for the target VP.

[0070] The results are as follows Figure 5As shown, the T1 of the sample containing VP has a significant change, while the signal response of the detection sample containing other pathogenic bacteria is similar to that of the blank control group. The above results show that the MRSw sensor has high specificity for VP, and in addition, the ΔT1 of the sample containing VP mixture is similar to that of the single VP sample, which means that other pathogenic bacteria hardly affect the specific recognition of the sensor for VP, reflecting the excellent specificity of the MRSw sensor for VP detection.

[0071] Application Examples

[0072] Table 1 is the seawater and fish meat spiked sample of East China Sea, and the VP concentration is detected by low-field nuclear magnetic resonance method , n = 5).

[0073] .

[0074] The standard addition recovery experiment of VP was carried out with real seawater and fish sample. The pretreatment method of real sample is described in detail in SI. As shown in Table 1, the recovery rate of seawater sample is 99.3% ~ 106.2%, and the RSD is 2.3% ~ 6.5%. For fish samples, the recovery rate is 98.4% ~ 104.9%, and the RSD is 1.8% ~ 9.2%. The inter-group t test was carried out with the detection results of plate counting method. The calculated t values of seawater and fish samples are 1.02 and 1.10, respectively, which are less than t 0.10,3 = 2.35, which shows that there is no significant difference between the two methods. This method has good precision and accuracy, and can be used for reliable detection of actual samples.

[0075] The above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by ordinary skilled in the art within the essential scope of the present application shall also fall within the protection scope of the present application.

Claims

1. A method for homogeneous low-field nuclear magnetic resonance immunoassay of foodborne pathogens based on explosive amplification of gadolinium quantum dots, not intended for diagnosis or treatment, characterized in that The method comprises the following steps: (1) synthesis of a capture unit MGO@Ab A. Magnetic graphene oxide is prepared by a double solvent method, and the specific process is as follows: 200-520 mg of FeCl3·6H2O, 7 mL of ethylene glycol and 13 mL of diethylene glycol are mixed, and after stirring at room temperature for 10-30 min to dissolve, 0.5-2.5 g of polyvinylpyrrolidone is added, and after stirring in an oil bath at 120℃ until the PVP is dissolved, 1-3 g of sodium acetate and 5-30 mg of graphene oxide powder are added, and stirred at room temperature for 30 min. The mixture is transferred to a polytetrafluoroethylene reaction kettle, and reacted at 200℃ for 8-10 h. The black precipitate obtained is washed with water and ethanol, and dried in a vacuum drying oven at 60℃. Thus, magnetic graphene oxide is obtained; B. Foodborne pathogenic bacteria polyclonal antibodies are coupled to the surface of magnetic graphene oxide through EDC / NHS coupling reaction, and non-specific binding sites are blocked with bovine serum albumin. Thus, the capture unit MGO@Ab is obtained, and the specific process is as follows: 12.2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 7.2 mg of N-hydroxysuccinimide are dissolved in a 10 mL 0.5 mg / mL MGO dispersion, and the pH is adjusted to 5.0 with 0.01 M hydrochloric acid. Stir at room temperature for 30 min, add 100 μL of 1.5 mg / mL foodborne pathogenic bacteria polyclonal antibody solution, and react at room temperature for 3 h. Through EDC / NHS coupling reaction, foodborne pathogenic bacteria polyclonal antibodies are assembled on MGO. Then, 200 μL of 2 wt% BSA solution is added to block the non-specific binding sites. After magnetic cleaning, the obtained precipitate is dispersed in a pH = 7.4, 10 mL, 0.01 M PBS solution. Thus, the capture unit MGO@Ab dispersion is obtained; (2) synthesis of a signal unit PS@Gd-CQDs@Ab A. 0.8-1.5 g of diethylenetriamine pentaacetic acid, 0.1-0.3 g of GdCl3·6H2O and 10 mL of water are mixed, and ultrasonic dispersion is performed for 10 min. The mixture is transferred to a polytetrafluoroethylene reaction kettle, and heated at 200℃ for 6-8 h. The product is dialyzed with water for 12 h, and the water is replaced every 3 h. After freeze-drying, Gd-CQDs are obtained; B. L-cysteine is coupled to Gd-CQDs through EDC / NHS coupling reaction. Thus, thiol-modified Gd-CQDs are obtained; C. 500~1000 µL 2.5 mg / mL carboxyl-modified polystyrene beads were added to 12.2 mg EDC, 7.2 mg NHS in 10 mL ethanol, stirred at room temperature for 30 min, 200 µL of ethylenediamine-capped polyethyleneimine was added, and the mixture was reacted by EDC / NHS at room temperature for 3 h. The mixture was centrifuged at 3405 ×g for 5~10 min, washed, and then dispersed in 10 mL of a pH = 7.5, 0.1 M PBS solution containing 10wt% N,N-dimethylformamide. 150~300 µL of 0.01 M succinimidyl 3-(2-pyridyldithio)propionate was added, and the reaction was carried out at room temperature for 1~3 h. After centrifugation and washing, the product was redispersed in 10 mL of a pH = 8.0, 0.1 M PBS solution containing 10% DMF. 1 mL of a thiol Gd-CQDs dispersion was added, and the reaction was carried out at 4 ℃ for 8~10 h. After centrifugation and washing, the product was dispersed in 10~20 mL of water to obtain a spherical bush-like PS@Gd-CQDs composite dispersion; D. Foodborne pathogenic bacteria polyclonal antibodies were coupled to the spherical bush-like PS@Gd-CQDs composite by EDC / NHS coupling reaction to obtain a biologically functionalized spherical bush-like PS@Gd-CQDs@Ab composite; (3) Low-field nuclear magnetic resonance homogeneous immunoassay 200 μL of the PS@Gd-CQDs@Ab dispersion and 50~200 μL of the MGO@Ab dispersion, 1 mL of the foodborne pathogenic bacteria sample to be detected were mixed in a sample bottle, shaken and incubated for 30~40 min, and the capture unit-foodborne pathogenic bacteria-signal unit sandwich structure was obtained by magnetic separation. The capture unit-foodborne pathogenic bacteria-signal unit sandwich structure was added to 800 μL of a 50 mM dithiothreitol solution, reacted for 20 min, washed magnetically, 200 μL of 0.01 M hydrochloric acid was added, reacted for 5 min, and placed in a low-field nuclear magnetic resonance contrast agent relaxation analyzer. T1 was collected at 35℃, the IR pulse sequence was used for measurement, the difference ΔT1 in the transverse relaxation time of water protons corresponding to a series of different concentrations of foodborne pathogenic bacteria was determined, and the quantitative relationship between the longitudinal relaxation time difference of water protons and the concentration of foodborne pathogenic bacteria was established. According to the quantitative relationship, the concentration of foodborne pathogenic bacteria in the unknown sample can be determined. The value of ΔT1 is calculated by the following formula: ΔT1 = T1(negative) – T1(positive), wherein T1 is the longitudinal relaxation time of water protons, T1(negative) is the average T1 without foodborne pathogenic bacteria, and T1(positive) is the average T1 with foodborne pathogenic bacteria.

2. The method for the detection of foodborne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous immunoassay based on gadolinium quantum dots explosion amplification strategy according to claim 1, which is not for the purpose of diagnosis or treatment, characterized in that The specific process of step (2)B is as follows: 1 mg of Gd-CQDs, 12.2 mg of EDC, and 7.2 mg of NHS are dissolved in 10 mL of water, the pH is adjusted to 5.0 with 0.01 M hydrochloric acid, and stirring is performed at room temperature for 30 min, 1 mL of 0.1 M L-cysteine is added, and reaction is performed at room temperature for 4 h, then dialysis is performed for 12 h by using a dialysis membrane with a molecular weight cut-off of 1000 Da to remove excess L-cysteine, and the product is dispersed in 5 mL of 0.1 M PBS solution at pH = 7.5 to obtain a thiol-modified Gd-CQDs dispersion.

3. The method for the detection of foodborne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous immunoassay based on gadolinium quantum dots explosion amplification strategy according to claim 1, which is not for the purpose of diagnosis or treatment, characterized in that The specific process of step (2)D is as follows: 12.2 mg of EDC and 7.2 mg of NHS are added to the spherical bush-like PS@Gd-CQDs composite dispersion obtained in step (2)C, and reaction is performed at room temperature for 30 min, 200 μL of a 1.5 mg / mL foodborne pathogenic bacteria polyclonal antibody solution is added, and reaction is performed at room temperature for 1-3 h, then 200 μL of a 2 wt% BSA solution is added to block non-specific binding sites, the mixture is centrifuged at 3405 ×g for 7 min, and after washing to remove unbound foodborne pathogenic bacteria polyclonal antibodies, the mixture is dispersed in 10 mL of 0.01M PBS solution at pH = 7.4 to obtain a biologically functionalized spherical bush-like PS@Gd-CQDs@Ab composite.

4. The method for the detection of foodborne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous immunoassay based on gadolinium quantum dots explosion amplification strategy according to claim 1, which is not for the purpose of diagnosis or treatment, characterized in that: T1 is measured at 35 ℃ by using an IR pulse sequence measurement with the following parameters: main frequency 19.00 MHz, sampling frequency 100 kHz, sampling delay 0.08 ms, sampling points 2048, waiting time 20000 ms, inversion number 20, number of accumulations 2 times, digital gain 3, and analog gain 15.0 dB.

5. The method for the detection of foodborne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous immunoassay based on gadolinium quantum dots explosion amplification strategy according to any one of claims 1-4, which is not for the purpose of diagnosis or treatment, characterized in that: The foodborne pathogenic bacteria include Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli, and Salmonella. The foodborne pathogenic bacteria include Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli, and Salmonella.

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