CN114966012B - Method for detecting food-borne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous phase immunity based on superparamagnetism two-dimensional material - Google Patents
Method for detecting food-borne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous phase immunity based on superparamagnetism two-dimensional material Download PDFInfo
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Abstract
The invention discloses a method for detecting food-borne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous phase immunity based on superparamagnetic two-dimensional materials, which is characterized by comprising the following steps: taking 0.1mL functionalized superparamagnetic two-dimensional material GO@SPIONS&Ab dispersion and 1.0-1.4. 1.4mL to-be-detected samples containing different concentrations of food-borne pathogenic bacteria are added into a sample bottle to be mixed, shaken and incubated for 30min, and then placed into a low-field nuclear magnetic resonance contrast agent relaxation analyzer to collect T at 35 DEG C 2 Measuring the transverse relaxation time difference delta T of water protons corresponding to a series of food-borne pathogenic bacteria with different concentrations by using CPMG pulse sequence measurement 2 Establishing a quantitative relationship between the transverse relaxation time difference of water protons and the concentration of food-borne pathogenic bacteria; the concentration of the food-borne pathogenic bacteria in the unknown sample can be determined according to the quantitative relationship, and the method has the advantages of high sensitivity and accuracy, strong specificity and simple and rapid operation.
Description
Technical Field
The invention relates to a method for detecting pathogenic bacteria in low-field nuclear magnetic resonance homogeneous phase, in particular to a method for detecting food-borne pathogenic bacteria in low-field nuclear magnetic resonance homogeneous phase based on superparamagnetic two-dimensional materials.
Background
Food-borne pathogenic bacteria refer to pathogenic bacteria that can cause food poisoning or be a vehicle for food transmission. Common food-borne pathogens are: the traditional method for detecting the food-borne pathogenic bacteria is a biochemical culture identification method, and the method has the advantages of complicated steps, long detection period, time consumption and labor consumption. Along with the rapid development of molecular biology technology, methods such as Polymerase Chain Reaction (PCR), DNA hybridization, loop-mediated isothermal amplification (LAMP), biochip and the like are also applied to detecting food-borne pathogenic bacteria, so that good accuracy and sensitivity are obtained, but in practical application, the methods have a plurality of problems: the probability of false positive is high, the instrument is expensive, the detection cost is high, the detection steps are complex, the detection time is long, and the like. Therefore, development of a sensitive, accurate, simple and rapid method for detecting food-borne pathogenic bacteria is an urgent need. Vibrio Parahaemolyticus (VP), a gram-negative halophilic bacterium, is widely distributed in estuary, coastal and marine environments and in marine organisms such as zooplankton, fish and shellfish. VP is recognized as one of the most important food-borne pathogens in the world, and when eating raw, uncooked or improperly processed seafood (particularly shellfish), it can induce a range of diseases such as acute gastroenteritis, wound infection, sepsis, and even death in severe cases. In order to cope with the increasing public health and medical diagnosis demands, it is significant to develop a rapid, sensitive, accurate and nondestructive VP on-site detection method.
The VP is conventionally detected by adopting a plate colony counting method, and the method is long in time consumption and complex in steps and cannot meet the high-efficiency field detection requirement. Methods based on molecular biological detection of VP, including enzyme-linked immunoassay (ELISA), polymerase Chain Reaction (PCR), loop-mediated isothermal amplification (LAMP), etc., although the detection speed is high, the detection accuracy and sensitivity are high, expensive instruments, complex detection steps and skilled operators are required, and the method is not suitable for on-site rapid detection. Other methods such as colorimetry, fluorescence (FIA), electrochemiluminescence (ECL) and the like can detect VP, but the requirements on samples are high, analysis on turbid real samples cannot be directly carried out, detection can be carried out only by complex pretreatment, and field usability is reduced.
The detection principle of the low-field nuclear magnetic resonance MRsw sensor is as follows: in a uniform magnetic field, the precession frequency of the water proton is closely related to the environment where the water proton magnet is located, and if the magnetic field intensity of the environment where the water proton magnet is located at different positions is inconsistent, the precession frequency is also changed, the phase consistency is lost, and the relaxation time is shortened. The influencing magnetic field comprises two main magnetic fields, a local magnetic field formed by superparamagnetic materials, T if the main magnetic fields are uniform and consistent 2 Depending on the local magnetic field. When the sample is positioned outside the magnetic field, hydrogen protons in water molecules in the sample are in a spin disorder state, when the sample is positioned in the magnetic field, the hydrogen protons are oriented and arranged under the action of the magnetic field, a radio frequency pulse with the same spin frequency is applied to the hydrogen protons, the hydrogen protons can absorb the energy of the radio frequency pulse, when the radio frequency field is removed, the hydrogen protons can release the energy of the absorbed radio frequency field, and a specific coil can detect the energy and convert the energy into a signal. The detection of the low-field nuclear magnetic resonance technology can be applied to the fields of industries such as agricultural foods, energy exploration, high polymer materials, textile industry, life science and the like, such as food detection, fiber oil-up rate detection, core oil-up rate analysis, seed oil-up rate analysis, medicine curative effect analysis in biological medicine, petroleum exploration, unconventional energy development, determination of rubber high polymer materials and the like, relaxation time measurement of tumor targeted contrast agent, porous medium pore diameter and distribution research thereof and the like.
Superparamagnetism is a phenomenon of super spin with no or weak interactions. Because most two-dimensional materials are not magnetic, a great deal of work is focused on creating vacancies, doping elements, and the like to introduce magnetism into non-magnetic graphene or molybdenum disulfide materials, and in recent years, intrinsic secondary two-dimensional materials have been discovered by mechanical exfoliation, chemical vapor deposition, molecular beam epitaxy, and the like, and exhibit superparamagnetism under certain specific conditions. However, the intrinsic magnetic two-dimensional material is still in the theoretical research stage at present, but a magnetic nano two-dimensional composite material formed by combining nano ions can be manufactured. Two-dimensional materials are limited in two-dimensional planes due to both their carrier transport and thermal diffusion, so that such materials exhibit many unique properties. The band gap adjustable characteristic is widely applied to the fields of field effect transistors, photoelectric devices, thermoelectric devices and the like; the controllability of the spin degree of freedom and the valley degree of freedom thereof has led to intensive studies in the fields of spintronics and valley electronics. At present, no related research report on a preparation method of an immunosensor for detecting food-borne pathogenic bacteria based on superparamagnetic two-dimensional materials exists at home and abroad.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for detecting food-borne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous phase immunity based on superparamagnetic two-dimensional materials, which has high sensitivity and accuracy, strong specificity and simple and rapid operation.
The technical scheme adopted for solving the technical problems is as follows: a method for detecting food-borne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous phase immunity based on superparamagnetic two-dimensional materials, which does not aim at diagnosis or treatment, comprises the following steps:
(1) Synthesis of functionalized superparamagnetic two-dimensional material GO@SPIONS & Ab
A. Centrifuging 0.5-1.5-mg/mL Graphene Oxide (GO) dispersion 15-25mL at 12000rpm for 5min, discarding supernatant, dispersing in 15-25mL anhydrous ethanol again for 6min, adding 15-25 μL 0.0426M 3-aminopropyl triethoxysilane (APTES) solution, magnetically stirring at 65-75deg.C for 3-5h, repeatedly washing with ethanol and water, dispersing in 20mL water to obtain aminoSingle-layer graphene oxide (NH) 2 -GO) dispersion;
B. 1-3mL of 25wt% glutaraldehyde solution is added to 8-12mL NH 2 In GO dispersion (deionized water as solvent), magnetically stirring at room temperature for 2-5h, centrifuging at 5000rpm, washing with ethanol, dispersing in 8-12mL water, adding 150-250 [ mu ] L0.5-mg/mL superparamagnetic nano-iron oxide particles (SPIONs) and 150-250 [ mu ] L100 [ mu ] g/mL food-borne pathogenic bacteria polyclonal antibody (Ab) solution into the dispersion, oscillating for 4h, SPIONs, ab and NH 2 GO is fully bonded to form functionalized GO@SPIONs&Ab, after further addition of 150-250 μL 2wt% Bovine Serum Albumin (BSA) to block non-specific binding sites and washing with PBS solution pH=7.4, 0.01M to remove unbound BSA and Ab, was dispersed in 2mL PBS solution, 1737×gCentrifuging for 3min to remove free SPIONs, and dispersing the obtained product in 10mL PBS solution to obtain functionalized superparamagnetic two-dimensional material GO@SPIONs&Ab dispersion, stored in a refrigerator at 4 ℃;
(2) Low-field nuclear magnetic resonance homogeneous phase immunoassay for food-borne pathogenic bacteria
Taking 0.1mLGO@SPIONS&Ab dispersion and 1.0-1.4. 1.4mL to-be-detected samples containing different concentrations of food-borne pathogenic bacteria are added into a sample bottle to be mixed, shaken and incubated for 30min, and then placed into a low-field nuclear magnetic resonance contrast agent relaxation analyzer to collect T at 35 DEG C 2 Using CPMG pulse sequence measurement (Carr-Purcell-Meiom-Gill) to determine transverse relaxation time difference DeltaT of water protons corresponding to a series of different concentrations of food-borne pathogenic bacteria 2 Establishing a quantitative relationship between the transverse relaxation time difference of water protons and the concentration of food-borne pathogenic bacteria; and determining the concentration of the food-borne pathogenic bacteria in the unknown sample according to the quantitative relationship.
The measurement parameters of the step (2) are as follows: the main frequency is 19.00MHz, the number of echoes is 18000, the echo time is 0.6ms, the accumulation times are 2 times, the waiting time is 5000ms, the digital gain is 3, and the analog gain is 15.0dB.
Step (2) DeltaT 2 The value of (2) is calculated by the following formula: delta T 2 = T 2N (negative) – T 2P (positive)，Wherein T is 2 Is the transverse relaxation time of water proton, T 2N (negative) is the average T in the absence of food-borne pathogenic bacteria 2 ，T 2P (positive) is the average T when the pathogen is food-borne 2 . The greater the concentration of food-borne pathogenic bacteria, the corresponding T 2 The shortening is about small.
The food-borne pathogenic bacteria comprise vibrio parahaemolyticus, vibrio vulnificus, staphylococcus aureus, escherichia coli and salmonella.
The principle of the invention: the invention constructs a superparamagnetism two-dimensional material MRSw detection VP sensor based on a magnetic relaxation switch sensor (MRSw) sensor principle and combined with a GO sheet structure with an ultra-large specific surface area. The functionalized superparamagnetism GO is used as a capturing unit and a signal unit of the sensor, and a capturing antibody Ab immobilized on the GO through glutaraldehyde has the function of capturing VP; the detection antibody Ab can form a space network aggregate structure by the superparamagnetism GO and VP of the multivalent ligand through the specific combination of antigen and antibody, and change the T of surrounding water molecules 2 Time, T 2 The amount of change in time can be quantified to detect the target concentration.
According to the immunological principle of the invention, SPIONs and Ab are fixed on aminated GO through glutaraldehyde, so that a magnetic label with superparamagnetism and specificity is formed. When VP is not present, the flaky magnetic labels are uniformly dispersed in water to form a relatively uniform and stable magnetic fluid; when VP exists in the system, ab can capture VP by utilizing the specific binding of antigen and antibody, and is mutually connected with a magnetic target to form a space network polymer structure, the dosage of VP is adjusted, the regulation and control of the size and the quantity of the space network polymer can be realized, and therefore the regulation and control of water molecules T in the system is controlled 2 The intensity of the influence. Water molecule T in system 2 Is reduced by an amount DeltaT 2 And the sample and the VP concentration show a certain relation, and under a specific working curve, the detection of the unknown concentration of VP in the sample can be realized.
Compared with the prior art, the invention has the advantages that:
1. sample pretreatment is simple: the signal of LF-NMR is derived from magnetic rather than photoelectric properties, so that LF-NMR is almost free of background interference and even a turbid sample can be detected directly, considering that there is almost no magnetic substance in the detection environment. The detection object is a pathogenic individual, DNA is not required to be extracted for amplification, and the sample can be directly detected.
2. The steps are simple, and the detection efficiency is improved: the prepared MRsw sensor is only required to be mixed with pathogenic bacteria, and the mixture is incubated and reacted for 30min under the shaking condition, so that the mixture can be placed into a low-field nuclear magnetic resonance detector for detection, and the detection time is 2-3min. When detecting a plurality of sample sets, incubation time is parallel to detection time, and detection efficiency is improved.
3. Triple amplified signals improve detection sensitivity: (1) magnetic susceptibility enhancement. The SPIONs are uniformly dispersed on the surface of the GO, so that the contact area with water is increased, the magnetic susceptibility of the magnetic material per unit mass is increased, the affected water molecule range is enlarged, and the primary enhancement of signals is realized. (2) the number of magnetic signal tags SPIONs increases. The binding sites on each target are not a corresponding SPIONs, but a large number of SPIONs bound on a GO sheet, so that the secondary enhancement of signals is realized. (3) synergistic effect is enhanced. The multivalent SPIONs have larger size difference with micron-level pathogenic bacteria, when immune complexes are formed, the steric effect is obviously difficult to form a larger space network structure, the superparamagnetic two-dimensional material depends on a larger surface area and excellent plasticity, and the cross section area of the space network structure formed by the target object is larger, so that the synergistic effect formed by interaction is also larger, the local magnetic field is more uneven, and T is the same as that of the target object 2 Shorter, realize the signal tertiary enhancement. The triple amplified signal greatly improves the detection sensitivity.
In conclusion, the invention prepares the method for detecting the food-borne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous phase immunity based on the superparamagnetism two-dimensional material for the first time, wherein the material is GO surface and simultaneously carries superparamagnetism nanometer Fe 3 O 4 Particles (SPIONs) that generate a signal and VP antibodies that specifically recognize and capture VP. By adopting the technical framework, the triple signal amplification can be realized through the following paths: (1) increasing the number of magnetic signal tags SPIONs. (2) enhancing magnetic susceptibility. (3) Forming a space network polymer structure with excellent superparamagnetismCompared with the traditional MRsw, the two-dimensional material MRsw performs triple signal amplification, and improves detection sensitivity. In addition, the novel MRsw biosensor can directly detect turbid real samples, is simple to operate, simple in sample pretreatment and short in detection time, and has great potential of being used as a food-borne pathogenic bacteria on-site instant detection tool.
Drawings
FIG. 1 is a flow chart of the preparation of a functionalized superparamagnetic two-dimensional material of the present invention;
FIG. 2 is an electron microscope image of the functionalized superparamagnetic two-dimensional material of the present invention;
FIG. 3 is a schematic diagram of a detection scheme of a VP sensor for detecting MRsw of the functionalized superparamagnetic two-dimensional material;
FIG. 4 is a graph showing the linear relationship between VP concentration and VP concentration for low-field NMR detection;
FIG. 5 is a diagram of sensor specificity for low field nuclear magnetic resonance detection of different species of bacteria at the same concentration.
Detailed Description
The invention is described in further detail below with reference to the embodiments of the drawings.
Detailed description of the preferred embodiments
Example 1
A method for detecting vibrio parahaemolyticus by low-field nuclear magnetic resonance homogeneous phase immunity based on superparamagnetic two-dimensional materials is shown in figure 1, and comprises the following steps:
(1) Synthesis of functionalized superparamagnetic two-dimensional material GO@SPIONS & Ab
A. Centrifuging 0.5-1.5mg/mL Graphene Oxide (GO) dispersion 20mL at 12000rpm for 5min, discarding supernatant, dispersing in 20mL anhydrous ethanol again, performing ultrasonic treatment for 6min, adding 25 μL 0.0426M 3-aminopropyl triethoxysilane (APTES) solution, magnetically stirring at 70deg.C for 4h, repeatedly washing with ethanol and water to remove excessive APTES, dispersing in 20mL water to obtain aminated single-layer graphene oxide (NH) 2 -GO) dispersion;
B. 2mL of a 25wt% glutaraldehyde solution was added to 10mL NH 2 GO dispersion (solvent is de-oIon water), after magnetically stirring 3 h at room temperature, centrifuging at 5000rpm and washing with ethanol, dispersing in 10mL water, adding 200 [ mu ] L of 0.5mg/mL superparamagnetic nano-iron oxide particles (SPIONs) and 200 [ mu ] L of 100 [ mu ] g/mL Vibrio parahaemolyticus polyclonal antibody (Ab) solution into the dispersion, oscillating 4h, SPIONs, ab and NH 2 GO is fully bonded to form functionalized GO@SPIONs&Ab, 200 [ mu ] L2 wt% Bovine Serum Albumin (BSA) was added to block non-specific binding sites and washed with PBS solution at pH=7.4, 0.01M to remove unbound BSA and Ab, dispersed in 2mL PBS solution, 1737×gCentrifuging for 3min to remove free SPIONs, and dispersing the obtained product in 10mL PBS solution to obtain functionalized superparamagnetic two-dimensional material GO@SPIONs&Ab dispersion, stored in a refrigerator at 4 ℃;
the morphological characteristics of the superparamagnetism two-dimensional material are shown in figure 2, and the figure 2 shows that the surface of the two-dimensional material is modified with a large number of SPIONs, the particle sizes of the SPIONs are uniform, and the SPIONs are relatively evenly distributed on the surface of GO, so that the superparamagnetism two-dimensional material synthesized by the method has good stability and good reproducibility. In addition, the combination amount of the GO surface magnetic ball is controlled and the magnetic moment of the whole material is adjusted by adjusting the amount of the SPIONs.
(2) Low-field nuclear magnetic resonance homogeneous immunoassay of vibrio parahaemolyticus
Taking 0.1mLGO@SPIONS&Adding Ab dispersion and 1.2 mL sample containing Vibrio parahaemolyticus at different concentrations into sample bottle, mixing, shaking and incubating for 30min, placing into low-field nuclear magnetic resonance contrast agent relaxation analyzer, collecting T at 35deg.C 2 CPMG pulse sequence measurement (Carr-Purcell-Meiom-Gill), deltaT was used 2 The value of (2) is calculated by the following formula: delta T 2 = T 2N (negative) – T 2P (positive), wherein T 2 Is the transverse relaxation time of water proton, T 2N (negative) is the average T in the absence of Vibrio parahaemolyticus 2 ，T 2P (positive) is the average T in the presence of Vibrio parahaemolyticus 2 Measuring the transverse relaxation time difference delta T of a series of water protons corresponding to different concentrations of food-borne pathogenic bacteria 2 Establishing the transverse direction of water protonsQuantitative relation between relaxation time difference and food-borne pathogenic bacteria concentration, and according to the quantitative relation, the concentration of vibrio parahaemolyticus in an unknown sample can be determined, and the greater the concentration of vibrio parahaemolyticus is, the corresponding T 2 The shortening is about small, i.e. DeltaT 2 The larger the basis weight.
The measurement parameters were as follows: the main frequency is 19.00MHz, the number of echoes is 18000, the echo time is 0.6ms, the accumulation times are 2 times, the waiting time is 5000ms, the digital gain is 3, and the analog gain is 15.0dB.
The detection principle of the low-field nuclear magnetic resonance sensor in the study is shown in fig. 3, and it can be known from fig. 3 that in a uniform magnetic field, the precession frequency of water protons is closely related to the environment where the water proton magnets are located, and if the magnetic field strengths of the environments where the water proton magnets are located at different positions are inconsistent, the precession frequency is also changed, the phase consistency is lost, and the relaxation time is shortened. The influencing magnetic field comprises two main magnetic fields, a local magnetic field formed by superparamagnetic materials, T if the main magnetic fields are uniform and consistent 2 Depending on the local magnetic field. When marine pathogens exist, superparamagnetism two-dimensional materials are agglomerated into clusters, the more marine pathogens are, the larger the clusters are, the more uneven local magnetic fields are, and T is 2 The shorter the time, the more quantitative.
Example 2
The difference from the above embodiment 1 is that: (1) Functionalized superparamagnetic two-dimensional material GO@SPIONS&Synthesis of Ab: centrifuging 15mL of graphene oxide dispersion liquid with the concentration of 0.5mg/mL at 12000rpm for 5min, discarding supernatant, dispersing in 15mL of absolute ethyl alcohol again for 6min by ultrasonic treatment, adding 15 mu L of 0.0426M 3-aminopropyl triethoxysilane solution, magnetically stirring at 65 ℃ for 5h, repeatedly washing with ethanol and water, dispersing in 20mL of water to obtain aminated single-layer graphene oxide (NH) 2 -GO) dispersion; 1mL of a 25wt% glutaraldehyde solution was added to 8mL NH 2 In the GO dispersion liquid, after magnetically stirring for 2 hours at room temperature, centrifuging at 5000rpm, washing with ethanol, dispersing in 8mL of water, adding 150 mu L of 0.5mg/mL superparamagnetic nano-iron oxide particles (SPIONs) and 150 mu L of 100 mu g/mL vibrio parahaemolyticus polyclonal antibody (Ab) solution into the dispersion liquid, oscillating for 4h to form the functionalized GO@SPIONs&Ab, after further addition of 150. Mu.L 2wt% Bovine Serum Albumin (BSA) to block non-specific binding sites and washing with PBS solution at pH=7.4, 0.01M, was dispersed in 2mL PBS solution 1737×gCentrifuging for 3min to remove free SPIONs, and dispersing the obtained product in 10mL PBS solution to obtain functionalized superparamagnetic two-dimensional material GO@SPIONs&Ab dispersion, stored in a refrigerator at 4 ℃;
(2) Low-field nmr homogeneous immunodetection of vibrio parahaemolyticus: 0.1 mLGO@SPIONs&Ab dispersion and 1.0 mL samples to be tested containing different concentrations of food-borne pathogenic bacteria are taken and added into a sample bottle for mixing.
Example 3
The difference from the above embodiment 1 is that: (1) Functionalized superparamagnetic two-dimensional material GO@SPIONS&Synthesis of Ab: centrifuging 1.5. 1.5mg/mL Graphene Oxide (GO) dispersion liquid 25mL at 12000rpm for 5min, discarding supernatant, dispersing in 25mL anhydrous ethanol again for 6min by ultrasonic treatment, adding 25 μL 0.0426M 3-aminopropyl triethoxysilane (APTES) solution, magnetically stirring at 75deg.C for 5h, repeatedly washing with ethanol and water, dispersing in 20mL water to obtain aminated single-layer graphene oxide (NH) 2 -GO) dispersion; 3mL of a 25wt% glutaraldehyde solution was added to 12mL NH 2 In the GO dispersion liquid, after magnetically stirring 5h at room temperature, centrifuging at 5000rpm, washing with ethanol, dispersing in 12mL of water, adding 250 [ mu ] L0.5 mg/mL superparamagnetic nano-iron oxide particles (SPIONs) and 250 [ mu ] L100 [ mu ] g/mL Vibrio parahaemolyticus polyclonal antibody (Ab) solution into the dispersion liquid, oscillating 4h to form functionalized GO@SPIONs&Ab, after addition of 250. Mu.L 2wt% Bovine Serum Albumin (BSA) to block non-specific binding sites and washing with PBS solution at pH=7.4, 0.01M, was dispersed in 2mL PBS solution 1737×gCentrifuging for 3min to remove free SPIONs, and dispersing the obtained product in 10mL PBS solution to obtain functionalized superparamagnetic two-dimensional material GO@SPIONs&Ab dispersion, stored in a refrigerator at 4 ℃;
(2) Low-field nmr homogeneous immunodetection of vibrio parahaemolyticus: 0.1 mLGO@SPIONs&Ab dispersion and 1.4mL samples to be tested containing different concentrations of food-borne pathogenic bacteria are taken and added into a sample bottle for mixing.
In addition to the above embodiments, vibrio parahaemolyticus may be replaced with vibrio vulnificus, staphylococcus aureus, escherichia coli or salmonella, and corresponding antibodies, thereby achieving the purpose of detecting different food-borne pathogenic bacteria.
Second embodiment
Sensitivity detection
FIG. 4 is a graph showing the linear relationship between VP concentration and VP concentration for low-field NMR detection; taking 30min as the optimal incubation time, the sensitivity of MRsw for detecting vibrio parahaemolyticus VP is examined, and quantitative detection is carried out by 1.0X10 1 CFU/mL~1.0×10 6 CFU/mL of pathogenic bacteria at different concentrations. The results are shown in FIG. 4A, where DeltaT is between the different samples 2 Increasing with increasing VP concentration. As shown in FIG. 4B, the concentration is 1.0X10 2 CFU/mL~1.0×10 5 Within the CFU/mL concentration interval, deltaT 2 (y) shows good linear relation with the logarithm of VP concentration (x), and the linear regression equation is y= 48.533x-64.495, R 2 0.993. According to FIG. 4B, the limit of quantitation of MRsw in detecting VP is 1.0X10 2 CFU/mL, therefore demonstrated that VP detection with MRsw is sensitive.
Detailed description of the preferred embodiments
Specific detection
Specificity experiments of MRsw sensor, in order to simulate seawater complex bacterial environment, the concentration was measured to be 1.0X10 under the optimal experimental conditions 6 CFU/mL Vibrio vulnificusVibrio Vulnificus, VV) Vibrio harveyi (Vibrio harveyi)Vibrio harveyi, VH) Listeria strainListeria monocytogenes, LM) Salmonella (Salmonella)Salmonella, SM) Staphylococcus aureus @ sStaphylococcus aureus,SA) Coli @Escherichia Coli, E.coli) Vibrio parahaemolyticusVibrio Parahemolyticus, VP) And contains 1.0X10 6 Mixed samples of VP at CFU/mL concentration, blank samples were added to PBS to verify the specificity of MRSw sensor for target detector VP. As a result, as shown in FIG. 5, T of VP-containing sample 2 With obvious changes, but detection of other pathogenic bacteriaThe signal response of the sample was similar to that of the blank. The above results indicate that MRsw sensor has higher specificity to VP, and in addition, at the ΔT of VP mixture sample 2 Similar to the signal of the VP alone sample, this means that other pathogens hardly affect the specific recognition of VP by the sensor, reflecting the excellent specificity of VP detection by MRSw sensors.
Application examples
To verify the value of the method in practical application, the standard solution of vibrio parahaemolyticus is added into the sea water of the east sea as a practical sample, the vibrio parahaemolyticus with different concentrations in the sea water is detected by a low-field nuclear magnetic resonance method by adopting a method of adding a standard and recycling, the results are shown in table 1,
TABLE 1
。
As shown in Table 1, the Relative Standard Deviation (RSD) of the method is less than 8.9%, the recovery rate is 93.5-108.4%, and the result is satisfactory, which shows that the method is accurate and reliable for detecting the vibrio parahaemolyticus in the seawater.
The above description is not intended to limit the invention, nor is the invention limited to the examples described above. Variations, modifications, additions, or substitutions will occur to those skilled in the art and are therefore within the spirit and scope of the invention.
Claims (3)
1. A method for detecting food-borne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous phase immunity based on superparamagnetic two-dimensional materials, which does not aim at diagnosis or treatment, and is characterized by comprising the following steps:
(1) Synthesis of functionalized superparamagnetic two-dimensional material GO@SPIONS & Ab
A. Centrifuging 15-25mL of graphene oxide GO dispersion liquid with the concentration of 0.5-1.5mg/mL at 12000rpm for 5min, discarding supernatant, dispersing in 15-25mL of absolute ethyl alcohol again, performing ultrasonic treatment for 6min, adding 15-25 mu L of 0.0426M 3-aminopropyl triethoxysilane solution with the concentration of 0.0426M, magnetically stirring at 65-75 ℃ for 3-5h,repeatedly washing with ethanol and water, dispersing in 20ml water to obtain aminated monolayer graphene oxide NH 2 -GO dispersion;
B. 1-3mL of 25wt% glutaraldehyde solution is added to 8-12mL NH 2 In the GO dispersion liquid, after magnetically stirring for 2-5h at room temperature, centrifuging at 5000rpm, washing with ethanol, dispersing in 8-12mL of water, adding 150-250 mu L of 0.5mg/mL superparamagnetic nano ferric oxide particle SPIONs and 150-250 mu L of 100 mu g/mL polyclonal antibody Ab solution of food-borne pathogenic bacteria into the dispersion liquid, oscillating for 4h to obtain functionalized GO@SPIONs&Ab, adding 150-250 mu L of 2wt% bovine serum albumin BSA solution to block non-specific binding sites, washing with PBS solution with pH= 7.4,0.01M to remove unbound BSA and Ab, dispersing in 2mL of PBS solution, centrifuging at 1737 Xg for 3min to remove free SPIONs, and dispersing the obtained product in 10mL of PBS solution to obtain functionalized superparamagnetic two-dimensional material GO@SPIONs&Ab dispersion;
(2) Low-field nuclear magnetic resonance homogeneous phase immunoassay for food-borne pathogenic bacteria
Taking 0.1mL of O@SPIONS&Adding Ab dispersion and 1.0-1.4mL of sample to be tested containing different concentrations of food-borne pathogenic bacteria into a sample bottle, mixing, shaking and incubating for 30min, placing into a low-field nuclear magnetic resonance contrast agent relaxation analyzer, and collecting T at 35deg.C 2 Measuring transverse relaxation time difference delta T of water protons corresponding to a series of food-borne pathogenic bacteria with different concentrations by using CPMG pulse sequence measurement method 2 Establishing a quantitative relationship between the difference in transverse relaxation time of water protons and the concentration of food-borne pathogenic bacteria, and determining the concentration of food-borne pathogenic bacteria in an unknown sample according to the quantitative relationship, wherein the delta T 2 The value of (2) is calculated by the following formula: delta T 2 ＝T 2N –T 2P Wherein T is 2 Is the transverse relaxation time of water proton, T 2N Is the average T of food-borne pathogenic bacteria 2 ，T 2P Is the average T when the pathogenic bacteria are food-borne 2 。
2. The method for low-field nuclear magnetic resonance homogeneous phase immunoassay of food-borne pathogenic bacteria based on superparamagnetic two-dimensional materials according to claim 1, which is not for diagnosis or treatment purposes, characterized in that the measurement parameters of step (2) are as follows: the main frequency is 19.00MHz, the number of echoes is 18000, the echo time is 0.6ms, the accumulation times are 2 times, the waiting time is 5000ms, the digital gain is 3, and the analog gain is 15.0dB.
3. The method for low-field nuclear magnetic resonance homogeneous phase immunoassay of food-borne pathogenic bacteria based on superparamagnetic two-dimensional materials according to claim 1, which is not for diagnostic or therapeutic purposes, characterized in that: the food-borne pathogenic bacteria are selected from the group consisting of vibrio parahaemolyticus, vibrio vulnificus, staphylococcus aureus, escherichia coli and salmonella.
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