CN114966012A - Method for detecting food-borne pathogenic bacteria based on low-field nuclear magnetic resonance homogeneous phase immunoassay of superparamagnetic two-dimensional material - Google Patents
Method for detecting food-borne pathogenic bacteria based on low-field nuclear magnetic resonance homogeneous phase immunoassay of superparamagnetic 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 immunoassay based on a superparamagnetic two-dimensional material, which is characterized by comprising the following steps of: taking 0.1 mL of functionalized superparamagnetic two-dimensional material GO @ SPIONs&Ab dispersion liquid and 1.0-1.4 mL of samples to be detected containing food-borne pathogenic bacteria with different concentrations are added into a sample bottle for mixing, shaking and incubating for 30min, and the samples are placed in a low-field nuclear magnetic resonance contrast agent relaxation analyzer to collect T at 35 DEG 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 2 Establishing a quantitative relation between the transverse relaxation time difference of the water proton and the concentration of the food-borne pathogenic bacteria; the concentration of the food-borne pathogenic bacteria in the unknown sample can be determined according to the quantitative relation, and the method has the advantages of high sensitivity and accuracy, strong specificity and simple and quick operation.
Description
Technical Field
The invention relates to a method for detecting pathogenic bacteria by low-field nuclear magnetic resonance homogeneous phase, in particular to a method for detecting food-borne pathogenic bacteria by low-field nuclear magnetic resonance homogeneous phase immunoassay based on a superparamagnetic two-dimensional material.
Background
Food-borne pathogenic bacteria refer to pathogenic bacteria that can cause food poisoning or that are food-borne vehicles. Common food-borne pathogenic bacteria include: vibrio parahaemolyticus, vibrio vulnificus, staphylococcus aureus, escherichia coli, salmonella and the like, and the traditional method for detecting food-borne pathogenic bacteria is a biochemical culture identification method, so that the method has the disadvantages of complicated steps, long detection period and time and labor consumption. With the rapid development of molecular biology technology, methods such as Polymerase Chain Reaction (PCR), DNA hybridization and loop-mediated isothermal amplification (LAMP), biochip, etc. are also applied to detecting food-borne pathogenic bacteria, obtaining better accuracy and sensitivity, but there are many problems in practical application: the false positive probability 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, the development of a sensitive, accurate, simple, convenient and rapid method for detecting food-borne pathogenic bacteria is an urgent need. Vibrio Parahaemolyticus (VP) is a gram-negative halophilic bacterium, widely distributed in the environment of estuaries, coastal areas, oceans and the like, and in marine organisms such as zooplankton, fish, shellfish and the like. VP is recognized as one of the most important food-borne pathogens in the world, and a series of diseases, such as acute gastroenteritis, wound infection, septicemia, etc., even death, can be induced by eating raw, uncooked or improperly processed marine products (especially shellfish). In order to meet the growing public health and medical diagnosis demands, it is significant to develop a rapid, sensitive, accurate, nondestructive VP in-situ detection method.
VP is detected by adopting a plate colony counting method in the prior art, and the method is long in time consumption, complex in steps and incapable of meeting the requirement of efficient on-site detection. The method for detecting VP based on molecular biology comprises enzyme-linked immunosorbent assay (ELISA), Polymerase Chain Reaction (PCR), loop-mediated isothermal amplification (LAMP) and the like, and 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 rapid field detection. There are other methods such as colorimetry, Fluorescence (FIA) and Electrochemiluminescence (ECL) which can detect VP, but the requirements for samples are high, the analysis of turbid real samples cannot be directly performed, complicated pretreatment is required for detection, and the field availability 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 water protons is closely related to the environment, and if the magnetic field strength of the environment of the water proton magnet at different positions is inconsistent, the precession frequency of the water proton magnet is changed, the phase consistency is lost, and the relaxation time is shortened. The magnetic field influenced comprises two main magnetic fields, a local magnetic field formed by a superparamagnetic material, T if the main magnetic fields are uniform and coincident 2 Depending on the local magnetic field. When the sample is 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 directionally 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 absorbed energy of the radio frequency field, and a specific coil can detect the energy and convert the energy into a signal. The low-field nuclear magnetic resonance technology can be applied to agricultural food, energy exploration,The method is applied to the fields of industries such as high polymer materials, textile industry, life science and the like, such as food detection, fiber oiling rate detection, core oil content analysis, seed oil content analysis, drug efficacy analysis in biomedicine, oil exploration, unconventional energy development, measurement of rubber high polymer materials and the like, relaxation time measurement of tumor-targeted contrast agents, pore size and distribution research of porous media.
Superparamagnetism is a phenomenon of superspin with no or weak interaction. Since most two-dimensional materials do not have magnetism, a great deal of work is focused on introducing magnetism into non-magnetic graphene or molybdenum disulfide materials by means of vacancy production, element doping and the like, and in recent years, intrinsic secondary two-dimensional materials are found through methods such as mechanical stripping, 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, but a magnetic nano two-dimensional composite material formed by combining nano ions can be manufactured. Two-dimensional materials are confined to two-dimensional planes due to their carrier transport and thermal diffusion, making such materials exhibit many unique properties. The adjustable band gap characteristic of the band gap is widely applied in the fields of field effect tubes, photoelectric devices, thermoelectric devices and the like; the controllability of its spin degree of freedom and valley degree of freedom has led to intensive research in the fields of spintronics and valley electronics. At present, no relevant research report about a preparation method of an immunosensor for detecting food-borne pathogenic bacteria based on a superparamagnetic two-dimensional material exists at home and abroad.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for low-field nuclear magnetic resonance homogeneous immunoassay of food-borne pathogenic bacteria based on a superparamagnetic two-dimensional material, which has high sensitivity and accuracy, strong specificity, and simple and rapid operation.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for detecting food-borne pathogenic bacteria based on low-field nuclear magnetic resonance homogeneous phase immunoassay of a superparamagnetic two-dimensional material, 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 15-25mL of Graphene Oxide (GO) dispersion liquid of 0.5-1.5 mg/mL at 12000 rpm for 5 min, discarding the supernatant, re-dispersing in 15-25mL of absolute ethanol, performing ultrasonic treatment for 6 min, adding a solution of 0.0426M 3-Aminopropyltriethoxysilane (APTES) of 15-25 muL concentration, magnetically stirring at 65-75 ℃ for 3-5 h, repeatedly washing with ethanol and water, and dispersing in 20mL of water to obtain aminated single-layer graphene oxide (NH) 2 -GO) dispersion;
B. 1-3 mL of a 25 wt% glutaraldehyde solution was added to 8-12mL of NH 2 Magnetic stirring is carried out on GO dispersion liquid (deionized water is used as a solvent) at room temperature for 2-5 h, centrifugation is carried out at 5000 rpm, the GO dispersion liquid is cleaned by ethanol, the GO dispersion liquid is dispersed in 8-12mL of water, 150 plus 250 muL 0.5mg/mL superparamagnetic nano iron oxide particles (SPIONs) and 150 plus 250 muL 100 mug/mL foodborne pathogenic bacterium polyclonal antibody (Ab) solution are added into the dispersion liquid, oscillation is carried out for 4 h, and the SPIONs, the Ab and NH are carried out 2 -GO is fully bonded to form functionalized GO @ SPIONs&Ab, after adding 150-250 μ L of 2 wt% bovine serum albumin solution (BSA) to block non-specific binding sites and washing with PBS solution of pH =7.4, 0.01M to remove unbound BSA and Ab, dispersed in 2mL of PBS solution, 1737×gCentrifuging for 3min to remove free SPIONs, and dispersing the obtained product in 10 mL PBS solution to obtain functionalized superparamagnetic two-dimensional material GO @ SPIONs&Storing the Ab dispersion liquid in a refrigerator at 4 ℃;
(2) low-field nuclear magnetic resonance homogeneous immunoassay for food-borne pathogenic bacteria
Take 0.1 mLGO @ SPIONs&Ab dispersion liquid and 1.0-1.4 mL of samples to be tested containing food-borne pathogenic bacteria with different concentrations are added into a sample bottle for mixing, shaking and incubating for 30min, the sample bottle is placed in a low-field nuclear magnetic resonance contrast agent relaxation analyzer, and T is collected at 35 DEG 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 (Carr-Purcell-Meiom-Gill) 2 Establishing a quantitative relation between the transverse relaxation time difference of the water proton and the concentration of the food-borne pathogenic bacteria; according to the quantitative amountThe relationship can determine the concentration of the food-borne pathogenic bacteria in the unknown sample.
The measurement parameters in the step (2) are as follows: the main frequency is 19.00 MHz, the echo number is 18000, the echo time is 0.6 ms, the accumulation times are 2 times, the waiting time is 5000 ms, the digital gain is 3, and the analog gain is 15.0 dB.
Step (2) Δ T 2 Is calculated by the following formula: delta T 2 = T 2N (negative) – T 2P (positive) wherein T 2 Transverse relaxation time, T, of water protons 2N (negative) is the mean T in the absence of food-borne pathogenic bacteria 2 ，T 2P (Positive) is the average T in the presence of food-borne pathogenic bacteria 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 invention principle is as follows: the invention constructs a superparamagnetic two-dimensional material MRSw detection VP sensor based on a magnetic relaxation switch sensor (MRSw) sensor principle and by combining a GO sheet structure with an ultra-large specific surface area. The functionalized superparamagnetic GO is used as a capture unit and a signal unit of the sensor, and a capture antibody Ab immobilized on GO through glutaraldehyde has the function of capturing VP; the detection antibody Ab can be specifically combined through an antigen antibody, and superparamagnetic GO and VP of a multivalent ligand form a space network aggregate structure to change T of surrounding water molecules 2 Time, T 2 The change in time allows quantitative detection of the target concentration.
According to the immunological principle of the invention, SPIONs and Ab are fixed on aminated GO through glutaraldehyde to form a magnetic label with superparamagnetism and specificity. When VP is not present, the flaky magnetic labels are uniformly dispersed in water to form relatively uniform and stable magnetic fluid; when VP exists in the system, the Ab can capture VP by utilizing specific binding of antigen and antibody, and is mutually connected with the magnetic target to form a space network polymer structure, the size of the VP dosage is adjusted, the size and the quantity of the space network polymer can be regulated, and therefore the regulation on the water molecules T in the system is controlled 2 The strength of the effect. Water molecule T in the system 2 Reduction amount of (delta T) 2 And the concentration of the VP presents a certain relation, and the detection of the unknown concentration of the VP in the sample can be realized under a specific working curve.
Compared with the prior art, the invention has the advantages that:
1. the sample pretreatment is simple: the signal of the LF-NMR comes from magnetic properties rather than photoelectric properties, and the LF-NMR almost has no background interference considering that magnetic substances hardly exist in the detection environment, and even a turbid sample can be directly detected. The detection object is a pathogenic bacterium individual, DNA does not need to be extracted for amplification, and a sample can be directly detected.
2. Simple steps and improved detection efficiency: the prepared MRSw sensor is mixed with pathogenic bacteria, and the mixture is incubated for 30min under the shaking condition, and then the mixture can be put into a low-field nuclear magnetic resonance detector for detection, wherein the detection time is 2-3 min. During detection of a plurality of sample sets, incubation time and detection time are parallel, and detection efficiency is improved.
3. Triple amplification signals improve detection sensitivity: (1) the magnetic susceptibility is enhanced. The SPIONs are uniformly dispersed on the surface of the GO, so that the contact area of the SPIONs and water is increased, the magnetic susceptibility of the magnetic material with unit mass is increased, the range of affected water molecules is enlarged, and the first-level enhancement of signals is realized. (2) The number of magnetic signal tags SPIONs increases. The binding site on each target corresponds to a large number of SPIONs bound on one GO sheet, instead of one SPIONs, so that secondary enhancement of the signal is realized. (3) The synergistic effect is enhanced. The size difference between the polyvalent SPIONs and the micron-level pathogenic bacteria is large, when an immune complex is formed, a large space network structure is obviously difficult to form due to the steric hindrance effect, the cross-sectional area of the space network structure formed by the superparamagnetic two-dimensional material and a target object is larger due to the large surface area and the excellent plasticity, so that the synergistic effect formed by the interaction is larger, the local magnetic field is more uneven, and the T is more uniform 2 And the signal is enhanced in three stages. The triple amplification signal greatly improves the detection sensitivity.
In conclusion, the invention firstly prepares the low-field core based on the superparamagnetic two-dimensional materialThe magnetic resonance homogeneous immunoassay method for food-borne pathogenic bacteria comprises the step that the surface of GO is simultaneously loaded with superparamagnetic nano Fe 3 O 4 Particles (SPIONs) that produce a signal and VP antibodies that specifically recognize and capture VP. With the technical framework, triple signal amplification can be realized through the following paths: (1) the number of magnetic signal tags SPIONs is increased. (2) The magnetic susceptibility is enhanced. (3) And a space network polymer structure is formed, and the superparamagnetic two-dimensional material MRSw with excellent properties is subjected to triple signal amplification compared with the traditional MRSw, so that the detection sensitivity is improved. 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 to become a tool for on-site instant detection of food-borne pathogenic bacteria.
Drawings
FIG. 1 is a flow chart of the preparation of the functionalized superparamagnetic two-dimensional material of the present invention;
FIG. 2 is an electron microscope image of a functionalized superparamagnetic two-dimensional material of the present invention;
FIG. 3 is a schematic diagram of the detection of a VP sensor for detecting a functionalized superparamagnetic two-dimensional material MRSw according to the present invention;
FIG. 4 is a linear relationship diagram of low-field nuclear magnetic resonance detection at different VP concentrations;
FIG. 5 is a diagram showing the specificity of sensors for detecting different types of bacteria at the same concentration by low-field NMR.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Detailed description of the preferred embodiment
Example 1
A method for detecting Vibrio parahaemolyticus by low-field nuclear magnetic resonance homogeneous immunoassay based on a superparamagnetic two-dimensional material, as shown in figure 1, 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 20mL at 12000 rpm for 5 min, discarding supernatant, re-dispersing in 20mL anhydrous ethanol, ultrasonic treating for 6 min, and dispersingAdding 25 muL of 0.0426M 3-Aminopropyltriethoxysilane (APTES) solution, magnetically stirring at 70 ℃ for 4 h, repeatedly washing with ethanol and water to remove excessive APTES, and dispersing in 20ml of water to obtain aminated monolayer graphene oxide (NH) 2 -GO) dispersion;
B. 2mL of a 25 wt% glutaraldehyde solution was added to 10 mL of NH 2 Magnetic stirring is carried out on GO dispersion liquid (deionized water is used as a solvent) at room temperature for 3 hours, centrifugation is carried out at 5000 rpm, the GO dispersion liquid is cleaned by ethanol, the centrifugation is dispersed in 10 mL of water, 200 muL of 0.5mg/mL superparamagnetic nano iron oxide particles (SPIONs) and 200 muL of 100 mug/mL vibrio parahaemolyticus polyclonal antibody (Ab) solution are added into the dispersion liquid, oscillation is carried out for 4 hours, and the SPIONs, Ab and NH are added into the dispersion liquid 2 -GO is fully bonded to form functionalized GO @ SPIONs&Ab, after adding 200 μ L of 2 wt% bovine serum albumin solution (BSA) to block non-specific binding sites and washing with PBS solution of pH =7.4, 0.01M to remove unbound BSA and Ab, dispersed in 2mL of PBS solution, 1737×gCentrifuging for 3min to remove free SPIONs, and dispersing the obtained product in 10 mL PBS solution to obtain the functionalized superparamagnetic two-dimensional material GO @ SPIONs&Storing the Ab dispersion liquid in a refrigerator at 4 ℃;
the shape characteristics of the superparamagnetic two-dimensional material are shown in fig. 2, and it can be known from fig. 2 that a large number of SPIONs are modified on the surface of the two-dimensional material, and the SPIONs have uniform particle size, and are relatively uniformly distributed on the surface of GO, which shows that the superparamagnetic two-dimensional material synthesized by the method has good stability and good reproducibility. In addition, the bonding amount of the magnetic spheres on the GO surface is controlled by adjusting the amount of the added SPIONs, and the size of the magnetic moment of the overall material is adjusted.
(2) Low-field nuclear magnetic resonance homogeneous immunoassay for vibrio parahaemolyticus
Take 0.1 mLGO @ SPIONs&Ab dispersion liquid and 1.2 mL of samples to be detected containing vibrio parahaemolyticus with different concentrations are added into a sample bottle for mixing, shaking for incubation for 30min, placed in a low-field nuclear magnetic resonance contrast agent relaxation analyzer, and T is collected at 35 DEG C 2 Measured using a CPMG pulse sequence (Carr-Purcell-Meiom-Gill), Δ T 2 Is calculated by the following formula: delta T 2 = T 2N (negative) – T 2P (positive) wherein T 2 Transverse relaxation time, T, of water protons 2N (negative) is the average T without 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 water protons corresponding to a series of food-borne pathogenic bacteria with different concentrations 2 Establishing a quantitative relation between the transverse relaxation time difference of the water proton and the concentration of the food-borne pathogenic bacteria, and determining the concentration of the vibrio parahaemolyticus in the unknown sample according to the quantitative relation, wherein the larger the concentration of the vibrio parahaemolyticus is, the corresponding T is 2 The shortening being about small, i.e. Δ T 2 The larger the amount was quantified accordingly.
The measurement parameters were as follows: the main frequency is 19.00 MHz, the echo number is 18000, the echo time is 0.6 ms, the accumulation times are 2 times, the waiting time is 5000 ms, the digital gain is 3, and the analog gain is 15.0 dB.
The detection principle of the low-field nuclear magnetic resonance sensor in the present study is shown in fig. 3, and it can be known from fig. 3 that 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 strength of the environment where the water proton magnet is located at different positions is inconsistent, the precession frequency will also change, the phase consistency is lost, and the relaxation time is shortened. The magnetic field influenced comprises two main magnetic fields, a local magnetic field formed by a superparamagnetic material, T if the main magnetic fields are uniform and coincident 2 Depending on the local magnetic field. When marine pathogens exist, the superparamagnetic two-dimensional material is caused to be agglomerated into clusters, the more the marine pathogens are, the larger the clusters are, the more the local magnetic field is uneven, and T 2 The shorter the length, the more quantitative the result.
Example 2
The difference from the above example 1 is that: (1) functionalized superparamagnetic two-dimensional material GO @ SPIONs&Ab synthesis: centrifuging 15mL of 0.5mg/mL graphene oxide dispersion liquid at 12000 rpm for 5 min, discarding the supernatant, dispersing again in 15mL absolute ethyl alcohol for 6 min by ultrasonic treatment, then adding 15 muL of 0.0426M 3-aminopropyltriethoxysilane solution, magnetically stirring for 5 h at 65 ℃, repeatedly washing with ethanol and water, and dispersing in 20mL of water to obtain aminated monolayer graphene oxide (NH) 2 -GO) dispersion; 1 mL of a 25 wt% glutaraldehyde solution was added to 8mL of NH 2 Dispersing the GO in 8mL of water after magnetically stirring for 2h at room temperature, centrifuging at 5000 rpm and cleaning with ethanol, adding 150 muL 0.5mg/mL superparamagnetic nano iron oxide particles (SPIONs) and 150 muL 100 mug/mL vibrio parahaemolyticus polyclonal antibody (Ab) solution into the dispersion, and oscillating for 4 h to form functionalized GO @ SPIONs&Ab, further added with 150 μ L of 2 wt% bovine serum albumin solution (BSA) to block non-specific binding sites, and after washing with PBS solution of pH =7.4, 0.01M, dispersed in 2mL of PBS solution, 1737×gCentrifuging for 3min to remove free SPIONs, and dispersing the obtained product in 10 mL PBS solution to obtain the functionalized superparamagnetic two-dimensional material GO @ SPIONs&Storing the Ab dispersion liquid in a refrigerator at 4 ℃;
(2) low-field nuclear magnetic resonance homogeneous immunoassay of vibrio parahaemolyticus: and adding 0.1 mL of the @ SPIONs & Ab dispersion liquid and 1.0 mL of samples to be detected containing different concentrations of food-borne pathogenic bacteria into a sample bottle for mixing.
Example 3
The difference from the above example 1 is that: (1) functionalized superparamagnetic two-dimensional material GO @ SPIONs&Ab synthesis: centrifuging 25mL of 1.5 mg/mL Graphene Oxide (GO) dispersion liquid at 12000 rpm for 5 min, discarding the supernatant, redispersing the dispersion liquid in 25mL of absolute ethyl alcohol for 6 min by ultrasonic treatment, then adding 25 muL of 0.0426M 3-Aminopropyltriethoxysilane (APTES) solution, magnetically stirring the solution at 75 ℃ for 5 h, repeatedly washing the solution with ethanol and water, and dispersing the solution in 20mL of water to obtain aminated single-layer graphene oxide (NH) 2 -GO) dispersion; 3 mL of a 25 wt% glutaraldehyde solution was added to 12mL of NH 2 Dispersing the GO in 12mL of water after magnetically stirring for 5 h at room temperature, centrifuging at 5000 rpm and cleaning with ethanol, adding 250 muL 0.5mg/mL superparamagnetic nano iron oxide particles (SPIONs) and 250 muL 100 mug/mL vibrio parahaemolyticus polyclonal antibody (Ab) solution into the dispersion, and oscillating for 4 h to form functionalized GO @ SPIONs&Ab, 250 μ L of 2 wt% bovine serum albumin solution (BSA) was added to block non-specific binding sites and dissolved in PBS at pH =7.4, 0.01MAfter washing, the solution was dispersed in 2mL of PBS solution 1737×gCentrifuging for 3min to remove free SPIONs, and dispersing the obtained product in 10 mL PBS solution to obtain the functionalized superparamagnetic two-dimensional material GO @ SPIONs&Storing the Ab dispersion liquid in a refrigerator at 4 ℃;
(2) low-field nuclear magnetic resonance homogeneous immunoassay of vibrio parahaemolyticus: adding 0.1 mLGO @ SPIONs & Ab dispersion liquid and 1.4 mL of samples to be tested containing food-borne pathogenic bacteria with different concentrations into a sample bottle for mixing.
In addition to the above embodiments, Vibrio parahaemolyticus can 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.
Detailed description of the preferred embodiment
Sensitivity detection
FIG. 4 is a linear relationship diagram of low-field nuclear magnetic resonance detection at different VP concentrations; with 30min as the optimal incubation time, the sensitivity of MRSw for detecting the VP of the vibrio parahaemolyticus is considered, and the quantitative detection is 1.0 multiplied by 10 1 CFU/mL~1.0×10 6 CFU/mL of different concentrations of pathogen. The results are shown in FIG. 4A, Δ T between different samples 2 Increases with increasing VP concentration. As shown in FIG. 4B, the concentration was 1.0X 10 2 CFU/mL~1.0×10 5 Delta T within the CFU/mL concentration interval 2 (y) shows a good linear relationship with the logarithm of the VP concentration (x), and the linear regression equation is y =48.533x-64.495, R 2 Is 0.993. According to FIG. 4B, the limit of quantitation of MRSw in detecting VP is 1.0X 10 2 CFU/mL, thus demonstrating sensitivity for VP detection with MRSw.
Detailed description of the preferred embodiment
Specificity detection
Specific experiment of MRSw sensor, in order to simulate the complicated bacterial environment of seawater, under the optimal experimental condition, the detection concentration is 1.0 multiplied by 10 6 CFU/mL Vibrio vulnificus (II) ((III))Vibrio Vulnificus, VV) Vibrio harveyi: (Vibrio harveyi, VH) Listeria (Listeria monocytogenes)Listeria monocytogenes, LM) Salmonella bacteria (I), (II)Salmonella, SM) Staphylococcus aureus (1)Staphylococcus aureus,SA) Escherichia coli (E.coli)Escherichia Coli, E.coli) Vibrio parahaemolyticus: (Vibrio Parahemolyticus, VP) And contains 1.0X 10 6 CFU/mL concentration of VP mixed samples, blank samples added PBS to verify the MRSw sensor on target detection of VP specificity. The results are shown in FIG. 5, T for VP-containing samples 2 There was a significant change, while the test samples containing other pathogens responded similarly to the signal from the placebo. The above results indicate that the MRSw sensor has a high specificity for VP, and in addition, the Δ T in samples containing VP mixtures 2 Similar to the signal of the VP sample alone, this means that other pathogens hardly affect the specific recognition of VP by this sensor, reflecting the excellent specificity of the MRSw sensor for VP detection.
Application examples
In order to verify the value of the method in practical application, the standard solution of the vibrio parahaemolyticus is added into the eastern sea water 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 labeling recovery method, the result is shown in table 1,
TABLE 1
As can be seen from 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 in detection result of vibrio parahaemolyticus in seawater.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.
Claims (4)
1. A method for detecting food-borne pathogenic bacteria based on low-field nuclear magnetic resonance homogeneous immunoassay of a superparamagnetic two-dimensional material is not used for 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 0.5-1.5 mg/mL graphene oxide dispersion liquid at 12000 rpm for 5 min, removing a supernatant, dispersing again in 15-25mL of absolute ethanol, performing ultrasonic treatment for 6 min, adding a solution of 0.0426M 3-aminopropyltriethoxysilane with a concentration of 15-25 mu L, magnetically stirring for 3-5 h at 65-75 ℃, repeatedly washing with ethanol and water, and dispersing in 20mL of water to obtain an aminated single-layer graphene oxide dispersion liquid;
B. 1-3 mL of a 25 wt% glutaraldehyde solution was added to 8-12mL of NH 2 Magnetic stirring is carried out on the GO dispersion liquid for 2-5 h at room temperature, centrifugation is carried out at 5000 rpm, the GO dispersion liquid is cleaned by ethanol, the GO dispersion liquid is dispersed in 8-12mL of water, 150 plus glass L of 0.5mg/mL superparamagnetic nano iron oxide particles and 150 plus glass L of 100 plus glass G/mL food-borne pathogenic bacterium polyclonal antibody solution are added into the dispersion liquid, and the functionalized GO @ SPIONs are obtained after oscillation for 4 h&Ab, after adding 150-250 μ L of 2 wt% bovine serum albumin solution to block non-specific binding sites and washing with PBS solution of pH =7.4, 0.01M to remove unbound BSA and Ab, was dispersed in 2mL of PBS solution 1737×gCentrifuging for 3min to remove free SPIONs, and dispersing the obtained product in 10 mL PBS solution to obtain functionalized superparamagnetic two-dimensional material GO @ SPIONs&Ab dispersion liquid;
(2) low-field nuclear magnetic resonance homogeneous immunoassay for food-borne pathogenic bacteria
Take 0.1 mLGO @ SPIONs&Adding Ab dispersion liquid and 1.0-1.4 mL of samples to be tested containing food-borne pathogenic bacteria with different concentrations into a sample bottle, mixing, incubating for 30min with shaking, placing in a low-field nuclear magnetic resonance contrast agent relaxation analyzer, and collecting T at 35 deg.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 2 And establishing a quantitative relation between the transverse relaxation time difference of the water protons and the concentration of the food-borne pathogenic bacteria, and determining the concentration of the food-borne pathogenic bacteria in the unknown sample according to the quantitative relation.
2. The method for the homogeneous immunoassay of food-borne pathogenic bacteria based on the low-field nuclear magnetic resonance of superparamagnetic two-dimensional material as set forth in claim 1, which is not intended for diagnosis or treatment, and is characterized in that the parameters measured in the step (2) are as follows: the main frequency is 19.00 MHz, the echo number is 18000, the echo time is 0.6 ms, the accumulation times are 2 times, the waiting time is 5000 ms, the digital gain is 3, and the analog gain is 15.0 dB.
3. The method for low-field NMR homogeneous immunoassay of food-borne pathogenic bacteria based on superparamagnetic two-dimensional material as claimed in claim 1, which is not for diagnosis or treatment purpose, wherein said Δ T in step (2) 2 Is calculated by the following formula: delta T 2 = T 2N – T 2P Wherein T is 2 Transverse relaxation time, T, of water protons 2N Mean T in the absence of food-borne pathogenic bacteria 2 ，T 2P Mean T in the presence of food-borne pathogenic bacteria 2 。
4. The method for the homogeneous immunoassay of food-borne pathogenic bacteria based on the low-field nuclear magnetic resonance of superparamagnetic two-dimensional material as claimed in claim 1, which is not intended for diagnosis or treatment, characterized in that: the food-borne pathogenic bacteria comprise vibrio parahaemolyticus, vibrio vulnificus, staphylococcus aureus, escherichia coli and salmonella.
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