CN111007252B - Method for detecting pesticide residue by magnetic relaxation time sensor based on quantity and state change of nano magnetic particles - Google Patents

Method for detecting pesticide residue by magnetic relaxation time sensor based on quantity and state change of nano magnetic particles Download PDF

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CN111007252B
CN111007252B CN201911389247.6A CN201911389247A CN111007252B CN 111007252 B CN111007252 B CN 111007252B CN 201911389247 A CN201911389247 A CN 201911389247A CN 111007252 B CN111007252 B CN 111007252B
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陈翊平
董永贞
曾令文
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Huazhong Agricultural University
Wuhan Academy of Agricultural Sciences
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Abstract

The invention discloses a biological sensing method for changing the quantity and the state of nano magnetic particles based on a biological orthogonal reaction so as to detect pesticide residues.

Description

Method for detecting pesticide residue by magnetic relaxation time sensor based on quantity and state change of nano magnetic particles
Technical Field
The invention belongs to the technical field of food safety detection, and particularly relates to a method for detecting pesticide residues by a magnetic relaxation time sensor based on the quantity and state change of nano magnetic particles.
Background
Food safety is an important civil engineering which is related to the health of the masses, and pesticide residue in food is an important food safety problem which is harmful to human health. The pesticide residue exceeds the standard due to illegal use of high-toxicity pesticides, enhanced drug resistance of diseases and pests, improper use of pesticides and the like, and the pesticide residue has great harm to human health, can cause acute poisoning or chronic poisoning, reduces human immunity, can cause cancer, teratogenesis and mutation, and even causes death of individuals. In view of the great harm to health caused by pesticide residue, reducing and reasonably using pesticide is a fundamental measure for solving the problem, and the rapid and accurate detection of pesticide residue in food is a precondition for ensuring the reasonable use and a last line of defense for ensuring the safety of the tongue tips of people.
At present, the main means for qualitative and quantitative analysis of pesticide residue in food are instrumental analysis, enzyme inhibition and immunoassay. The instrument analysis method has the advantages of high sensitivity, good accuracy and the like, but the pretreatment of the sample is complicated, the detection cost is high, a high-level professional technician is required, and the instrument analysis method is not suitable for on-site rapid detection. The enzyme inhibition method mainly aims at organophosphorus pesticides, has the advantages of simple operation, low cost and the like, but is easily interfered by a sample matrix, has more false positives and is not accurate enough. The traditional immunoassay mainly comprises enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography test strip methods. ELISA has the advantages of relatively simple operation, high flux and the like, but the concentration of some pesticide residues can be very low, and the traditional ELISA method cannot meet the detection requirement. The colloidal gold immunochromatographic test strip method has the advantages of simple operation, high detection speed and the like, but has low sensitivity, is easily interfered by matrix and the like. Thus, conventional immunoassays cannot meet the actual requirements for detection sensitivity. Screening for antibodies with higher affinity binding can improve sensitivity, but is often more expensive. Therefore, it is highly desirable to improve the sensitivity of immunoassay methods without compromising the specificity of the methods.
Various nanomaterials have been used to improve the analytical performance of traditional immunoassay methods due to their unique physicochemical properties. Nanoparticles have large specific surface area and diverse surface chemistry, and are widely used for enrichment and separation in immunoassays to simplify pretreatment. The optical, electrochemical and magnetic characteristics of the nanoparticles also greatly enhance signal readout and amplification in conventional immunoassays. Among the various nanoparticles, magnetic Nanoparticles (MNPs) have attracted great interest because MNPs can be easily separated not only in a magnetic field, used as a carrier for sample pretreatment, but also as magnetic signal sensing probes because MNPs can cause an inhomogeneous magnetic field that significantly shortens the transverse relaxation time (T) of the protons of the surrounding water molecules 2 ). In addition, because the magnetic signal in the sample matrix is negligible, the magnetic relaxation time immunosensor based on the MNPs has high signal-to-noise ratio and is widely applied to the fields of food safety, clinical diagnosis and the like. The sensing mechanism of the conventional magnetic immunosensor is mainly divided into two aspects: (1) change in the state of MNPs: after MNPs are coupled with antibodies, originally dispersed MNPs can be changed into an aggregated state due to the immune recognition effect between the antibodies and antigens, and then an uneven magnetic field can be caused, so that T is caused 2 The signal is changed, and the signal of the traditional magnetic immunosensorMost depend on changes in the state of MNPs, are not sensitive enough to detect trace amounts of target, and are susceptible to interference from non-specific aggregation. (2) variation in the number of MNPs: there is research to show that the magnetic signal T 2 The quantity change of the nanometer magnetic particles MNPs is more sensitive, so that researchers can realize one-to-one correspondence of the quantity change of the target object and the magnetic particles by combining immunomagnetic separation, and further construct a magnetic relaxation time immunosensor based on the quantity change of the nanometer magnetic particles, and the immunosensor is used for detecting biomacromolecules, such as food-borne pathogenic bacteria, viruses and the like. However, small molecule chemicals like pesticides only have one antibody binding site and cannot effectively cause the change of the number of magnetic particles, so that other amplification means are needed to apply the magnetic relaxation time immunosensor to the highly sensitive detection of trace pesticide residue small molecules.
The Biorthogonal Reactions (BRs) have the advantages of high reaction speed, good specificity and the like, and the quantity of MNPs combined on a target object can be greatly enhanced through the biorthogonal reactions assembled layer by layer, so that T is amplified 2 Signal and enhancement of conventional magnetic transverse relaxation time immunosensor (T) 2 MRS). Thus, the bio-orthogonal reaction of layer-by-layer assembly is to overcome T 2 The sensitivity of MRS to detect trace small molecule targets provides an attractive tool. More importantly, on one hand, the coupling amount of the nano magnetic particles can be increased through the bioorthogonal reaction, so that the quantity of the nano magnetic particles is changed; meanwhile, we find that the nano-magnetic particles originally in a dispersed state can also be changed into an aggregated state through a bio-orthogonal reaction, namely, the state of the nano-magnetic particles is changed, so that T is further amplified 2 A signal. Therefore, based on the cascade bioorthogonal reaction, the state change of MNPs is organically combined on the basis of the change of the quantity of MNPs, the cascade amplification of magnetic signals is realized, and an ultrasensitive magnetic relaxation time immunosensor is further constructed and used for the rapid detection of pesticide micromolecules.
Disclosure of Invention
The invention aims to provide a biosensor method for detecting pesticide residues based on bioorthogonal reaction, which can simultaneously change the aggregation state and the number of nano magnetic particles, and the nano magnetic particles are organically combined to carry out magnetic signal cascade amplification, thereby improving the accuracy and the sensitivity of pesticide residue detection.
A method for detecting pesticide residues by a magnetic relaxation time sensor based on the quantity and state changes of nano magnetic particles comprises the following steps:
1) Coupling a pesticide to be detected with carrier protein, and then reacting with carboxyl magnetic beads to obtain magnetic beads coupled with complete antigens of the pesticide to be detected;
2) The monoclonal antibody for identifying the pesticide to be detected and diphenyl cyclooctyne-tetraethylene glycol-active ester (DBCO-PEG) 4 -NHS ester) reaction to obtain diphenyl cyclooctyne (DBCO) modified monoclonal antibody;
3) Adding magnetic beads coupled with complete pesticide antigen to be detected and a DBCO modified monoclonal antibody into a sample solution to be detected, carrying out an immune competition reaction, and carrying out magnetic separation to obtain a magnetic bead-complete antigen-antibody-DBCO immune compound;
4) 15-azido-4,7,10,13-tetraoxypentadecanoic acid-N-succinimidyl ester (Azide-PEG) 4 -NHS ester) reacts with the amino nano-magnetic particles to obtain Azide (Azide) -labeled nano-magnetic particles (Azide-MNPs), the product is subjected to magnetic separation and then is resuspended by PBS buffer solution, and then is added into the immune complex obtained in the step 3), so that the Azide and DBCO are subjected to bio-orthogonal reaction, the nano-magnetic particles are coupled to magnetic beads, the supernatant is collected after the magnetic separation, the supernatant contains unreacted Azide-MNPs, and the amount of the unreacted Azide-MNPs is in positive correlation with the content of the substance to be detected;
5) Adding an azide cross-linking agent into the supernatant obtained after the magnetic separation in the step 4), triggering a bioorthogonal reaction, and forming a nano magnetic particle-azide cross-linking agent-azide-nano magnetic particle compound, so that the nano magnetic particles in the original dispersion state are changed into an aggregation state, the transverse relaxation time signal of the magnetic immunosensor is amplified in a cascade manner, and the transverse relaxation time is measured to quantitatively analyze the pesticide residue.
Preferably, the particle size of the carboxyl magnetic beads is 500-3000nm.
Preferably, the particle size of the amino nano-magnetic particle is 10-100nm.
Preferably, the azide crosslinker is diphenylcyclooctyne-tetraethylene glycol-diphenylcyclooctyne (DBCO-PEG) 4 -DBCO)。
Preferably, the weight ratio of the 15-azido-4,7,10,13-tetraoxypentadecanoic acid-N-succinimidyl ester to the amino nano-magnetic particles is 1:5.
preferably, the molar ratio of the reaction of the monoclonal antibody and the diphenyl cyclooctyne-tetraethylene glycol-active ester is 1:10.
preferably, the reaction time of bioorthogonal reaction of the nano-magnetic particles labeled with Azide (Azide) and DBCO-PEG4-DBCO is 20min.
Preferably, the pesticide is a small molecule compound pesticide, such as chlorpyrifos.
Preferably, the carrier protein of the pesticide is bovine serum albumin.
In the invention, the controllable regulation of the quantity and the state of Magnetic Nanoparticles (MNPs) is realized based on cascade Bioorthogonal Reactions (BRs), and an ultrasensitive magnetic relaxation time immunosensor is constructed on the basis and is used for detecting pesticide residues. By utilizing a rapid and highly selective bio-orthogonal reaction and combining a competitive immune reaction, the amplification of a magnetic relaxation signal can be realized through the change of the quantity of MNPs; on the basis, different amounts of MNPs are further subjected to bio-orthogonal reaction, and the state of the MNPs is changed (from dispersion to aggregation), so that the multi-stage amplification of magnetic relaxation signals is realized, and the high-sensitivity detection of pesticide residues in the sample is finally realized. The signal amplification caused by the change of the quantity of MNPs and the signal amplification caused by the change of the states of MNPs are organically combined to realize cascade signal amplification, and the sensor is a core technology capable of realizing ultra-sensitive detection. As shown in FIG. 1, a single small molecule target can not only increase MNP by two-step bioorthogonal reaction 30 And can assemble MNP 30 The state of the particles is changed from dispersion to aggregation. When the content of pesticide molecules (in the case of chlorpyrifos) is high, relatively less diphenyl cyclooctyne (DBCO) is modified due to competition effectThe antibody (Ab-DBCO) is capable of binding to MNP 1000 -MNP formed by BSA-chlorpyrifos binding 1000 The amount of BSA-Chlorpyrifos-Ab-DBCO "complexes is lower. When joining MNP 30 After Azide, it is able to bio-orthogonally react with DBCO (multiple DBCO sites on Ab, binding multiple MNP) 30 Azide, effecting amplification of the signal), due to "MNP 1000 The complex BSA-Chlorpyrifos-Ab-DBCO is less and therefore coupled to the "MNP 1000 MNP on the BSA-Chlorpyrifos-Ab-DBCO "complex 30 The less. Cause MNP 1000 With MNP 30 Magnetic separation efficiency is significantly different, 1000nm MNP 1000 The MNP of 30nm is easy to separate magnetically due to large grain size and large magnetic saturation intensity 30 Because the particle size is small, the magnetic saturation intensity is small, and the magnetic separation is not easy. Thus, MNP 1000 -MNP 30 The complexes can be readily complexed with MNP 30 Separating with magnetic separation rack. MNP remaining in the supernatant after magnetic separation 30 The more azides (the change in the number of magnetic particles causes a change in the magnetic signal). On the contrary, if the content of pesticide molecules (chlorpyrifos as an example) in the sample is less, the MNP remained in the supernatant fluid 30 Less Azide. To further amplify the magnetic signal of the sensor, we added DBCO-PEG to the above supernatant 4 DBCO, further triggering a bioorthogonal reaction between DBCO and Azide, MNP in dispersed state 30 Azide changes to the aggregate state (the magnetic particles change from dispersed to aggregate state, the magnetic signal of which increases), further amplifying the signal.
The transverse relaxation time immunosensor prepared by the invention based on the state and the quantity change has the advantages that:
1. the signal amplification caused by the change of the quantity of MNPs and the signal amplification caused by the change of the state of MNPs are organically combined through the bioorthogonal reaction for the first time, the cascade amplification of magnetic signals is realized, the sensitivity and the detection range of the magnetic biosensor are effectively improved, and the method belongs to the innovation from the aspect of methodology.
2. The bio-orthogonal reaction of Azide (Azide) and DBCO has the characteristics of rapidness and high selectivity, and is an important means for realizing signal amplification without introducing cross reaction, and the advantage is particularly obvious in complex samples.
3. The detection method of the magnetic immunosensor has the advantages of simple pretreatment, simple and convenient operation, low detection cost and high detection speed, avoids using large instruments such as GC-MS and the like, and effectively improves the detection efficiency.
Drawings
FIG. 1 is the operation principle of the magnetic relaxation time immunosensor for detecting pesticide residues according to the present invention.
FIG. 2 shows Azide-PEG 4 -NHS ester with MNP 30 The effect of the mass ratio on the sensor signal.
FIG. 3 is the change in Molecular Weight (MWs) of a monoclonal antibody before and after conjugation to DBCO, where A is before conjugation; and B is after coupling.
FIG. 4 is the effect of Ab/DBCO ratio on sensor signal intensity.
FIG. 5 shows Azide-MNP during the phase of change of state of magnetic particles 30 With DBCO-PEG 4 The effect of the reaction time of DBCO on the signal intensity.
FIG. 6 is an Azide-MNP 30 And comparing the magnetic relaxation performance before and after the magnetic particle state change stage with the change of the microscopic state. In the figure, A is T before and after the change of the state of the nano-magnetic particles with different concentrations 2 Comparing the change of the value; b is comparison of the change of the relaxation rate before and after the change of the state of the nano-magnetic particles with different concentrations; c is a transmission electron microscope image before the state of the nano magnetic particles is changed; d is a transmission electron microscope image after the state of the nano magnetic particles is changed.
Fig. 7 shows the linear range of the MRS sensor of the present invention to the conventional MRS sensor and the MRS sensor based on the change in the number of magnetic particles to detect chlorpyrifos. In the figure, a is the detection principle of the conventional MRS; d is MRS detection principle based on the change of the number of the magnetic particles; g is the detection principle of the MRS sensor; B. e, H is the sample concentration (ng/mL) -T of three detection modes respectively 2 A linear relationship of values; C. f, I is the logarithm of the concentration of the sample in three detection modes-T 2 The values are linear.
FIG. 8 shows the specific result of the MRS sensor of the present invention for detecting chlorpyrifos.
FIG. 9 is a comparison of the MRS sensor of the present invention detecting chlorpyrifos in apple and cabbage.
FIG. 10 is a comparison of the results of MRS sensor of the present invention detecting imidacloprid in apple and cabbage.
Detailed Description
The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as limiting the present invention.
The reagent instrument equipment sources used:
1. carboxyl magnetic beads (MNP for short) with particle size of 1000nm 1000 ): invitrogen (USA)
2. Amino nano magnetic particle (MNP for short) with particle size of 30nm 30 ): ocean NanoTech corporation (USA)
3.15-azido-4,7,10,13-tetraoxypentadecanoic acid-N-succinimidyl ester (Azide-PEG) 4 -NHS ester), diphenylcyclooctyne-tetrapolyethylene glycol-active ester (DBCO-PEG) 4 -NHS ester), diphenylcyclooctyne-tetrapolyethylene glycol-diphenylcyclooctyne (DBCO-PEG) 4 -DBCO): click Chemistry Tools corporation (USA)
4. chlorpyrifos-BSA, chlorpyrifos monoclonal antibody, chlorpyrifos ELISA kit, imidacloprid-BSA, imidacloprid monoclonal antibody and imidacloprid ELISA kit: shandong Lvdu Biotech Ltd
5. Bovine serum albumin, chlorpyrifos, imidacloprid, dimethoate, triazophos, glyphosate, acephate: shanghai Sigma-Aldrich Co
6. Magnetic separation frame: shanghai Mo Run nanotechnology
7.0.47T nuclear magnetic resonance apparatus (PQ 001): shanghai Newmai science and technology
8. Random apple and cabbage samples: purchased from local supermarket
Example 1 construction of the present magnetic relaxation immunosensor
(1)Azide-MNP 30 Preparation of conjugates
mu.L of 15-azido-4,7,10,13-tetraoxypentadecanoic acid-N-succinimidyl ester (Azide-PEG) 4 -NHS ester solution (1)mg/mL) was added to 200. Mu.L of NH 2 -MNP 30 The solution (2.5 mg/mL) was slowly stirred at room temperature for 1 hour. After the reaction was completed, magnetic separation was carried out at 4 ℃ for 12 hours, and the supernatant was removed to obtain Azide-MNP 30 Conjugate, then 500 μ L PBST (pH =7.4,0.01M with 0.05% tween 20) was used to resuspend the conjugate, and this procedure was repeated three times. Finally, azide-MNP 30 The conjugate was resuspended in 100 μ L PBS solution (pH =7.4,0.01M) and stored at 4 ℃ until use.
In the embodiment, the amount of Azide used in the coupling process is optimized, and the specific method is as follows: taking Azide-PEG with different masses 4 -NHS with 1mg MNP 30 Coupling to obtain Azide-MNP 30 With DBCO-Ab-chlorpyrifos-BSA-MNP 1000 Performing bioorthogonal reaction, and measuring T of supernatant 2 Value, T 2 The larger the value, the evidence of Azide-MNP remaining in the supernatant 30 The less, the coupling to DBCO-Ab-Chlorpyrifos-BSA-MNP 1000 Azide-MNP of surfaces 30 The more. As shown in FIG. 2, when Azide-PEG 4 0.2mg of-NHS, i.e. Azide-PEG 4 -NHS with MNP 30 The mass ratio is 1: at 5, T 2 The value is the largest, which proves that the coupling rate is the highest. It is therefore selected as the final amount.
(2) Preparation of DBCO-Ab conjugates
10 μ L of diphenylcyclooctyne-tetraethyleneglycol-active ester (DBCO-PEG) 4 -NHS ester) (2 mg/mL) was dissolved in DMF and mixed with 100 μ L Ab (3 mg/mL) in 10mM PBS (pH = 7.4) and reacted at room temperature for 1 hour. Then, 50mM Tris-HCl (pH = 8.0) buffer was added to the mixed solution to terminate the reaction for 10 minutes. After completion of the reaction, excess DBCO-PEG was removed by centrifugation at 9000rpm for 20 minutes at 4 ℃ using a clean centrifugal ultrafiltration device (10 kDa filter) 4 -NHS molecule. After 3 centrifugal washes, DBCO-Ab was resuspended in PBS (pH 7.4, 10 mM) and stored at-20 ℃.
In the embodiment, matrix-assisted laser desorption ionization time-of-flight mass spectrometry is used to compare the change of the Molecular Weight (MWs) of the monoclonal antibody (Ab) before and after DBCO coupling, as shown in FIG. 3, the molecular weight of Ab and DBCO-Ab is changed from 146985 to 149135, which proves that DBCO-Ab coupling is successful. And calculating the coupling rate of the DBCO-Ab conjugate through the following formula. It was calculated that 4 DBCO-PEG4-NHS molecules could be coupled per antibody molecule.
Coupling ratio = (MWs) (DBCO-Ab) -MWs (Ab) )/MWs (DBCO-PEG4-NHS)
In this example, the ratio of DBCO to Ab in the coupling process was optimized, and the specific method was as follows: ab/DBCO with different proportions is taken for coupling, and the successfully coupled DBCO-Ab and MNP are coupled 1000 -BSA-Chlorpyrifos coupling and further one-time bioorthogonal reaction with Azide-MNP 30 Coupling, after magnetic separation, different ratios of Ab/DBCO can cause Azide-MNP in the supernatant 30 By measuring T 2 Quantitative comparison of values, see fig. 4, when Ab/DBCO molar ratio is 1:10 th, T 2 The value is the largest and the signal amplification is the best, so it is chosen as the final ratio.
(3)MNP 1000 Preparation of-BSA-Chlorpyrifos conjugates
First, 200. Mu.L of carboxylated MNP was activated by the EDC/NHS method 1000 (5 mg/mL), to which 0.2mg BSA-chlorpyrifos conjugate was then added, along with activated MNP 1000 Mix and shake the reaction at room temperature for 2h. After completion of the reaction, 100. Mu.L of 3% BSA solution was added to the solution mixture, and shaken at room temperature for 0.5h. After magnetic separation and washing 3 times with PBST, MNP was separated 1000 The BSA-chlorpyrifos conjugate was resuspended in 200. Mu.L PBS solution and stored at 4 ℃ until use.
(4) Construction of the magnetic relaxation immunosensor
Mixing 100 μ L of chlorpyrifos solution with 100 μ L of MNP 1000 -BSA-Ag solution, 100. Mu.L DBCO-Ab conjugate, and slowly vortexing at room temperature for 30-60min. After completion of the reaction, MNP was obtained after magnetic separation and washing 3 times with PBST 1000 -BSA-Ag-Ab-DBCO conjugate. In the obtained MNP 1000 Adding 100 μ L Azide-MNP to the BSA-Ag-Ab-DBCO conjugate 30 Reacting the solution at room temperature, and obtaining unreacted Azide-MNP in supernatant fluid after magnetic separation 30 . To the resulting supernatant was added 100. Mu.L of DBCO-PEG 4 -DBCO solution and in the chamberStanding and reacting for 15min at the temperature. Finally, the mixture was collected and its T was measured 2 The value is obtained.
In the embodiment, azide-MNP in the magnetic particle state change stage is optimized 30 With DBCO-PEG 4 -reaction time of DBCO, the specific method is as follows: uniformly mixing Azide-MNP 30 With DBCO-PEG 4 DBCO and reacting for various times, determining the T of the reaction solution 2 Values, see FIG. 5, T when the time is 20min 2 The smallest value demonstrates the most obvious change in state. It is therefore chosen as the final reaction time.
In this example, azide-MNP was studied 30 The changes of the magnetic signal and the relaxation efficiency before and after the phase of the change of the state of the magnetic particles were characterized by TEM. As shown in FIG. 6, after bioorthogonal reaction, the system T 2 The value is obviously reduced, the relaxation efficiency is obviously improved, and the change of the state of the magnetic particles is proved.
Example 2 comparison of sensitivity of the present magnetic relaxation immunosensor to conventional magnetic relaxation time Sensors
According to example 1, chlorpyrifos in gradient concentration was detected with conventional MRS (fig. 7A), MRS based on changes in the number of magnetic particles (fig. 7D), and MRS based on changes in the number and state of magnetic particles (fig. 7G), respectively. The specific method comprises the following steps: (1) legacy MRS: coupling of antibodies (MNP) to magnetic particles 30 Ab) and coupling of a complete antigen (MNP) to another magnetic particle 30 BSA-Chlorpyrifos), 100. Mu.L of Chlorpyrifos solutions at various concentrations were mixed with 100. Mu.L of MNP 30 -BSA-Chlorpyrifos solution, 100. Mu.L MNP 30 Ab conjugate mix, and a competitive immune reaction occurs, causing aggregation of magnetic particles (MNP) 30 Ab-Chlorpyrifos-BSA-MNP 30 State change) to cause T 2 A change in value. (2) MRS based on changes in the number of magnetic particles: preparation of conjugates As in example 1, 100. Mu.L of chlorpyrifos solutions at various concentrations were mixed with 100. Mu.L of MNP 1000 -BSA-chlorpyrifos solution, 100. Mu.L DBCO-Ab conjugate, mixed and vortexed slowly at room temperature for 30-60min. After the reaction is completed, the MNP obtained 1000 Adding 100 mu LAzide-MNP into the BSA-Ag-Ab-DBCO conjugate 30 Reacting the solution at room temperature, and magnetically separating to obtain the productUnreacted Azide-MNP in the clear liquid 30 . Magnetic separation and T-treatment of the supernatant 2 Assay (MNP) 30 The number changes). (3) MRS based on magnetic particle number and state change: the construction method was the same as in example 1.
Sample concentration (ng/mL) is plotted as abscissa and T 2 The values are plotted on the ordinate as shown in FIG. 7B, E, H. The logarithm of the sample concentration (ng/mL) is plotted as the abscissa and T 2 The values are plotted on the ordinate as shown in FIG. 7C, F, I. LOD was calculated from a 3S/M calibration curve, where S is the standard for the blank sample and M is the slope of the standard curve in the low concentration range. The results indicate that (1) legacy MRS: LOD =4.1ng/mL (S =8.8, m = 6.4), linear range (5-500 ng/mL); (2) MRS based on changes in the number of magnetic particles: LOD =0.36ng/mL (S =14.1, m = 117.9), linear range (1-500 ng/mL); (3) LOD =0.05ng/mL (S =18.2, m = 1096), linear range (0.1-1000 ng/mL) based on the number of magnetic particles and the change of state MRS. Therefore, MRS based on the number and state change of magnetic particles has the characteristics of higher sensitivity, better linear range, high sensitivity and wide detection range.
Example 3 study on specificity and recovery of the magnetic relaxation immunosensor
(1) In a specificity test, four chlorpyrifos analogs of dimethoate, triazophos, glyphosate and acephate are respectively added to determine the specificity of the sensor for detecting the chlorpyrifos so as to verify the specificity of the sensor. Wherein, the concentration of the chlorpyrifos is 10ng/mL, and the concentration of the analogues is 100ng/mL. As shown in FIG. 8, only chlorpyrifos was able to cause T 2 Significant changes in value, other analogs have negligible effect on the magnetic signal.
(2) Recovery was studied using a standard addition method, where varying concentrations of chlorpyrifos (0.5, 1, 5, 10, 50 and 100 ng/mL) were added to blank apple samples and then measured using the present sensor. As shown in table 1, the recovery rate (76% -119%) of chlorpyrifos detection also indicates the accuracy of the method.
TABLE 1 recovery rate of chlorpyrifos in apple sample detected by the sensor
Chlorpyrifos spiking concentration (ng mL) -1 ) Concentration detected (ng mL) -1 ) Recovery (%)
0.5 0.38 76
1 1.19 119
5 4.55 91
10 9.06 90.6
50 50.14 100.28
100 96.5 96.5
Embodiment 4 the magnetic relaxation immunosensor is used for quantitative detection of chlorpyrifos residues in fruits and vegetables
50.00g apple and cabbage samples were sliced and homogenized. A certain amount of the slurry was separately weighed and sonicated with a methanol/water solution (3/17, V/V). Analysis was performed by the MRS sensor in this example 1 and methodological alignment was performed by Gas Chromatography (GC) and ELISA. As shown in fig. 9, the detection result of the sensor has good agreement with the GC method, which proves that the sensor has good accuracy.
Embodiment 5 the magnetic relaxation immunosensor is used for quantitative detection of imidacloprid residues in fruits and vegetables
The method also detects an actual sample of imidacloprid which is a common pesticide in food, and compares the actual sample with Gas Chromatography (GC) and ELISA to further verify the accuracy and applicability of the sensor. The fruit and vegetable processing method is the same as that of the embodiment 4, and the steps of detection by the sensor are similar to those of the embodiment 1, except that the target substance is defined as imidacloprid. As shown in fig. 10, the detection result of the sensor is well matched with the GC method, which proves that the sensor has good accuracy and applicability.

Claims (9)

1. A method for detecting pesticide residues by a magnetic relaxation time sensor based on the quantity and state changes of nano magnetic particles is characterized by comprising the following steps:
1) Coupling a pesticide to be detected with carrier protein, and then reacting with carboxyl magnetic beads to obtain magnetic beads coupled with complete antigens of the pesticide to be detected;
2) Reacting the monoclonal antibody for identifying the pesticide to be detected with diphenyl cyclooctyne-tetraethylene glycol-active ester to obtain a diphenyl cyclooctyne modified monoclonal antibody;
3) Adding magnetic beads coupled with complete antigens of pesticides to be detected and monoclonal antibodies modified by diphenyl cyclooctyne into a sample solution to be detected, carrying out immune competition reaction, and carrying out magnetic separation to obtain immune complexes of the magnetic beads, the complete antigens, the antibodies and the diphenyl cyclooctyne;
4) Reacting 15-azido-4,7,10,13-tetraoxypentadecanoic acid-N-succinimidyl ester with amino nano-magnetic particles to obtain azido-labeled nano-magnetic particles, carrying out magnetic separation on the product, then carrying out resuspension by using a PBS (phosphate buffer solution), then adding the product into the magnetic bead-complete antigen-antibody-diphenylcyclooctyne immune complex obtained in the step 3), carrying out bio-orthogonal reaction on azido and diphenylcyclooctyne, so that the azido-labeled nano-magnetic particles are coupled to the magnetic beads, collecting supernatant after magnetic separation, and changing the number of the azido-labeled nano-magnetic particles in the supernatant;
5) Adding an azide cross-linking agent into the supernatant obtained after the magnetic separation in the step 4), triggering a bioorthogonal reaction, changing the azide-nano magnetic particles in the original dispersion state into a gathering state, enabling transverse relaxation time signals of the magnetic immunosensor to be amplified in a cascade mode, and measuring the transverse relaxation time signals to further carry out quantitative analysis on pesticide residues.
2. The method for detecting pesticide residue according to claim 1, characterized in that: the particle size of the carboxyl magnetic beads is 500-3000nm.
3. The method for detecting pesticide residue according to claim 1, characterized in that: the particle size of the amino nano magnetic particles is 10-100nm.
4. The method for detecting pesticide residue according to claim 1, characterized in that: the azide cross-linking agent is diphenyl cyclooctyne-tetraethylene glycol-diphenyl cyclooctyne.
5. The method for detecting pesticide residue according to claim 1, characterized in that: the weight ratio of the 15-azido-4,7,10,13-tetraoxypentadecanoic acid-N-succinimidyl ester to the amino nano magnetic particles is 1:5.
6. the method for detecting pesticide residue according to claim 1, characterized in that: the molar ratio of the reaction of the monoclonal antibody and diphenyl cyclooctyne-tetraethylene glycol-active ester is 1:10.
7. the method for detecting pesticide residue according to claim 1, characterized in that: and 5) the reaction time of the bioorthogonal reaction is 20min.
8. The method for detecting pesticide residue according to claim 1, characterized in that: the pesticide is a small molecular compound pesticide.
9. The method for detecting pesticide residue according to claim 1, characterized in that: the carrier protein of the pesticide is bovine serum albumin.
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