CN109507151B - Tetracycline detection method based on surface plasma resonance technology - Google Patents
Tetracycline detection method based on surface plasma resonance technology Download PDFInfo
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Abstract
The invention relates to the technical field of molecular detection, and discloses a tetracycline detection method based on a surface plasmon resonance technology, which comprises the following steps: (1) adding magnetic molecularly imprinted nanoparticles (MMIPPs NPs) having specific adsorption on tetracycline into a sample solution to be detected, oscillating and standing, and separating by using an external magnetic field to obtain the tetracycline MMIPPs NPs; (2) and (3) fixing the volume of the tetracycline MMIPPs NPs to the initial volume of the sample solution to be detected by using a buffer solution, and directly injecting the buffer solution into the SPR chip surface modified by mercaptoethylamine to detect the tetracycline content in the sample solution to be detected. The method combines MMIPs and SPR technology, effectively improves the sensitivity and accuracy of detection, has good specificity and repeatability, meets the requirement on recovery rate, is simple and quick to operate, and has important practical application value.
Description
Technical Field
The invention relates to the technical field of molecular detection, in particular to a tetracycline detection method based on a surface plasmon resonance technology.
Background
Tetracycline antibiotics are widely used in the animal husbandry and clinical treatment, and after people take food containing tetracycline antibiotic residues for a long time, the tetracycline antibiotics can cause potential harm to health. The residue of tetracycline antibiotics in the environment is mainly expressed in soil environment, water environment, meat, dairy products, eggs and other foods, and the detection method mainly comprises a microbiological method, an enzyme-linked immunosorbent assay, a fluorescence spectrophotometry, an HPLC method and the like. Because the food sample matrix is complex and the content of the target substance is low, effective sample pretreatment must be adopted before measurement. The existing detection method for tetracycline antibiotic residues mainly comprises high performance liquid chromatography, chromatography/mass spectrometry combined technology, enzyme-linked immunoassay and the like, wherein chromatographic analysis instruments and equipment are expensive, sample pretreatment is complex, and operation of professionals is needed; enzyme-linked immunoassay methods are relatively complex, have low sensitivity and high cost, and all require complex sample pretreatment. Compared with the detection method, the Surface Plasmon Resonance (SPR) technology is a real-time and label-free modern analysis technology based on the change of the refractive index of a substance, and has the advantages of no need of labeling a sample, less consumption, simple operation, high detection sensitivity, real-time monitoring reaction dynamics, reusable chip and the like.
The analysis of tetracycline residues is the detection of trace components in a complex matrix, and the adoption of a proper purification method is the key point for establishing accurate, rapid and high-sensitivity tetracycline residue detection. At present, Magnetic Nanoparticles (MNPs) have the advantages of simple preparation method, low price, easy obtainment, low toxicity, controllable motion under an external magnetic field and the like, and are widely used as magnetic carriers for preparing molecularly imprinted polymers. Magnetic Molecularly Imprinted Polymers (MMIPs) are polymers having specific molecular recognition sites, which are carried by magnetic materials. Fe3O4Nanoparticles (Fe)3O4NPs) is simple in preparation and low in cost, is often used as a carrier of a molecularly imprinted polymer, and can directly and selectively enrich and separate an object to be detected under the action of an external magnetic field, so that the problems of long time consumption, difficult separation and the like in food sample pretreatment can be effectively solved, and the NPs can be combined with an analysis method to be applied to detection, so that the object to be detected can be quickly, accurately and highly sensitively detected.
Surface Plasmon Resonance (SPR) technology is an analytical technique based on physical optical phenomena. When polarized light is incident on the interface of metal and dielectric medium at resonance angle, total internal reflection occurs, evanescent wave of incident light is coupled with surface plasma wave, surface plasma resonance is caused, and reflected light with sharply reduced light intensity is generated. When the measurement is carried out by using the optical SPR technology, an SPR sensing chip is generally modified, and when a sample to be measured is added and interacts with a modified object on the surface of the sensing chip, the refractive index of a system is changed, so that the optical signal of the SPR is changed. Due to the advantages of high sensitivity, rapidness, convenience and the like of the SPR technology, the SPR technology is widely applied to the fields of life science research, drug screening, food environment detection and the like.
At present, no research and report of combining magnetic molecular imprinting nanoparticles (MMIPs NPs) with specific adsorption on tetracycline with SPR detection technology is seen at home and abroad.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a tetracycline detection method based on a surface plasmon resonance technology, which combines magnetic molecular imprinting nanoparticles (MMIPs NPs) with specific adsorption to tetracycline with an SPR technology, thereby not only improving the sensitivity and accuracy of detection, but also having the advantages of rapid detection, simple operation, high selectivity, good reproducibility and reproducibility, and qualified recovery rate, and having important practical application value.
In order to achieve the above object, the present invention provides a tetracycline detection method based on surface plasmon resonance technology, comprising the following steps:
(1) adding MMIPPs NPs into a sample solution to be detected, oscillating and standing, and separating by using an external magnetic field to obtain tetracycline MMIPPs NPs;
(2) and (3) fixing the volume of the tetracycline MMIPPs NPs to the initial volume of the sample solution to be detected by using a buffer solution, and directly injecting the buffer solution into the SPR chip surface modified by mercaptoethylamine to detect the tetracycline content in the sample solution to be detected.
Preferably, the oscillating comprises: carrying out vortex oscillation on the sample solution to be detected for 30-50 min; the standing time is 5-10 min.
Preferably, the tetracycline MMIPs NPs are washed 2-5 times with the buffer before the volume fixing.
Preferably, the SPR chip modified with mercaptoethylamine is prepared by a method comprising the steps of:
(1) preprocessing an SPR chip;
(2) and (3) mounting the pretreated SPR chip on an SPR sensor, introducing a buffer solution, and introducing a mercaptoethylamine solution after the baseline is stable so as to perform self-assembly on the surface of the SPR chip.
Preferably, the flow rate of the buffer is 8-15 μ L/min, the concentration of the mercaptoethylamine solution is 0.5-3.5mg/mL, the flow rate is 8-15 μ L/min, and the volume is 100-.
Preferably, the pre-treatment comprises: soaking the SPR chip in absolute ethyl alcohol for 3-8min, washing with water for 5-8 times, blowing with nitrogen gas, burning with hydrogen flame for 15-30s, and cooling.
Preferably, the buffer is a phosphate buffer, preferably having a pH of 7.2-7.4.
Preferably, the MMIPS NPs are prepared by a method comprising the following steps:
(i) dissolving ferric chloride hexahydrate in water, introducing nitrogen to remove oxygen, stirring, adding ferrous chloride tetrahydrate, a sodium hydroxide solution and citric acid, and reacting for 1-2 hours at the temperature of 75-95 ℃ and the rotating speed of 500-;
(ii) cooling the solution prepared in the step (1) to room temperature, separating by using a magnet, collecting a product A, washing the product A by using water until the pH value is 6-7, and drying in vacuum to prepare surface carboxylated Fe3O4Nanoparticles (Fe)3O4@CA NPs)；
(iii) Adding tetracycline and methacrylic acid into an ethanol-water solution, and stirring for 20-40min to obtain a pre-assembled solution; the surface carboxylation Fe prepared in the step (2)3O4Adding the nano particles and ethylene glycol dimethacrylate into the pre-assembly solution, and carrying out ultrasonic treatment for 20-40min to obtain a pre-polymerization solution; adding the prepolymerization solution into a polyvinylpyrrolidone-ethanol solution, adding azobisisobutyronitrile, introducing nitrogen for protection, and reacting for 20-30h at the temperature of 50-70 ℃ and the rotation speed of 250-350 r/min; and (3) enriching, separating and collecting a product B by using a magnet, washing the product B with a methanol-acetic acid solution for 6-8 times, then washing with water for 2-3 times, and drying in vacuum to obtain the magnetic molecularly imprinted nanoparticles with specific adsorption to tetracycline, namely MMIPs NPs.
Preferably, the mass ratio of ferric chloride hexahydrate, ferrous chloride tetrahydrate, sodium hydroxide and citric acid in the step (i) is (4.5-5.5): (1.5-2.5): (3-3.5): 1, the mass to volume ratio of the ferric chloride hexahydrate to the water is (4.5-5.5): 100 g/mL.
Preferably, the volume ratio of ethanol to water in the ethanol-water solution in the step (iii) is 1: 7-10, wherein the volume ratio of methanol to acetic acid in the methanol-acetic acid solution is 4-9: 1; the tetracycline, the methacrylic acid and the surface carboxylated Fe3O4The mass ratio of the nano particles to the ethylene glycol dimethacrylate to the polyvinylpyrrolidone to the azobisisobutyronitrile is (4-5): (2-9): (2.5-10): (37-42): (3.5-4.5): 1.
through the technical scheme, the invention has the beneficial effects that:
(1) the invention utilizes the characteristics of MMIPPs NPs with magnetic rapid separation and molecular specificity identification, effectively simplifies the pretreatment steps, reduces background interference and shortens the detection time. Meanwhile, as MMIPPs have the characteristic of large refractive index, response signals of SPR are effectively improved, so that the newly constructed SPR sensor has high sensitivity, and the detection limit is 0.98 pg/mL.
(2) The invention utilizes specific combination of MMIPPs NPs and tetracycline, directly carries out SPR detection on the obtained combination after separation by an external magnetic field, combines the sample pretreatment technology with the detection technology, effectively improves the sensitivity and accuracy of the detection, and has simple operation and high detection speed. In addition, the SPR sensor has good specificity, reproducibility and recovery rate, can directly detect the tetracycline residue in animal derived food, and has important practical application value.
Drawings
FIG. 1 is a graph showing that MMIPs NPs enhance the response signal of SPR sensors of the present invention, a MMIPs NPs at a tetracycline concentration of 0pg/mL, b MMIPs NPs at a tetracycline concentration of 50pg/mL, c represents MNIPs NPs at a tetracycline concentration of 50pg/mL, and d represents MMIPs NPs at a tetracycline concentration of 1000 pg/mL;
FIG. 2 is the specificity of SPR sensors of the present invention for tetracycline detection;
FIG. 3 is a kinetic curve of the SPR sensor of the invention for real-time tetracycline detection, wherein the concentration of tetracycline a is 5pg/mL, the concentration of tetracycline b is 7.5pg/mL, the concentration of tetracycline c is 10pg/mL, the concentration of tetracycline d is 25pg/mL, the concentration of tetracycline e is 50pg/mL, and the concentration of tetracycline f is 100 pg/mL.
FIG. 4 is a working curve of the SPR sensor of the present invention for detecting tetracycline.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention provides a tetracycline detection method based on a surface plasmon resonance technology, which comprises the following steps:
(1) adding MMIPPs NPs into a sample solution to be detected, oscillating and standing, and separating by using an external magnetic field to obtain tetracycline MMIPPs NPs;
(2) and (3) fixing the volume of the tetracycline MMIPPs NPs to the initial volume of the sample solution to be detected by using a buffer solution, and directly injecting the buffer solution into the SPR chip surface modified by mercaptoethylamine to detect the tetracycline content in the sample solution to be detected.
Preferably, the oscillating comprises: carrying out vortex oscillation on the sample solution to be detected for 30-50 min; the standing time is 5-10 min.
Preferably, the tetracycline MMIPs NPs are washed 2-5 times with the buffer before the volume fixing.
Preferably, the SPR chip modified with mercaptoethylamine is prepared by a method comprising the steps of:
(1) preprocessing an SPR chip;
(2) and (3) mounting the pretreated SPR chip on an SPR sensor, introducing a buffer solution, and introducing a mercaptoethylamine solution after the baseline is stable so as to perform self-assembly on the surface of the SPR chip.
Preferably, the flow rate of the buffer is 8-15 μ L/min, the concentration of the mercaptoethylamine solution is 0.5-3.5mg/mL, the flow rate is 8-15 μ L/min, and the volume is 100-.
Preferably, the pre-treatment comprises: soaking the SPR chip in absolute ethyl alcohol for 3-8min, washing with water for 5-8 times, blowing with nitrogen gas, burning with hydrogen flame for 15-30s, and cooling.
Preferably, the buffer is phosphate buffered saline (PBS buffer), and the pH of the PBS buffer is preferably 7.2 to 7.4.
Preferably, the MMIPS NPs are prepared by a method comprising the following steps:
(i) dissolving ferric chloride hexahydrate in water, introducing nitrogen to remove oxygen, stirring, adding ferrous chloride tetrahydrate, a sodium hydroxide solution and citric acid, and reacting for 1-2 hours at the temperature of 75-95 ℃ and the rotating speed of 500-;
(ii) cooling the solution prepared in the step (1) to room temperature, separating by using a magnet, collecting a product A, washing the product A by using water until the pH value is 6-7, and drying in vacuum to prepare surface carboxylated Fe3O4Nanoparticles (Fe)3O4@CA NPs)；
(iii) Adding tetracycline and methacrylic acid into an ethanol-water solution, and stirring for 20-40min to obtain a pre-assembled solution; the surface carboxylation Fe prepared in the step (2)3O4Adding the nano particles and ethylene glycol dimethacrylate into the pre-assembly solution, and carrying out ultrasonic treatment for 20-40min to obtain a pre-polymerization solution; adding the prepolymerization solution into a polyvinylpyrrolidone-ethanol solution, adding azobisisobutyronitrile, introducing nitrogen for protection, and reacting for 20-30h at the temperature of 50-70 ℃ and the rotation speed of 250-350 r/min; and (3) enriching, separating and collecting a product B by using a magnet, washing the product B with a methanol-acetic acid solution for 6-8 times, then washing with water for 2-3 times, and drying in vacuum to obtain the magnetic molecularly imprinted nanoparticles with specific adsorption to tetracycline, namely MMIPs NPs.
Magnetic non-molecular engram nano particles (MNIPs NPs) are synthesized by the same preparation method under the condition of not adding tetracycline.
In the invention, the water can be ultrapure water, distilled water, deionized water or any other experimental water.
Preferably, the mass ratio of ferric chloride hexahydrate, ferrous chloride tetrahydrate, sodium hydroxide and citric acid in the step (i) is (4.5-5.5): (1.5-2.5): (3-3.5): 1, the mass to volume ratio of the ferric chloride hexahydrate to the water is (4.5-5.5): 100 g/mL.
Preferably, the volume ratio of ethanol to water in the ethanol-water solution in the step (iii) is 1: 7-10, wherein the volume ratio of methanol to acetic acid in the methanol-acetic acid solution is 4-9: 1; the tetracycline, the methacrylic acid and the surface carboxylated Fe3O4The mass ratio of the nano particles to the ethylene glycol dimethacrylate to the polyvinylpyrrolidone to the azobisisobutyronitrile is (4-5): (2-9): (2.5-10): (37-42): (3.5-4.5): 1.
the present invention will be described in detail below by way of examples.
In the following examples, the SPR chip used was manufactured by Biosensing Instrument Inc. of U.S. A.A. and the SPR Instrument used was BI-SPR2000 manufactured by Biosensing Instrument Inc. of U.S. A.A.; mercaptoethylamine was purchased from Sigma-Aldrich, USA; tetracycline is available from Yongsheng Biotech, Inc., Shanghai Rui; methacrylic acid was purchased from Shanghai Michelin Biotechnology, Inc.; ethylene glycol dimethacrylate was purchased from Shanghai Aladdin Biotechnology Ltd; polyvinylpyrrolidone is available from national pharmaceutical group chemical agents, ltd; azobisisobutyronitrile was purchased from Shanghai Shanpu chemical Co., Ltd; aureomycin was purchased from Shanghai-derived leaf Biotech, Inc.; sulfadiazine was purchased from Yongsheng Biotech limited, Shanghai; norfloxacin was purchased from shanghai source leaf biotechnology limited; other reagents and milk samples were all commercially available.
Preparation example 1
(1) Dissolving 4.5g of ferric chloride hexahydrate in 100mL of ultrapure water, introducing nitrogen, removing oxygen, and stirring; adding 1.5g of ferrous chloride tetrahydrate, 37.5mL of 2mol/L sodium hydroxide solution and 1g of citric acid, and reacting for 1h at the temperature of 75 ℃ and the rotating speed of 500 r/min;
(2) cooling the solution prepared in the step (1) to room temperature, separating by using a magnet, collecting a product A, repeatedly washing by using ultrapure water to be neutral, performing ultrasonic treatment for 3 times, and performing vacuum drying to obtain the surface carboxylated Fe3O4Nanoparticles (Fe)3O4@CA NPs)；
(3) 0.4g of tetracycline, 0.35g of methacrylic acid were added to 10mL of the ethanol-water solution (V)Water (W)：VEthanol7:1) and stirring for 20min to obtain a preassembly solution; 0.25g of surface carboxylated Fe prepared in the step (2) is taken3O4Adding nanoparticles and 3.7g of ethylene glycol dimethacrylate into the pre-assembly solution, and carrying out ultrasonic treatment for 20min to obtain a pre-polymerization solution; adding the prepolymerization solution into 100mL of ethanol solution containing 0.35g of polyvinylpyrrolidone, adding 0.1g of azobisisobutyronitrile, and reacting for 20h under the conditions of 50 ℃ and 250r/min of rotation speed under the protection of nitrogen; collecting product B by enrichment and separation with magnet, and treating with methanol-acetic acid solution (V)Methanol：VAcetic acid7:1) and washing for 6 times, then washing for 2 times by using ultrapure water, and drying in vacuum to obtain the magnetic molecularly imprinted nanoparticles (MMIPs NPs) with specific adsorption to tetracycline.
Magnetic non-molecular engram nano particles (MNIPs NPs) are synthesized by the same preparation method under the condition of not adding tetracycline.
Preparation example 2
(1) Dissolving 5g of ferric chloride hexahydrate in 100mL of ultrapure water, introducing nitrogen to remove oxygen, and stirring; adding 2g of ferrous chloride tetrahydrate, 40mL of sodium hydroxide solution with the concentration of 2mol/L and 1g of citric acid, and reacting for 1.5h under the conditions that the temperature is 85 ℃ and the rotating speed is 550 r/min;
(2) cooling the solution prepared in the step (1) to room temperature, separating by using a magnet, collecting a product A, repeatedly washing by using ultrapure water to be neutral, carrying out ultrasonic treatment for 4 times, and carrying out vacuum drying to obtain the surface carboxylated Fe3O4Nanoparticles (Fe)3O4@CA NPs)；
(3) 0.45g of tetracycline and 0.65g of methacrylic acid were added to 10mL of an ethanol-water solution (V)Water (W)：VEthanol9:1) and stirring for 30min to obtain a preassembly solution; 0.5g of the surface carboxylated Fe prepared in the step (2) is taken3O4Adding nanoparticles and 3.9g of ethylene glycol dimethacrylate into the pre-assembly solution, and carrying out ultrasonic treatment for 30min to obtain a pre-polymerization solution; adding the prepolymerization solution into 100mL of ethanol solution containing 0.4g of polyvinylpyrrolidone, adding 0.1g of azobisisobutyronitrile, and reacting for 24h under the protection of nitrogen at the temperature of 60 ℃ and the rotating speed of 300 r/min; collecting product B by enrichment and separation with magnet, and treating with methanol-acetic acid solution (V)Methanol：VAcetic acidWashing 7 times with ultrapure water, washing 3 times with ultrapure water, and vacuum drying to obtain magnetic molecularly imprinted nanoparticles (MMIPs NPs) with tetracycline specific adsorption.
Preparation example 3
(1) Dissolving 5.5g of ferric chloride hexahydrate in 100mL of distilled water, introducing nitrogen to remove oxygen, and stirring; adding 2.5g of ferrous chloride tetrahydrate, 44mL of sodium hydroxide solution with the concentration of 2mol/L and 1g of citric acid, and reacting for 2 hours at the temperature of 95 ℃ and the rotating speed of 600 r/min;
(2) cooling the solution prepared in the step (1) to room temperature, separating by using a magnet, collecting a product A, repeatedly washing to be neutral by using distilled water, carrying out ultrasonic treatment for 5 times, and carrying out vacuum drying to obtain surface carboxylated Fe3O4Nanoparticles (Fe)3O4@CA NPs)；
(3) 0.5g of tetracycline, 0.85g of methacrylic acid were added to 10mL of an ethanol-water solution (V)Water (W)：VEthanol10:1) and stirring for 40min to obtain a preassembly solution; 0.75g of surface carboxylated Fe prepared in the step (2) is taken3O4Adding nanoparticles and 4.2g of ethylene glycol dimethacrylate into the pre-assembly solution, and carrying out ultrasonic treatment for 40min to obtain a pre-polymerization solution; adding the prepolymerization solution into 100mL of ethanol solution containing 0.45g of polyvinylpyrrolidone, adding 0.1g of azobisisobutyronitrile, introducing nitrogen, and reacting for 30h at the temperature of 70 ℃ and the rotating speed of 350 r/min; collecting product B by enrichment and separation with magnet, and treating with methanol-acetic acid solution (V)Methanol：VAcetic acid9:1) washing for 8 times, and distillingWashing with water for 2 times, and vacuum drying to obtain magnetic molecularly imprinted nanoparticles (MMIPs NPs) with specific adsorption to tetracycline.
Magnetic non-molecular engram nano particles (MNIPs NPs) are synthesized by the same preparation method under the condition of not adding tetracycline.
Example 1
(1) Soaking the SPR chip in absolute ethyl alcohol for 5min, washing with water for 3 times, drying with nitrogen, and burning with hydrogen flame for 20 s;
(2) installing the cooled SPR chip on an SPR sensor, introducing PBS buffer solution (10mM, pH7.4) at the flow rate of 10 muL/min, introducing 100 muL of mercaptoethylamine solution with the concentration of 3.5mg/mL at the flow rate of 10 muL/min after the base line is stable, and carrying out self-assembly on the mercaptoethylamine solution on the surface of the SPR chip to prepare the SPR chip modified by mercaptoethylamine;
(3) preparing tetracycline-PBS buffer solution with the concentration of 50pg/mL and 1000pg/mL respectively;
(4) 5mg of NPs of MMIPs prepared in preparation example 1 were added to 1mL of tetracycline solutions having final concentrations of 0pg/mL, 50pg/mL and 1000pg/mL, respectively, to form samples a, b and d, and 5mg of MNIPs prepared in preparation example 1 were added to 1mL of tetracycline solution having a concentration of 50pg/mL to form sample c. Respectively carrying out vortex oscillation on the sample solutions for 40min, standing for 5min, then separating by an external magnetic field to obtain a combination, repeatedly washing the combination for 3 times by using PBS (phosphate buffer solution), and then fixing the volume to 1mL by using the PBS;
(5) and (3) introducing the solution obtained in the step (4) into the SPR chip modified by mercaptoethylamine prepared in the step (2) at the flow rate of 10 mu L/min, so that the SPR chip is combined with the mercaptoethylamine and fixed on the surface of the sensing chip, and directly detecting tetracycline (each sample is subjected to three parallel detections). After each sample detection, the SPR chip is regenerated by water or NaOH (1mM-5mM, specific concentration, depending on response signal), and then the next sample detection is carried out. The ordinate Δ θ (mDeg) in fig. 1 represents the change in resonance angle (i.e., change in SPR signal), which is the stable resonance angle achieved after the sample has flowed through the chip minus the stable resonance angle before the sample entered the instrument.
As shown in FIG. 1, after MMIPS NPs were added to a solution without tetracycline (0pg/mL) (sample a), the change in resonance angle was only 1.10mDeg, which is probably due to non-specific adsorption; when the SPR sensor detection is carried out after tetracycline enrichment by using MMIPs NPs, the change value of the SPR resonance angle is very obvious and is increased along with the increase of the tetracycline concentration (b: the tetracycline concentration is 50pg/mL, the Delta theta is 19.39 mDeg; d: the tetracycline concentration is 1000pg/mL, and the Delta theta is 46.21 mDeg). In addition, after the tetracycline solution (50pg/mL) with the same concentration is enriched and separated by using MMIPs NPs and MNIPs NPs respectively, the resonance angle change values caused by SPR detection are 19.39mDeg and 4.47mDeg respectively, and the SPR resonance angle change value of the MMIPs NPs is 4.34 times of that of the MNIPs NPs, which shows that the method established by the invention has good specificity.
Example 2
(1) Soaking the SPR chip in absolute ethyl alcohol for 3min, washing with water for 8 times, drying with nitrogen, and igniting with hydrogen flame for 15 s;
(2) installing the cooled SPR chip on an SPR sensor, introducing PBS buffer solution (10mM, pH7.2) at the flow rate of 8 muL/min, introducing 150 muL of mercaptoethylamine solution with the concentration of 0.5mg/mL at the flow rate of 8 muL/min after the base line is stable, and carrying out self-assembly on the mercaptoethylamine solution on the surface of the SPR chip to prepare the SPR chip modified by mercaptoethylamine;
(3) preparing a mixed solution a with the final concentrations of tetracycline, aureomycin, sulfadiazine and norfloxacin being 50pg/mL, a mixed solution b with the final concentrations of tetracycline and aureomycin being 50pg/mL, a mixed solution c with the final concentrations of tetracycline and sulfadiazine being 50pg/mL, a mixed solution d with the final concentrations of tetracycline and norfloxacin being 50pg/mL, a solution e with the final concentration of tetracycline being 50pg/mL, a solution f with the final concentration of aureomycin being 50pg/mL, a solution g with the final concentration of sulfadiazine being 50pg/mL and a solution h with the final concentration of norfloxacin being 50pg/mL, and enriching and separating the solutions a-h by respectively using Ps MMIs and MNIPs NPs: 5mg of MMIPS NPs prepared in preparation example 3 were added to 1mL of the solutions a-h, and 5mg of MNIPs NPs prepared in preparation example 3 were added to 1mL of the solutions a-h. Respectively carrying out vortex oscillation on the sample solutions for 30min, standing for 7min, then separating by an external magnetic field to obtain a conjugate, repeatedly washing the conjugate for 2 times by using PBS (phosphate buffer solution), and then fixing the volume to 1mL by using the PBS;
(4) and (3) introducing the solution obtained in the step (3) into the SPR chip modified by mercaptoethylamine prepared in the step (2) at the flow rate of 10 mu L/min, so that the SPR chip is combined with the mercaptoethylamine and is fixed on the surface of the sensing chip, and directly detecting tetracycline. After each sample detection, the SPR chip is regenerated by water or NaOH (1mM-5mM, specific concentration, depending on response signal), and then the next sample detection is carried out. Three replicates of each sample were performed to verify the specificity of the detection method.
As shown in figure 2, after various samples are enriched and separated by MMIPs NPs and MNIPs NPs, SPR sensor detection is directly carried out, and the resonance angle change value (delta theta) caused by the MMIPs NPs is obviously higher than the resonance angle change value (delta theta) caused by the MNIPs NPs; secondly, the resonance angle change value (Delta theta) caused by adding MMIPS NPs into the sample containing tetracycline is also obviously higher than that (Delta theta) caused by adding MMIPS NPs into the sample not containing tetracycline, which shows that the tetracycline MMIPS NPs coupling SPR sensor has good specificity for tetracycline detection. From the single-component sample solutions (e, f, g, h), it is seen that the resonance angle change value caused by tetracycline enrichment by MMPs NPs is 19.39mDeg, which is 5.71 times of the resonance angle change value caused by sulfadiazine enrichment by MMPs NPs, 4.32 times of the resonance angle change value caused by norfloxacin enrichment by MMPs NPs, but only 1.15 times of the resonance angle change value caused by aureomycin enrichment by MMPs NPs, because tetracycline and aureomycin both belong to tetracycline compounds, the structures are very similar, and the response signals are relatively high.
Example 3
(1) Soaking the SPR chip in absolute ethyl alcohol for 8min, repeatedly washing with water, drying with nitrogen, and burning with hydrogen flame for 30 s;
(2) installing the cooled SPR chip on an SPR sensor, introducing PBS buffer solution (10mM, pH7.4) at the flow rate of 15 muL/min, introducing 120 muL of mercaptoethylamine solution with the concentration of 2.5mg/mL at the flow rate of 15 muL/min after the base line is stable, and carrying out self-assembly on the mercaptoethylamine solution on the surface of the SPR chip to prepare the SPR chip modified by mercaptoethylamine;
(3) 5mg of MMIPPs prepared in preparation example 3 are weighed and added into 1mL of tetracycline-PBS buffer solution with different concentrations to obtain samples with the final concentrations of tetracycline of 0pg/mL, 1pg/mL, 2.5pg/mL, 5pg/mL, 7.5pg/mL, 10pg/mL, 25pg/mL, 50pg/mL, 75pg/mL, 100pg/mL, 250pg/mL, 500pg/mL and 1000pg/mL respectively, the samples are vortexed for 50min, the samples are kept still for 10min and then separated by an external magnetic field to obtain a conjugate, and the conjugate is repeatedly washed 5 times by PBS buffer (10mM, pH7.4) and then the volume of the conjugate is increased to 1mL by the PBS buffer.
(4) Introducing the sample solution obtained in the step (3) into the SPR chip modified by mercaptoethylamine prepared in the step (2) at the flow rate of 10 muL/min, enabling the sample solution to be combined with mercaptoethylamine and fixed on the surface of the sensing chip, performing direct tetracycline detection, regenerating the SPR chip by using water or NaOH (1mM-5mM, specific concentration and according to response signals) after each sample detection is finished, performing next sample detection, performing three parallel detections on the tetracycline solution at each concentration, recording SPR response signals, drawing an SPR kinetic curve and a working curve (S/N is 3), and obtaining results shown in FIGS. 3 and 4, the sample solution with the final concentration of 5pg/mL of tetracycline and mercaptoethylamine begin to be combined in about 200s, begin to be dissociated in about 500s and basically reach the dynamic equilibrium of combination-dissociation in 700 s; the sample solution with final concentration of 100pg/mL tetracycline begins to be combined with mercaptoethylamine at about 200s, begins to be dissociated at 515s, and basically reaches the dynamic equilibrium of combination-dissociation at 700 s. As is apparent from FIG. 3, when the dynamic equilibrium of binding-dissociation on the SPR chip surface is reached, the SPR signal (resonance angle) of the sample solution with the final tetracycline concentration of 100pg/mL is much higher than that of the equilibrium of the NPs of tetracycline MMPs with the concentration of 5pg/mL, and in the range of the final tetracycline concentration of 5-100pg/mL, the concentration of tetracycline is linearly related to the SPR signal, and the linear regression equation is that Y is 0.33031X +2.80487(R is)20.99549), the lowest detectable concentration was 5pg/mL, and the lowest detection limit was 0.98 pg/mL.
Example 4
(1) Soaking the SPR chip in absolute ethyl alcohol for 5min, repeatedly washing with water, drying with nitrogen, igniting with hydrogen flame for 15s,
(2) and (3) mounting the cooled SPR chip on an SPR sensor, introducing PBS buffer solution (10mM, pH7.4) at the flow rate of 12 mu L/min, introducing 150 mu L of mercaptoethylamine solution with the concentration of 2mg/mL at the flow rate of 12 mu L/min after the base line is stable, and carrying out self-assembly on the mercaptoethylamine solution on the surface of the SPR chip to prepare the SPR chip modified by mercaptoethylamine.
(3) Pretreatment of a milk sample: accurately weighing 5g of milk sample A, placing the milk sample A in a 50mL colorimetric tube, dissolving the milk sample A by using 0.1mol/L EDTA-Mclvaine buffer solution, fixing the volume to 50mL, carrying out vortex mixing for 1min, carrying out ice water bath ultrasound for 10min, transferring the milk sample A to a 50mL polypropylene centrifugal tube, cooling the milk sample A to 0-4 ℃, centrifuging the milk sample A for 10min under the condition of the rotating speed of 5000r/min, and filtering the supernatant through a 0.22 mu m filter membrane.
(4) And (3) taking the sample solution prepared in the step (3), and respectively adopting the method, the national standard (GBT21317-2007) and the ELISA method for detection, wherein tetracycline is not detected in the result, which indicates that the detection method provided by the invention has good accuracy.
(5) Taking the sample solution prepared in the step (3), adding tetracycline standard, adding 5mg of MMIPPs NPs prepared in the preparation example 2 into 1mL of tetracycline-milk solution to ensure that the final standard addition concentrations of the tetracycline in the sample solution are respectively 10pg/mL, 25pg/mL and 50pg/mL, carrying out vortex oscillation for 40min, standing for 6min, then carrying out external magnetic field separation to obtain a conjugate, repeatedly washing the conjugate for 3 times by using PBS (10mM, pH7.4), and then carrying out volume fixing to 1mL by using the PBS.
(6) And (3) introducing the solution obtained in the step (5) into the SPR chip modified by mercaptoethylamine prepared in the step (2) at the flow rate of 10 mu L/min, combining the solution with mercaptoethylamine and fixing the mercaptoethylamine on the surface of the sensing chip, directly detecting tetracycline, regenerating the SPR chip by using water or NaOH (1mM-5mM, specific concentration and according to response signals) after each sample detection is finished, then carrying out next sample detection, carrying out three parallel detections on the tetracycline-milk solution with each concentration, recording SPR response signals, and inspecting the accuracy of tetracycline detection by using the method.
The detection results are shown in table 1, the standard recovery rate of the milk sample A is 95.7-104.56%, the RSD range is 2.18-13.72%, the recovery rate is in an allowable range, and the requirement of the recovery rate is met.
TABLE 1 recovery of milk sample A with spiking
Example 5
(1) Soaking the SPR chip in absolute ethyl alcohol for 5min, washing with water for 3 times, drying with nitrogen, and burning with hydrogen flame for 20 s;
(2) installing the cooled SPR chip on an SPR sensor, introducing PBS buffer solution (10mM, pH7.4) at the flow rate of 10 muL/min, introducing 100 muL of mercaptoethylamine solution with the concentration of 3.5mg/mL at the flow rate of 10 muL/min after the base line is stable, and carrying out self-assembly on the mercaptoethylamine solution on the surface of the SPR chip to prepare the SPR chip modified by mercaptoethylamine;
(3) pretreatment of a milk sample: accurately weighing 5g of milk sample B, placing the milk sample B in a 50mL colorimetric tube, dissolving the milk sample B by using 0.1mol/L EDTA-Mclvaine buffer solution, fixing the volume to 50mL, carrying out vortex mixing for 1min, carrying out ice water bath ultrasound for 10min, transferring the milk sample B to a 50mL polypropylene centrifugal tube, cooling the milk sample B to 0-4 ℃, centrifuging the milk sample B for 10min under the condition of the rotating speed of 5000r/min, and filtering the supernatant through a 0.22 mu m filter membrane.
(4) And (3) taking the sample solution prepared in the step (3), and respectively adopting the method, the national standard (GBT21317-2007) and the ELISA method for detection, wherein tetracycline is not detected in the result, which indicates that the detection method provided by the invention has good accuracy.
(5) Taking the sample solution prepared in the step (3), adding tetracycline standard, adding 5mg of MMIPPs NPs prepared in the preparation example 2 into 1mL of tetracycline-milk solution to ensure that the final standard addition concentrations of the tetracycline in the sample solution are respectively 10pg/mL, 25pg/mL and 50pg/mL, carrying out vortex oscillation for 40min, standing for 6min, then carrying out external magnetic field separation to obtain a conjugate, repeatedly washing the conjugate for 3 times by using PBS (10mM, pH7.4), and then carrying out volume fixing to 1mL by using the PBS.
(6) And (3) introducing the solution obtained in the step (5) into the SPR chip modified by mercaptoethylamine prepared in the step (2) at the flow rate of 10 mu L/min, combining the solution with mercaptoethylamine and fixing the mercaptoethylamine on the surface of the sensing chip, directly detecting tetracycline, regenerating the SPR chip by using water or NaOH (1mM-5mM, specific concentration and according to response signals) after each sample detection is finished, then carrying out next sample detection, carrying out three parallel detections on the tetracycline-milk solution with each concentration, recording SPR response signals, and inspecting the accuracy of tetracycline detection by using the method.
The detection results are shown in Table 2, the standard recovery rate of the milk sample B is 96.26-105.69%, the RSD range is 2.67-6.28%, the recovery rate is in an allowable range, and the requirement of the recovery rate is met.
TABLE 2 recovery of milk sample B with spiking
Example 6
(1) Soaking the SPR chip in absolute ethyl alcohol for 5min, washing with water for 3 times, drying with nitrogen, and burning with hydrogen flame for 20 s;
(2) installing the cooled SPR chip on an SPR sensor, introducing PBS buffer solution (10mM, pH7.4) at the flow rate of 15 muL/min, introducing 100 muL of mercaptoethylamine solution with the concentration of 1.5mg/mL at the flow rate of 15 muL/min after the base line is stable, and carrying out self-assembly on the mercaptoethylamine solution on the surface of the SPR chip to prepare the SPR chip modified by mercaptoethylamine;
(3) pretreatment of a milk sample: accurately weighing 5g of milk sample C, placing the milk sample C in a 50mL colorimetric tube, dissolving the milk sample C by using 0.1mol/L EDTA-Mclvaine buffer solution, fixing the volume to 50mL, carrying out vortex mixing for 1min, carrying out ice water bath ultrasound for 10min, transferring the milk sample C to a 50mL polypropylene centrifugal tube, cooling to 0-4 ℃, centrifuging for 10min under the condition of the rotating speed of 5000r/min, and filtering the supernatant through a 0.22 mu m filter membrane.
(4) And (3) taking the sample solution prepared in the step (3), and respectively adopting the method, the national standard (GBT21317-2007) and the ELISA method for detection, wherein tetracycline is not detected in the result, which indicates that the detection method provided by the invention has good accuracy.
(5) Taking the sample solution prepared in the step (3), adding tetracycline standard substance, adding 5mg of MMIPPs NPs prepared in the preparation example 2 into 1mL of tetracycline-milk solution to ensure that the final standard concentration of tetracycline in the sample solution is respectively 10pg/mL, 25pg/mL and 50pg/mL, carrying out vortex oscillation for 40min, standing for 8min, then carrying out external magnetic field separation to obtain a conjugate, repeatedly washing the conjugate for 3 times by using PBS buffer solution (10mM, pH7.4), and then carrying out volume fixing to 1mL by using the PBS buffer solution.
(6) And (3) introducing the solution obtained in the step (5) into the SPR chip modified by mercaptoethylamine prepared in the step (2) at the flow rate of 10 mu L/min, combining the solution with mercaptoethylamine and fixing the mercaptoethylamine on the surface of the sensing chip, directly detecting tetracycline, regenerating the SPR chip by using water or NaOH (1mM-5mM, specific concentration and according to response signals) after each sample detection is finished, then carrying out next sample detection, carrying out three parallel detections on the tetracycline-milk solution with each concentration, recording SPR response signals, and inspecting the accuracy of tetracycline detection by using the method.
The detection results are shown in Table 3, the standard recovery rate of the milk sample C is 94.79-100.24%, the RSD range is 4.58-12.01%, the recovery rate is in an allowable range, and the requirement of the recovery rate is met.
TABLE 3 recovery of milk sample C with spiking
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (9)
1. A tetracycline detection method based on a surface plasmon resonance technology is characterized by comprising the following steps:
(1) adding the magnetic molecularly imprinted nanoparticles into a sample solution to be detected, oscillating and standing the sample solution, and separating the sample solution by using an external magnetic field to obtain tetracycline magnetic molecularly imprinted nanoparticles;
(2) fixing the volume of the tetracycline magnetic molecularly imprinted nanoparticles to the initial volume of the sample solution to be detected by using a buffer solution, and directly injecting the tetracycline magnetic molecularly imprinted nanoparticles to the surface of the SPR chip modified by mercaptoethylamine to detect the tetracycline content in the sample solution to be detected;
the magnetic molecularly imprinted nanoparticle is prepared by a method comprising the following steps:
(i) dissolving ferric chloride hexahydrate in water, introducing nitrogen to remove oxygen, stirring, adding ferrous chloride tetrahydrate, a sodium hydroxide solution and citric acid, and reacting for 1-2 hours at the temperature of 75-95 ℃ and the rotating speed of 500-;
(ii) (ii) cooling the solution prepared in step (i) to room temperature, separating with a magnet, collecting product A, washing product A with water until the pH value is 6-7, and drying in vacuum to obtain surface carboxylated Fe3O4Nanoparticles;
(iii) adding tetracycline and methacrylic acid into an ethanol-water solution, and stirring for 20-40min to obtain a pre-assembled solution; (iii) surface carboxylation of Fe obtained in step (ii)3O4Adding the nano particles and ethylene glycol dimethacrylate into the pre-assembly solution, and carrying out ultrasonic treatment for 20-40min to obtain a pre-polymerization solution; adding the prepolymerization solution into a polyvinylpyrrolidone-ethanol solution, adding azobisisobutyronitrile, introducing nitrogen for protection, and reacting for 20-30h at the temperature of 50-70 ℃ and the rotation speed of 250-350 r/min; collecting a product B by utilizing enrichment separation of a magnet, washing the product B with a methanol-acetic acid solution for 6-8 times, then washing with water for 2-3 times, and carrying out vacuum drying to prepare the magnetic molecularly imprinted nanoparticles with specific adsorption on tetracycline;
the SPR chip modified by mercaptoethylamine is prepared by a method comprising the following steps:
(1) preprocessing an SPR chip;
(2) and (3) mounting the pretreated SPR chip on an SPR sensor, introducing a buffer solution, and introducing a mercaptoethylamine solution after the baseline is stable so as to perform self-assembly on the surface of the SPR chip.
2. The method for detecting tetracycline based on surface plasmon resonance technology of claim 1, wherein the oscillating comprises: carrying out vortex oscillation on the sample solution to be detected for 30-50 min; the standing time is 5-10 min.
3. The tetracycline detection method based on surface plasmon resonance technology of claim 1, wherein before the volume fixing, the tetracycline magnetic molecularly imprinted nanoparticles are washed with the buffer solution for 2-5 times.
4. The method for detecting tetracycline based on surface plasmon resonance technology of claim 1, wherein the flow rate of the buffer is 8-15 μ L/min, the concentration of the mercaptoethylamine solution is 0.5-3.5mg/mL, the flow rate is 8-15 μ L/min, and the volume is 100-.
5. The method for detecting tetracycline based on surface plasmon resonance technique of claim 1, wherein the pre-treatment comprises: soaking the SPR chip in absolute ethyl alcohol for 3-8min, washing with water for 5-8 times, blowing with nitrogen gas, burning with hydrogen flame for 15-30s, and cooling.
6. The method for detecting tetracycline based on surface plasmon resonance technique of claims 1-5, wherein said buffer is phosphate buffer.
7. The method for detecting tetracycline based on surface plasmon resonance technique of claim 6, wherein the pH of the phosphate buffer is 7.2-7.4.
8. The method for detecting tetracycline based on surface plasmon resonance technology of claim 1, wherein the mass ratio of ferric chloride hexahydrate, ferrous chloride tetrahydrate, sodium hydroxide and citric acid in step (i) is (4.5-5.5): (1.5-2.5): (3-3.5): 1; the mass of the ferric chloride hexahydrate is 4.5-5.5g relative to 100mL of the water.
9. The method for detecting tetracycline based on surface plasmon resonance technique of claim 1, wherein the volume ratio of ethanol to water in said ethanol-water solution in step (iii) is 1: (7-10), wherein the volume ratio of methanol to acetic acid in the methanol-acetic acid solution is (4-9): 1; the tetracycline, the methacrylic acid and the surface carboxylated Fe3O4The mass ratio of the nano particles to the ethylene glycol dimethacrylate to the polyvinylpyrrolidone to the azobisisobutyronitrile is (4-5): (2-9): (2.5-10): (37-42): (3.5-4.5): 1.
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