Preparation method of magnetic molecular imprinting-aptamer sandwich fluorescence sensor for detecting kanamycin

Abstract

The invention designs a preparation method of a magnetic molecular imprinting-aptamer sandwich fluorescence sensor for detecting kanamycin. And the magnetic molecular imprinting composite material Fe3O4 (at) UiO-66 (at) MIP with a specific adsorption target object KANA is prepared. When a detection target object KANA exists, the Fe3O4-coated UiO-66-coated MIP can rapidly and specifically capture the KANA in a short time. Then, a DNA single-stranded aptamer marked with FAM is added, after the aptamer and KANA captured on the surface of the Fe3O4 at-UiO-66 at-MIP are subjected to specific recognition and magnetic separation again, an aptamer probe can enter sediment along with the magnetic composite material Fe3O4 at-UiO-66 at-MIP, the fluorescence intensity of supernate is reduced, and quantitative analysis is conducted according to the fluorescence difference value delta F. When the KANA does not exist, the aptamer cannot enter the precipitate, so that the fluorescence signal intensity is not changed. Double recognition is formed through specific binding of an MIP imprinting cavity and the aptamer, matrix interference is reduced by means of fluorescence characteristics and magnetic separation capacity, the method has the advantages of being high in adsorption capacity, specific in recognition, high in sensitivity, wide in detection range, good in selectivity and the like, and rapid and accurate detection of KANA in a complex sample can be achieved.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of food safety detection, in particular to a preparation method and application of a magnetic molecular imprinting-aptamer sandwich fluorescence sensor for detecting kanamycin. BACKGROUND

[0002] Since kanamycin (KANA) as an aminoglycoside antibiotic is widely used for treating gram-positive and gram-negative bacterial infections, its residual pollution has become a key issue threatening food safety and public health. The large-scale use of KANA in human medical treatment and livestock breeding results in residues of KANA in the form of original shape or metabolites in foods such as milk, honey, meat and water environment, which may enter the human body through the food chain and cause serious side effects such as ototoxicity and nephrotoxicity, and also induce bacterial resistance genes, thereby posing a long-term potential risk to public health. Therefore, in order to meet the regulatory requirements and control the residual risk, it is an important demand in the field of food and environmental analysis to develop a simple, efficient, sensitive and specific KANA detection method. At present, the methods for detecting KANA mainly include high performance liquid chromatography, liquid chromatography-mass spectrometry, capillary electrophoresis and the like. Although these methods can realize low-concentration residue detection, they have problems such as high equipment cost, complex sample pretreatment and long detection period, and are difficult to meet the needs of on-site rapid screening or large-scale sample detection. Although the electrochemical analysis method and the enzyme-linked immunoassay have improved the operation convenience, the former is easily disturbed by complex matrix, and the latter has the limitations of poor antibody stability and high preparation cost. The fluorescence analysis technology has a wide application prospect in the field of rapid detection due to its advantages such as simple operation, rapid response and strong visualization potential. The aptamer is a specific molecular probe selected by SELEX technology, and has the characteristics of high chemical stability, low preparation cost and easy modification, and has been widely used for specific detection of harmful substances in foods. The molecularly imprinted polymer (MIP) is a new type of functional polymer material prepared by molecular imprinting technology (MIT), which has a high selective recognition ability to specific target molecules, and the binding affinity and selectivity to specific target molecules can be comparable to natural antibodies.

[0003] Therefore, the application combines the magnetic porous composite material with the molecular imprinting technology, further introduces the aptamer fluorescence probe, constructs a “double recognition” system, realizes double targeting by the imprinting cavity of the molecularly imprinted polymer and the specific binding of the aptamer, reduces the matrix background interference by the optical characteristics of fluorescence and the magnetic separation capacity of MIP, enhances the detection sensitivity and specificity, and provides an innovative technical path for rapid and accurate detection of KANA. SUMMARY

[0004] The application designs a preparation method of a magnetic molecular imprinting-aptamer sandwich fluorescence sensor for detecting kanamycin, which is performed according to the following steps: (1) Preparation of magnetic material Fe3O4: Dissolve a certain mass ratio of ferric chloride hexahydrate and anhydrous sodium acetate in ethylene glycol and form a uniform solution under ultrasonic treatment, put it into a Teflon-lined stainless steel autoclave for high-temperature heating, and cool to room temperature after the reaction is completed. The solid product is washed several times with deionized water, the solid Fe3O4 is collected with a magnet, and vacuum constant temperature drying is carried out until the weight is constant; (2) Preparation of magnetic composite material Fe3O4@UiO-66: Stir Fe3O4 into a dilute solution of sodium polyacrylate, collect the solid with a magnet, wash several times with deionized water and N,N-dimethylformamide, disperse the collected solid in a mixture of a certain mass ratio of zirconium tetrachloride and diaminoterephthalic acid, add N,N-dimethylformamide solution and ultrasonic it to disperse uniformly, put the mixture into a high-pressure reaction kettle for reaction, collect it with a magnet and wash several times with N,N-dimethylformamide to obtain Fe3O4@UiO-66 and place it in a vacuum constant temperature dryer until the weight is constant; (3) Preparation of magnetic molecularly imprinted composite material Fe3O4@UiO-66@MIP: Under the action of ultrasonic waves, disperse Fe3O4@UiO-66 in Tris-HCl buffer solution, add KANA and a certain mass ratio of dopamine hydrochloride and 4-carboxyphenylboronic acid, stir overnight at room temperature, then separate with a magnet to obtain Fe3O4@UiO-66@MIP-KANA, elute the template KANA with a certain volume ratio of methanol-acetic acid mixture, repeat the washing with deionized water, magnetically separate Fe3O4@UiO-66@MIP and vacuum constant temperature dry; (4) Preparation of sandwich fluorescence sensor: Add an equal amount of Fe3O4@UiO-66@MIP to different concentrations of KANA standard solution, incubate at room temperature until the adsorption reaction reaches equilibrium, add an equal amount of FAM-labeled DNA single strand Apt and continue to incubate at room temperature, separate with a magnet after the reaction reaches equilibrium, collect the supernatant, and measure the change in fluorescence intensity using a fluorescence spectrophotometer for quantitative analysis by fluorescence difference ΔF.

[0005] Further limitation, in step (1), the mass ratio of ferric chloride hexahydrate and anhydrous sodium acetate is 1:2~1:3; the experimental reaction temperature and time are 200℃ and 12 h.

[0006] Further limitation, in step (2), the mass ratio of zirconium tetrachloride and diaminoterephthalic acid is 4:3~5:6; the experimental reaction temperature and time are 120℃ and 24 h.

[0007] Further limitation, in step (3), the mass ratio of dopamine hydrochloride and 4-carboxyphenylboronic acid is 2:1~3:1; the volume ratio of methanol-acetic acid mixture is 9:1~4:1.

[0008] Further limit, in step (4), the incubation time is 8-20 min; the mass of Fe3O4@UiO-66@MIP added is 0.02-0.03 mg; the concentration of DNA single-stranded Apt is 10-12 mM; the sequence of the DNA single-stranded Apt is 5'-TGG GGGTTG AGG CTA AGC CGA-3', wherein the 5' end is modified with FAM. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 It is a schematic diagram for preparation of a magnetic molecular imprinting-aptamer sandwich fluorescence sensor for detecting kanamycin.

[0010] Figure 2 It is the fluorescence detection result of the sensor constructed in Example 1 of the present application before (dotted line) and after (solid line) adding 50 ng / mL KANA.

[0011] Figure 3 It is the standard curve of KANA detected by the sensor constructed in Example 1 of the present application.

[0012] Figure 4 It is the specificity of KANA detected by the sensor constructed in Example 1 of the present application in the presence of other interfering substances. DETAILED DESCRIPTION

[0013] The specific embodiments of the present application will be described in detail below with reference to the accompanying drawings and specific examples, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are conventional methods unless otherwise specified. EMBODIMENT

[0014] A method for preparing a magnetic molecular imprinting-aptamer sandwich fluorescence sensor for detecting kanamycin, which is implemented as shown in Figure 1 .

[0015] A method for preparing a magnetic molecular imprinting-aptamer sandwich fluorescence sensor for detecting kanamycin, comprising the following steps: (1) Preparation of magnetic material Fe3O4: Dissolve 1.08 g of ferric chloride hexahydrate and 2.88 g of anhydrous sodium acetate in 40 mL of ethylene glycol to form a uniform solution under ultrasonic waves, then heat in a 100 mL Teflon-lined stainless steel autoclave at 200℃ for 12 h, cool to room temperature after the reaction is completed, wash the solid product with deionized water several times, and collect the solid Fe3O4 with a magnet.

[0016] (2) Preparation of magnetic composite Fe3O4@UiO-66: 0.64 mL of 30% sodium polyacrylate was dissolved in 11.36 mL of deionized water, then 0.2 g of Fe3O4 was added, the solution was stirred at 37°C for 24 h, the solid was collected by a magnet, washed with deionized water and N,N-dimethylformamide for several times, 0.2 g of functionalized Fe3O4 was dispersed in a mixture of 0.24 g of zirconium tetrachloride, 0.18 g of diaminoterephthalic acid and 30 mL of N,N-dimethylformamide, and was uniformly dispersed by shaking for 30 min, the mixture was placed in a high-pressure reaction kettle and reacted at 120°C for 24 h, and finally collected by a magnet and washed with N,N-dimethylformamide for several times to obtain Fe3O4@UiO-66 and placed in a vacuum constant-temperature dryer until the weight was constant.

[0017] (3) Preparation of magnetic molecularly imprinted composite Fe3O4@UiO-66@MIP: 0.1 g of Fe3O4@UiO-66 was dispersed in 50 mL of Tris-HCl buffer (50 mM, pH 8.5) under the action of ultrasonic waves, 0.05 g of KANA, 0.2 g of dopamine hydrochloride and 0.1 g of 4-carboxyphenylboronic acid were added, and stirred at room temperature for 12 h, and Fe3O4@UiO-66@MIP-KANA was obtained by magnetic separation, KANA template was eluted with a mixture of methanol-acetic acid (9:1, v / v), and the cycle was repeated for 10 times, and finally washed with water for 10 times, and the solid product was obtained by magnetic separation and vacuum constant-temperature drying to obtain Fe3O4@UiO-66@MIP, and non-imprinted polymer Fe3O4@UiO-66@NIP was prepared by the same method without KANA.

[0018] (4) Preparation of sandwich fluorescence sensor and identification of target: 10 uL of different concentrations of KANA standard solution was added to 10 uL of 2 mg / mL Fe3O4@UiO-66@MIP, and the adsorption reaction reached equilibrium after incubation at room temperature for 8 min, at this time, 10 uL of 10 mM labeled FAM DNA single strand Apt was added, and the incubation was continued at room temperature for 20 min, and then magnetic separation was performed, the supernatant was collected, and the change of fluorescence intensity was determined by a fluorescence spectrophotometer. The fluorescence difference ΔF = F0 - F was used for quantitative analysis.

[0019] (5) Establishment of standard curve: 10 μL of different concentrations of KANA standard solution was added to step (4) to obtain different gradient sample detection solutions, and different fluorescence signals were obtained after incubation, and the logarithmic value of KANA concentration was used as the abscissa, and the fluorescence signal difference corresponding to each concentration was used as the ordinate for linear fitting to establish the standard curve of the sensor for KANA.

[0020] AsFigure 2 As shown in the figure, the fluorescence detection results of the sensor constructed in Example 1 of the present application before (solid line) and after (dashed line) adding 50 ng / mL KANA.

[0021] As shown in the figure, the fluorescence detection results of the sensor constructed in Example 1 of the present application before (solid line) and after (dashed line) adding 50 ng / mL KANA. Figure 3 As shown in the figure, the standard curve of the sensor constructed in Example 1 of the present application for detecting KANA. Example

[0022] A preparation method and application of a magnetic molecular imprinting-aptamer sandwich fluorescence sensor for detecting kanamycin, and the actual application thereof, include the following steps: (1) In order to verify the specific recognition effect of the prepared double recognition fluorescence biosensor on KANA, KANA standard was added to the Tris-HCl buffer solution, so that the concentration of KANA in the sample was 50 ng / mL; other 7 kinds of interfering substances (ciprofloxacin, doxycycline hydrochloride, chloramphenicol, roxithromycin, amikacin sulfate, terramycin, tetracycline) were respectively configured into standard solutions with Tris-HCl buffer solution, and the concentration of each was 50 ng / mL; the detection system constructed in Example 1 was used to detect the above-mentioned 7 kinds of different interfering substance standard solutions and their mixed samples with KANA, and the detection results are shown in Figure 4 As shown in the figure, the selectivity of the method of the present application to KANA is good. Example

[0023] A preparation method of a magnetic molecular imprinting-aptamer sandwich fluorescence sensor for detecting kanamycin, and the actual application thereof, is carried out according to the following steps: (1) Food sample processing: for liquid sample milk, 1 μL milk sample was accurately measured and added into a centrifuge tube containing 2999 μL ultrapure water, and mixed well using a vortex oscillator to obtain a primary dilution of milk, 1 mL of the above primary dilution of milk was accurately measured and transferred into a new centrifuge tube containing 3 mL ultrapure water, and mixed again to prepare a secondary dilution of milk; 100 μL of the secondary dilution of milk was mixed with 100 μL of KANA standard sample of different concentrations to obtain a KANA spiked sample of milk; for viscous sample honey, due to the high viscosity of honey, it needs to be dissolved first: 5 mg of honey sample was accurately weighed and slowly added into 1 mL of ultrapure water, and placed in a 37℃ constant temperature water bath for oscillation for 10 min, and vortexed every 2 min during the oscillation to ensure complete dissolution of the honey to obtain an initial dissolution of honey; 2 μL of the initial dissolution of honey was accurately measured and added into a centrifuge tube containing 998 μL of ultrapure water, and mixed well to prepare a primary dilution of honey; 40 μL of the primary dilution of honey was further measured and transferred into a centrifuge tube containing 360 μL of ultrapure water, and mixed well to obtain a secondary dilution of honey; 100 μL of the secondary dilution of honey was mixed with 100 μL of KANA standard sample of different concentrations, and mixed gently to obtain a KANA spiked sample of honey.

[0024] (2) Sample detection: the fluorescence signal was measured according to the procedure of step (3) of Example 1, and the concentration of KANA in the sample was obtained by bringing in the standard curve.

[0025] (3) When milk and honey were used as food samples for determination, different concentrations of KANA standard were added into the samples, 10 μL of sample solution was taken, and the fluorescence signal was measured according to the procedure of step (1) to step (4) of Example 1, and the concentration of KANA in the sample was obtained by bringing in the standard curve detected in Example 1, each sample was measured for 3 times and the average value was taken, and the average recovery rate was calculated to be 98.4%-100.9%.

[0026] It has been verified that the prepared sandwich type fluorescence sensor for KANA detection has the advantages of high sensitivity, good selectivity, good reliability and stability, and the detection of actual samples shows that the prepared sensor has very good practical application value.

[0027] The above shows and describes the basic principles, main features and advantages of the present application. It should be understood by those skilled in the art that the present application is not limited by the above examples, the above examples and descriptions in the specification are only to illustrate the principles of the present application, and do not limit the present application in any way, various changes and improvements can be made to the present application without departing from the spirit and scope of the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for the preparation of a magnetic molecularly imprinted-aptamer sandwich fluorescence sensor for the detection of kanamycin, characterized by, Comprise the following steps: (1) Preparation of magnetic molecularly imprinted material Fe3O4@UiO-66@MIP: dissolve a certain mass ratio of ferric chloride hexahydrate and anhydrous sodium acetate in ethylene glycol and form a uniform solution under ultrasonic treatment, add a Teflon-lined stainless steel autoclave for high-temperature heating, cool to room temperature after the reaction is completed, wash the solid product with deionized water several times, collect the solid with a magnet to obtain Fe3O4; add Fe3O4 to a dilute solution of sodium polyacrylate and stir, collect the solid with a magnet, wash several times with deionized water and N,N-dimethylformamide, disperse the collected solid in a mixture of a certain mass ratio of zirconium tetrachloride and diaminoterephthalic acid, add N,N-dimethylformamide solution and shake to disperse uniformly, place the mixture in a high-pressure reaction kettle for reaction, collect with a magnet and wash several times with N,N-dimethylformamide to obtain Fe3O4@UiO-66, and place in a vacuum constant-temperature dryer until the weight is constant; disperse Fe3O4@UiO-66 in Tris-HCl buffer, add kanamycin and a certain mass ratio of dopamine hydrochloride and 4-carboxyphenylboronic acid, stir overnight at room temperature, then separate the product with a magnet, elute the template with a certain volume ratio of methanol-acetic acid mixture several times, repeat washing with deionized water, magnetically separate to obtain magnetic molecularly imprinted material Fe3O4@UiO-66@MIP and vacuum constant-temperature dry; the surface of the metal-organic framework UiO-66 is rich in amino groups, which enables the bifunctional monomers dopamine and 4-carboxyphenylboronic acid to be firmly combined on the surface and form a molecularly imprinted cavity, and the method of dopamine self-aggregation eliminates the complex steps of adding cross-linking agents and initiators in traditional molecular imprinting; (2) Preparation of a sandwich fluorescence sensor: add an equal amount of Fe3O4@UiO-66@MIP to kanamycin standard solutions of different concentrations, incubate at room temperature until the adsorption reaction reaches equilibrium, add FAM-labeled DNA single strand Apt and continue to incubate at room temperature, magnetically separate after the reaction reaches equilibrium, collect the supernatant, and determine the change in fluorescence intensity with a fluorescence spectrophotometer, and quantitatively analyze by fluorescence difference ΔF; this method introduces two recognition elements, molecularly imprinted cavities and Apt probes, to realize enrichment and detection of the target in the same system.

2. The method for preparing a magnetic molecularly imprinted-aptamer sandwich fluorescent sensor for detecting kanamycin according to claim 1, characterized in that, In step (1), the mass ratio of ferric chloride hexahydrate and anhydrous sodium acetate is 1:2-1:3; the mass ratio of zirconium tetrachloride to diaminoterephthalic acid is 4:3-5:6; the volume ratio of methanol-acetic acid mixture is 9:1-4:1; the mass ratio of dopamine hydrochloride to 4-carboxyphenylboronic acid is 2:1-3:1; the reaction temperature is 120-200℃; and the reaction time is 12-24 h.

3. The method for preparing a magnetic molecularly imprinted-aptamer sandwich fluorescent sensor for detecting kanamycin according to claim 1, characterized in that, In step (2), the incubation time is 8-20 min; the mass of Fe3O4@UiO-66@MIP added is 0.02-0.03 mg; and the concentration of DNA single strand Apt is 10-12 mM.

4. The method for preparing a magnetic molecularly imprinted-aptamer sandwich fluorescent sensor for detecting kanamycin according to claim 1, characterized in that, In step (2), the DNA single-stranded Apt sequence is 5'-TGG GGG TTG AGGCTA AGC CGA-3', wherein the 5' end is modified with FAM.

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