CN107084951B - Method for detecting zearalenone based on time-resolved fluorescence labeling-aptamer - Google Patents
Method for detecting zearalenone based on time-resolved fluorescence labeling-aptamer Download PDFInfo
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Abstract
The invention provides a method for identifying and detecting zearalenone based on a time-resolved fluorescent marker-aptamer, which is used for detecting the content of zearalenone in wheat, grains, feeds, products of the feeds and the like. The method fully utilizes the characteristic of long fluorescence life of the time-resolved fluorescence nanomaterial, effectively avoids the interference of biological background fluorescence of the sample, improves the detection sensitivity, and simultaneously combines the magnetic separation sample enrichment effect of the zearalenone specific aptamer functionalized magnetic nanomaterial to quickly concentrate a large number of samples, effectively improves the sample concentration by thousands of times, and can greatly shorten the detection period.
Description
Technical Field
The invention relates to a method for detecting zearalenone by utilizing a magnetic nano material and a time-resolved fluorescent nano material in combination with nucleic acid aptamer recognition, which is characterized in that the magnetic nano material functionalized by a zearalenone aptamer is utilized to separate and enrich zearalenone in samples such as corn wheat and the like, so that the detection is convenient and rapid, meanwhile, the time-resolved fluorescent nano material is used as a marker to further improve the detection sensitivity by detecting a time-resolved fluorescent signal, and the method belongs to the technical field of nano materials and molecular biology.
Background
Zearalenone (ZEN), also known as F-2 toxin, is a nonsteroidal estrogenic mycotoxin produced mainly by fusarium such as fusarium graminearum. The chemical name of the toxin is 6- (10-hydroxy-6-oxy-undecenyl) -beta-ranilic acid, and the molecular formula is as follows: c18H22O5Relative to molecular mass 318.4, it is insoluble in water and soluble in alkaline aqueous solution and alcohols, and its methanol solution exhibits green-blue fluorescence under ultraviolet light. ZEN is widely present in grains, mainly pollutes crops such as corn, barley, wheat, oat, sorghum, rice and the like, and is distributed to a certain extent in products such as flour, malt, beer, dairy products, meat products and the like. ZEN poses serious threat to human and animal health, and the harm is second to aflatoxin. ZEN has strong reproductive development toxicity and immunotoxicity to animals, can cause animal estrogen hyperemia, and cause infertility, fetal malformation and growth dysplasia, especially has large influence on pigs, cattle and sheep, and can be used for livestockThe animal husbandry brings huge economic loss. When people eat polluted crops or products such as meat, egg and milk and the like, the ZEN mainly harms the human body and shows reproductive development toxicity, immunotoxicity, genotoxicity, hepatotoxicity and hepatotoxicity, and the toxicity causes tumor (uterine tumor, breast cancer and testicular cancer).
Currently, Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), high performance liquid mass spectrometry (HPLC-MS/MS), established immunization methods based on antigen-antibody immune recognition and the like are mainly used for ZEN detection. Thin layer chromatography has more complicated operation steps and low sensitivity and specificity, and can not meet the requirements of modern detection. The common chromatographic analysis method has complex and tedious sample pretreatment method, needs to be skillfully mastered on the operation technology of the instrument, and is inconvenient for basic popularization. Although the immunoassay method for detecting ZEN has the advantages of high sensitivity, strong specificity and the like, the preparation process of the small molecular antigen ZEN is complicated, time-consuming and expensive, antibodies in different batches have differences, and the storage and use conditions are severe, so that the false positive rate is high and the repeatability is poor. Therefore, it is necessary to develop a rapid and convenient detection method with good stability, high sensitivity, strong specificity and low cost.
Aptamers (aptamers) are a cluster of small molecule DNA or RNA fragments that specifically bind to a target substance and are screened from random oligonucleotide libraries synthesized in vitro by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). The aptamer can recognize any type of target substance corresponding to the aptamer, such as proteins and low molecules, and has high affinity with the target substance. Compared with an antibody, the checking aptamer has a plurality of advantages, such as short in-vitro synthesis period, low preparation cost and no need of animal experiments; the stability is good, and the temperature is not sensitive; easy modification, etc., and aptamers have been widely used as recognition molecules in clinical diagnosis, clinical treatment, drug delivery, proteome research, and food safety.
Time-resolved fluorescence is a special fluorescence phenomenon, and is time-resolved by using the fluorescence lifetime of a substance, namely, by adopting a proper excitation light source and a detection system, a fluorescence intensity-time curve at a fixed wavelength and a fluorescence emission spectrum at a fixed time can be obtained, so that components with overlapped spectra but different lifetimes in a mixture can be distinguished and can be respectively measured; the interference of impurity autofluorescence and background fluorescence in the detection environment on the signal-to-noise ratio is eliminated by setting reasonable Delay time (Delay time) and Gate time (Gate time, also called detection time), thereby improving the sensitivity of the analysis method. The key to the development of time-resolved fluorescence analysis technology lies in the effective development and combined utilization of lanthanide fluorescent probes and analysis modes. With the vigorous development of nanotechnology, lanthanide nanomaterials with good water dispersibility as a new class of luminescent markers have their inherent time-resolved optical properties developed and utilized gradually. The lanthanide doped nano material has a plurality of excellent physicochemical properties, such as good water dispersibility, long fluorescence life, photobleaching resistance, wide Stokes shift, good biocompatibility, low cytotoxicity, multi-color controllability (doping element types and proportions), and is beneficial to simultaneous detection of multiple components in a biological system. In view of the advantages, the novel lanthanide doped fluorescent nano material has good time-resolved fluorescence analysis application prospect. The physical length of the magnetic nano material is just in nano magnitude, and the magnetic nano material has the characteristics of simple preparation method, low raw material cost, unique superparamagnetism, easy surface modification, good biocompatibility and the like, so that the magnetic nano material has wide application prospect in the field of biomedicine.
The method comprises the steps of firstly synthesizing a magnetic nano material with a biological functionalized surface and a time-resolved fluorescent nano material, assembling the magnetic nano material and the time-resolved fluorescent nano material into a composite nano structure through hybridization between a target nucleic acid aptamer combined with the magnetic nano material and a complementary chain of the nucleic acid aptamer combined with the time-resolved fluorescent nano material, exciting by ultraviolet light, inducing time-resolved fluorescence to be a detection signal, and establishing a standard curve through detection of zearalenone standard substances with different concentrations to achieve the purpose of quantitatively detecting a sample containing zearalenone. The invention can be used for detecting the content of zearalenone in samples such as corn, wheat, grains, feed and products thereof.
Disclosure of Invention
A kind ofThe method for detecting zearalenone by time-resolved fluorescence labeling-aptamer recognition comprises the following steps: respectively preparing aminated Fe3O4Magnetic nano material and time-resolved fluorescent nano material NaYF4Ce/Tb, surface Avidin (Avidin) modification is carried out on the magnetic nano material and the time fluorescence resolution nano material, and then the magnetic nano material and the time fluorescence resolution nano material are specifically combined with Biotin (Biotin) -modified aptamer DNA and aptamer complementary strand (cDNA) through the action between Avidin (Avidin) and Biotin (Biotin) to respectively form a fluorescent probe and a capture probe. And incubating the two for a period of time, and assembling the two into the composite nano material through complementary hybridization. Separating the nano material by using an external magnetic field, exciting by using ultraviolet light (273nm), recording the intensity of the fluorescence signal at the moment as the maximum value, and when a target analyte zearalenone is added, changing the spatial conformation of the aptamer and specifically combining the aptamer with zearalenone to melt the aptamer DNA and a complementary single strand (cDNA), so that the time-resolved fluorescence nano material is separated from the magnetic nano particles, and detecting after enrichment, so that the intensity of the obtained fluorescence signal is reduced. The content of the zearalenone in a certain range is related to the value of the decrease of the fluorescence signal intensity, and a standard curve is established on the basis of the value of the decrease of the fluorescence signal intensity to detect the zearalenone standard substance so as to achieve the aim of quantitatively detecting the zearalenone in an actual sample; the method comprises the following steps:
1. one-step solvothermal method is adopted to synthesize NaYF4Ce/Tb and surface modification thereof
1mmol of phosphoethanolamine (AEP) and 1mmol of NaCl are weighed out in 30mL of ethylene glycol and stirred continuously, after which 0.9mmol of Y (NO) is added3)3·6H2O、0.05mmol Ce(NO3)3·6H2O、0.05mmol Tb(NO3)3·6H2And O. Then, the solution containing 4mmol of NH4And (3) dropwise adding 10mL of ethylene glycol solution (fully dissolved by stirring in a water bath at 45 ℃) of the F into the transparent solution, continuously stirring for 30min at room temperature, transferring into a 50mL of teflon high-pressure reaction kettle, and reacting for 4h at 195 ℃. After the reaction, the reaction product is taken out and cooled to room temperature, washed by ethanol and ultrapure water alternately for three times, dissolved in ultrapure water and stored in a refrigerator at 4 ℃ for later use.
2. Preparation of aminated Fe3O4Magnetic nanoparticles
2.0g of anhydrous sodium acetate and 1.0g of ferric chloride hexahydrate are weighed, added into 30mL of ethylene glycol, 6.5g of warm-bath 1, 6-hexanediamine is added into the mixed solution, heated to 50 ℃, and stirred continuously to form a homogeneous colloidal solution with wine red and bright color. The solution was transferred to a 100mL autoclave and reacted at 198 ℃ for 6 h. And after the reaction is finished, taking out the reaction kettle, naturally cooling to room temperature, completely pouring into a beaker (washing and transferring residual powder in batches), separating and collecting by using a magnet, discarding the upper layer mixed liquid, ultrasonically dispersing black powder obtained by magnetic separation by using ultrapure water, then magnetically separating, discarding the solution, and collecting the powder. Washing with ultrapure water and anhydrous ethanol for three times in turn, drying the obtained black powder at 50 deg.C for 12h, taking out, grinding into fine powder, and storing for use.
3. Coupling the time-resolved fluorescent nano material and the magnetic nano material with Avidin (Avidin) by using a glutaraldehyde method
Because the surfaces of the time-resolved fluorescent nano material and the magnetic nano material contain amino groups, avidin can be covalently bonded to the surfaces of the materials through a glutaraldehyde method. The time fluorescence resolution nanometer material and the magnetic nanometer material are resuspended by 10mM Phosphate Buffer Solution (PBS) to be made into 1mg/mL solution, and the solution is fully dispersed in the buffer solution by ultrasonic for 15 min. The material and 5% glutaraldehyde are activated by gentle shaking at room temperature in the dark for 2h, the time-resolved fluorescence nano material is used for removing unreacted glutaraldehyde by a centrifugation method, the magnetic material is used for removing the unreacted glutaraldehyde by an external magnetic field, then the magnetic material is washed three times by PBS, and then avidin (1mg/mL) is added to the magnetic material and shaken at 37 ℃ overnight. After the reaction, the reaction mixture was washed several times and the supernatant was discarded.
4. The avidin-modified magnetic nano material and the time-resolved fluorescent nano material are coupled with the aptamer and the aptamer complementary strand respectively
The magnetic nano material and the time-resolved fluorescence nano material of the surface modified Avidin (Avidin) are connected with the Biotin (Biotin) modified zearalenone aptamer DNA single strand and the aptamer complementary strand cDNA by utilizing the specific binding between the Avidin (Avidin) and the Biotin (Biotin). The specific method comprises the following steps: dispersing 5mg of avidin-modified magnetic nano material and time-resolved fluorescent nano material in 5mL (10mM) of phosphate buffer, respectively adding a certain amount of biotinylated zearalenone aptamer and aptamer complementary strand, incubating for 12h in a shaking table at 37 ℃, centrifuging to collect the material, washing for three times by using the phosphate buffer, and dispersing in the phosphate buffer.
5. DNA single strand of zearalenone aptamer and cDNA hybridization of aptamer complementary strand
Connecting the time-resolved fluorescent nano-particles with the magnetic nano-material to form a nano-composite, wherein the specific implementation method comprises the following steps: and mixing 400 mu L of complementary DNA single-chain solution marked by the time-resolved fluorescent nano material with 100 mu L of aptamer functionalized magnetic nano material, reacting for 1h at 37 ℃ in a phosphate buffer solution system, acting for 1min in an external magnetic field, collecting the assembled nano material compound, and washing for 3 times by using phosphate buffer solution.
6. Detecting the zearalenone standard substance, and establishing a standard curve
Dissolving the assembled nano material compound in a phosphate buffer solution, preparing zearalenone with different concentrations, adding the zearalenone into the compound system, incubating for 40min at 37 ℃, performing magnetic separation, discarding supernatant, washing for three times to ensure that the time-resolved fluorescence nano material which is dissociated and dropped is not attached to the surface of the magnetic nano material, so that the sensitivity and accuracy of detection are ensured, and finally suspending the enriched mixture into 200 mu L of phosphate buffer solution for on-machine detection. An enzyme labeling instrument Synergy H1 and BioTek are adopted to set the excitation wavelength at 273nm, the delay time at 100 mus, the detection time at 1000 mus and a time-resolved fluorescence signal at 544nm in a time-resolved fluorescence mode for detecting the zearalenone. The time-resolved fluorescence signal gradually decreased with increasing zearalenone concentration. And establishing a standard curve according to the fluorescence value and the corresponding concentration of the zearalenone standard substance, wherein the experimental result obtains a good linear relation within the range of 0.001-10 ng/mL.
7. Detection of zearalenone sample
And (3) simply treating the sample, then directly adding the sample into the nano composite system, incubating for 40min at 37 ℃, exciting at 273nm to obtain a 544nm fluorescence signal in a time-resolved fluorescence mode of an enzyme-linked immunosorbent assay (ELSA), and obtaining the corresponding concentration of zearalenone from a standard curve.
The invention has the advantages that:
1. by utilizing the characteristic of long fluorescence life of lanthanide series doped nano materials, the fluorescence of the detection background is attenuated through time resolution, so that the detection sensitivity is greatly improved.
2. The detection accuracy and stability are effectively improved by utilizing the specific binding of the aptamer to the detection substance.
3. The magnetic nano material is used as a capture probe to enrich the target detection object, so that the detection period can be effectively shortened, and the operation is simple.
Drawings
FIG. 1 is a schematic diagram of the detection of zearalenone based on time-resolved fluorescence labeling-aptamer recognition
FIG. 2 is a time-resolved fluorescence intensity as a function of zearalenone concentration overlay (a); and (b) a zearalenone detection standard curve graph.
The specific implementation mode is as follows:
the present invention includes, but is not limited to, the following examples, and any equivalents or insubstantial modifications thereof, which are within the spirit and principles of the invention, are deemed to be within the scope of the invention.
Example 1: detection and standard recovery of zearalenone in corn actual sample
Sample pretreatment: pulverizing semen Maydis at high speed, sieving, weighing 5g in 100mL flask, adding 5g NaCl and methanol (7:3) water solution, mixing, extracting in homogenizer at high speed for 2min, standing for a moment, filtering, placing 10mL filtrate in 50mL flask, extracting in homogenizer at high speed for 2min, standing, filtering with superfine glass fiber filter paper until the filtrate is clarified, and collecting the filtrate. Respectively selecting zearalenone standard substances with the concentrations of 0.001, 0.01, 0.1, 0.5, 1, 5 and 10ng/mL, adding the zearalenone standard substances into the substance to be detected, and detecting the content of zearalenone again by using the method to obtain a detection value. Percent recovery is (detection value-background value)/addition amount × 100%. From the results in the table I, the recovery rate is 80.76-122.36%, which shows that the method is stable, sensitive and accurate, and is suitable for detecting zearalenone in a corn actual sample.
Table one: detection and standard recovery rate of zearalenone in actual corn sample
Example 2: detection and standard recovery of zearalenone in wheat actual sample
Sample pretreatment was the same as example 1. Respectively selecting zearalenone standard substances with the concentrations of 0.001, 0.01, 0.1, 0.5, 1, 5 and 10ng/mL, adding the zearalenone standard substances into the substance to be detected, and detecting the content of zearalenone again by using the method to obtain a detection value. Percent recovery is (detection value-background value)/addition amount × 100%. From the results in the table two, the recovery rate is 90.04-114.75%, which shows that the method is also suitable for detecting zearalenone in a wheat actual sample.
Table two: detection and standard recovery rate of zearalenone in wheat actual sample
Claims (3)
1. A method for identifying and detecting zearalenone based on time-resolved fluorescence labeling-aptamer is characterized in that a zearalenone aptamer functionalized magnetic nano material and a time-resolved fluorescence nano material NaYF4: Ce/Tb with an aptamer complementary single chain modified on the surface are obtained through assembly, surface Avidin (Avidin) modification is carried out on the magnetic nano material and the time-resolved fluorescence nano material, then the Avidin (Avidin) and Biotin (Biotin) are acted to be specifically combined with Biotin (Biotin) modified aptamer DNA and an aptamer complementary chain (cDNA), the two are assembled into a composite nano structure through DNA complementary hybridization, a time-resolved fluorescence detection signal is obtained through ultraviolet excitation in the detection process, when a target analyte zearalenone is added, the spatial conformation of the aptamer is changed to be specifically combined with zearalenone, and (3) melting the aptamer and the aptamer complementary single strand, so that a part of time-resolved fluorescent nano material is separated from the magnetic nano material, the fluorescence signal intensity of the time-resolved fluorescent nano material assembled on the surface of the magnetic nano material is reduced, and the zearalenone standard is detected based on the fluorescence signal intensity, so that a standard curve is established, and the content of the zearalenone sample is detected.
2. The method for detecting zearalenone based on time-resolved fluorescence labeling-aptamer recognition as claimed in claim 1, characterized in that Avidin (Avidin) is firstly modified on the surface of the aminated magnetic nanomaterial, and then the Avidin (Avidin) and Biotin (Biotin) are specifically combined with the Biotin (Biotin) modified aptamer DNA through the action between Avidin and Biotin, and the Avidin and the Biotin are used as a magnetic separation reagent.
3. The method for detecting zearalenone based on time-resolved fluorescence labeling-aptamer recognition as claimed in claim 1, wherein the Avidin (Avidin) modified time-resolved fluorescence nanomaterial is combined with Biotin (Biotin) modified aptamer complementary nucleic acid single strand to form a nucleic acid labeling probe.
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