CN112255214A - Method for simultaneously detecting three mycotoxins based on time-resolved fluorescence labeling-aptamer recognition - Google Patents
Method for simultaneously detecting three mycotoxins based on time-resolved fluorescence labeling-aptamer recognition Download PDFInfo
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Abstract
The invention provides a method for simultaneously detecting three mycotoxins based on time-resolved fluorescence labeling-aptamer recognition, and belongs to the technical field of nano materials and molecular biology. The specific method of the invention is as follows: respectively using biotinylated zearalenone aptamer Apt to time-resolved fluorescent nanoparticlesZENT-2toxin aptamer AptT‑2And aflatoxin B1 aptamer AptAFB1Modification to obtain KYF4:Dy3+‑AptZEN、KYF4:Tb3+‑AptT‑2And KYF4:Eu3+‑AptAFB1And detecting the fluorescence signal intensity of the mixture in the presence of mycotoxin by using a fluorescent probe and using tungsten disulfide nanosheets as fluorescence quenchers. The invention can be used for zearalenone, T-2toxin and aflatoxin B in samples such as corn, wheat, grain, feed and products thereof1Detection of (3).
Description
Technical Field
The invention belongs to the technical field of nano materials and molecular biology. In particular to a method for simultaneously detecting zearalenone, T-2toxin and aflatoxin B1 by utilizing the identification of a time-resolved fluorescent nano material and a tungsten disulfide nanosheet combined with an aptamer, which is characterized in that the strong fluorescence and long fluorescence life of the time-resolved fluorescent nano material, the high fluorescence quenching efficiency of the tungsten disulfide nanosheet and the high affinity and specificity of the aptamer to a target are fully utilized, the interference of biological background fluorescence of a sample is effectively avoided, the sensitivity and accuracy of detection are improved, and three mycotoxins can be simultaneously and correctly identified.
Background
Mycotoxins are chemical metabolites naturally produced during fungal growth and are considered to be the most important chronic dietary risk factors. Mycotoxin contamination can occur at any stage from the farm to the dining table. Mycotoxins often attack organs such as liver, kidney, and brain nervous system, and produce symptoms such as liver cirrhosis, hepatitis, hepatocyte necrosis, liver cancer, acute and chronic nephritis, severe hemorrhage of brain central nervous system, and nervous tissue degeneration. Therefore, it is important to monitor these mycotoxins regularly. Zearalenone (ZEN) has strong reproductive development toxicity and immunotoxicity to animals, and can cause animal estrogen hyperemia, resulting in infertility, sterility, fetal malformation and dysplasia. T-2toxin (T-2toxin) has wide toxicity, mainly destroys tissues and organs which are rapidly divided and actively proliferated, and can cause damage to multiple systems and multiple organs, especially the damage to lymphoid tissues such as bone marrow, thymus and the like is the most serious. Aflatoxin B1(AFB1) Is the most toxic and major mycotoxin, has the most extensive pollution in food and the most influence on the food safety, and is subjected to AFB by International Agency for Research on Cancer (IARC)1Is listed as a class I carcinogen. Since the mycotoxins are extremely destructive and toxic to human health and often coexist in grains, there is an urgent need to develop a sensitive, rapid and easy-to-use detection strategy for quantitatively determining multiple mycotoxins in food products.
Currently, mycotoxin detection methods mainly include Thin Layer Chromatography (TLC), High Performance Liquid Chromatography (HPLC), high performance liquid mass spectrometry (HPLC-MS/MS), and established immunological methods based on antigen-antibody immunological recognition. 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 the mycotoxin has the advantages of high sensitivity, strong specificity and the like, the preparation process of the small molecular antigen is complicated and time-consuming, the cost is high, the 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, convenient, stable, sensitive, specific and low-cost detection method.
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.
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 antibodies, the 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.
Tungsten Disulfide (WS) with S-W-S sandwich structure2) The nanosheets are recently emerging Transition Metal Dihalides (TMDC) having higher fluorescence quenching efficiency than graphene oxide, which would be highly beneficial for improving the sensitivity of biosensors based on fluorescence quenching and recovery. However, to date, only a few have been based on WS2Bioassay methods for nanoplatelets have been developed and are mostly based on WS2Quenching of fluorescent dyes, their use in time-resolved fluorescent aptamer sensors is extremely rare.
Disclosure of Invention
Aiming at the problems in the prior art, the applicant of the invention provides a method for simultaneously detecting zearalenone, T-2toxin and aflatoxin B based on time-resolved fluorescence labeling-aptamer recognition1The method of (1). The detection method has high sensitivity and higher stability. The invention firstly synthesizes three lanthanum-doped time-resolved fluorescent nanoparticles (KYF4: Dy)3+、KYF4:Tb3+And KYF4 Eu3+) And respectively biomarkers three mycotoxins (zearalenone, T-2toxin and aflatoxin B)1) Then WS is added2Used as a fluorescence quencher. Ultraviolet light is used for excitation, time-resolved fluorescence is induced to serve as a detection signal, a standard curve is established through detection of toxin standard substances with different concentrations, and the purpose of quantitative/qualitative detection of toxin-containing sample substances is achieved. The invention can be used in samples such as corn, wheat, grain, feed and products thereofZearalenone, T-2toxin and aflatoxin B1Detection of (3).
The technical scheme of the invention is as follows:
a method for simultaneously detecting three mycotoxins based on time-resolved fluorescence labeling-aptamer recognition, the detection method comprising: respectively using biotinylated zearalenone aptamer Apt to time-resolved fluorescent nanoparticlesZENT-2toxin aptamer AptT-2And aflatoxin B1Aptamer AptAFB1Modification to obtain KYF4:Dy3+-AptZEN、KYF4:Tb3+-AptT-2And KYF4:Eu3+-AptAFB1Detecting the fluorescence signal intensity of the mixture in the presence of mycotoxin by using a fluorescent probe and using tungsten disulfide nanosheets as a fluorescence quencher;
the three mycotoxins are zearalenone, T-2toxin and aflatoxin B1；
The time-resolved fluorescent nanoparticle is KYF4:Dy3+、KYF4:Tb3+And KYF4:Eu3+。
The KYF4:Dy3+-AptZEN、KYF4:Tb3+-AptT-2And KYF4:Eu3+-AptAFB1The preparation method of the fluorescent probe comprises the following steps:
The zearalenone aptamer AptZENThe sequence is 5 '-biotin-TCA TCT ATC TAT GGT ACA TTA CTA TCT GTA ATG TGA TAT G-3'; t-2toxin aptamer AptT-2The sequence is 5 '-biotin-CAG CTC AGA AGC TTG ATC CTG TAT ATC AAG CAT CGC GTG TTT ACA CAT GCG AGA GGT GAA GAC TCG AAG TCG TGC ATC TG-3'; aflatoxin B1Aptamer AptAFB1The sequence is 5 '-biotin AGC AGC ACA GAG GTC AGA TGG TGC TAT CAT GCG CTC AAT GGG AGA CTT TAG CTG CCC CCA CCT ATG CGT GCT ACC GTG AA-3'.
The detection method specifically comprises the following steps:
s1, establishing a standard curve: KYF with final concentration of 1mg/mL4:Dy3+-AptZEN、KYF4:Tb3+-AptT-2And KYF4:Eu3+-AptAFB1Mixing a fluorescent probe in a PBS (phosphate buffer solution), adding a tungsten disulfide nanosheet solution of 0.3mg/mL, incubating for 20min at room temperature, adding three mycotoxin solutions of different concentrations of 0.001-100 ng/mL into the mixture to enable the final volume to be 200 mu L, incubating for 40min at 37 ℃, detecting time-resolved fluorescence signal values of the solutions at 488nm, 544nm and 619nm after the incubation is finished, wherein the time-resolved fluorescence signal values respectively correspond to zearalenone, T-2toxin and aflatoxin B1The time-resolved fluorescence signal is gradually enhanced along with the increase of the toxin concentration, and a standard is established according to the fluorescence value and the corresponding standard substance concentrationThe curve:
s2, sample detection: adding the sample to be detected to KYF identical to that in step S14:Dy3+-AptZEN、KYF4:Tb3+-AptT-2And KYF4:Eu3+-AptAFB1Incubating the mixed solution of the fluorescent probe and the tungsten disulfide nanosheet for 40min at 37 ℃, detecting time-resolved fluorescence signal values of the solution at 488nm, 544nm and 619nm after the incubation is finished, and obtaining zearalenone, T-2toxin and aflatoxin B according to a standard curve1The concentration of (c).
The solution fluorescence detection adopts a BioTek Synergy H1 enzyme-labeling instrument, and the parameters are set as an excitation wavelength 273nm, the delay time is 100 mus, and the detection time is 1000 mus.
The method for simultaneously detecting three mycotoxins based on time-resolved fluorescence labeling-aptamer recognition is used for zearalenone, T-2toxin and aflatoxin B in corn, wheat, grains, feed and products thereof1Application in detection.
Simultaneous detection of zearalenone, T-2toxin and aflatoxin B based on time-resolved fluorescence labeling-aptamer recognition1The method of (1): respectively preparing three lanthanum-doped time-resolved fluorescent nano materials (KYF)4:Dy3+、KYF4:Tb3+And KYF4:Eu3+) Performing surface avidin modification, and respectively labeling three mycotoxins (zearalenone, T-2toxin and aflatoxin B) through the action between avidin and biotin1) Specific aptamer of (Apt)ZEN、AptT-2And AptAFB1) Time-resolved fluorescent nanoparticle KYF modified by three aptamers4:Dy3+-AptZEN、KYF4:Tb3+-AptT-2And KYF4:Eu3+-AptAFB1A fluorescent probe. WS2 was then added to the system to act as a fluorescence quencher, WS in the absence of target2The mixed bio-probe can be easily adsorbed by van der waals force, thereby causing fluorescence quenching of the bio-probe. In the presence of the target, the target will interact withAptamers bind and alter spatial conformation of aptamers, thereby attenuating DNA binding to WS2The interaction between the two biological probes enables the fluorescence of the biological probes to be recovered. Ultraviolet light is used for excitation, time-resolved fluorescence is induced to serve as a detection signal, a standard curve is established through detection of toxin standard substances with different concentrations, and the purpose of quantitative detection of toxin-containing sample substances is achieved.
The beneficial technical effects of the invention are as follows:
1, the characteristic of long fluorescence life of the lanthanide doped nano material is utilized, and the fluorescence of the detection background is attenuated through time resolution, so that the detection sensitivity is greatly improved.
2, use of WS having higher fluorescence quenching efficiency2As a fluorescence quencher, the sensitivity of detection is further improved.
3, the detection accuracy and stability are effectively improved by utilizing the specific binding of the aptamer to the detection substance.
Drawings
FIG. 1 is a graph showing the detection of zearalenone, T-2toxin and aflatoxin B based on time-resolved fluorescence labeling-aptamer recognition1Schematic diagram of (1);
FIG. 2 is a graph of time-resolved fluorescence intensity as a function of zearalenone, T-2toxin and aflatoxin B in example 11A concentration variation overlay (a); zearalenone, T-2toxin and aflatoxin B1And (c) detecting the standard curve chart (b).
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples.
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
Zearalenone, T-2toxin and aflatoxin B in corn sample1Detection and spiking recovery of
1. Amine functionalized Ln3+Doped KYF4Nano-particlesPreparation of pellets
Ln3+(Ln3+＝Dy3+，Tb3+And Eu3+) Doped KYF4The nanoparticles are prepared by a simple one-step solvothermal method: adding KCl (2.0mmol), YCl3(1.0mmol) and the required amount of LnCl3(0.01mmol Dy3+，0.1mmol Tb3+And 0.1mmol Eu3+) And PEI (1mL) was added to ethylene glycol (20mL) and mixed well. The solution was then added to a solution containing NH in a water bath at 45 deg.C4F (6.0mmol) in ethylene glycol (20 mL). The reaction was stirred at room temperature for a further 30min, after which the mixture was transferred into a 50mL autoclave and reacted at 200 ℃ for 4 h. And after the reaction is finished, taking out the reaction kettle, naturally cooling to room temperature, collecting nanoparticles through centrifugation, washing the nanoparticles three times by adopting ultrapure water and absolute ethyl alcohol in turn, drying the obtained powder at 50 ℃ for 12 hours, grinding the powder into fine powder by using a mortar, and storing the fine powder for later use.
2. Aptamer-modified KYF4:Ln3+Preparation of multicolor bioprobes
Distinguishing the time fluorescence from the nano material KYF4:Ln3+(Ln3+＝Dy3+、Tb3+And Eu3+) The nanoparticles were resuspended in 10mM Phosphate Buffer Solution (PBS) to 1mg/mL, and sonicated for 15min to disperse well in the buffer. The material was activated with 5% glutaraldehyde by gentle shaking at room temperature for 2h in the dark, and unreacted glutaraldehyde was removed by centrifugation, followed by washing three times with PBS, followed by addition of avidin (1mg/mL) and shaking at 37 ℃ overnight. After the reaction, the reaction mixture was washed several times and the supernatant was discarded.
KYF surface-modified Avidin (Biotin) by utilizing specific binding between Avidin (Avidin) and Biotin (Biotin)4:Ln3+(Ln3+＝Dy3+、Tb3+And Eu3+) The nanoparticles were linked to biotin-modified aptamers (AptZEN, AptT-2 and AptAFB1), respectively. The specific method comprises the following steps: dispersing 5mg of avidin-modified time-resolved fluorescent nano material in 5mL (10mM) of PBS, respectively adding a certain amount of biotinylated aptamer, incubating for 12h in a shaker at 37 ℃, centrifugally collecting and washing with PBS for three times to obtain the aptamer modifierDecorated time-resolved fluorescent nanoparticles KYF4:Dy3+-AptZEN、KYF4:Tb3+-AptT-2And KYF4:Eu3+-AptAFB1A fluorescent probe.
3. Detecting zearalenone, T-2toxin and aflatoxin B1 standard substances and establishing a standard curve:
and (2) simply treating the sample, directly adding the sample into the mixed solution containing the aptamer modified multiple biological probes and WS2, incubating for 40min at 37 ℃, exciting at 273nm to obtain fluorescence signals at 488nm, 544nm and 619nm in a time-resolved fluorescence mode of a microplate reader, and obtaining the corresponding concentrations of zearalenone, T-2toxin and aflatoxin B1 from a standard curve.
(a) 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.
(b) Preparation of standard curve and sample: respectively adding 0.05, 0.5, 5 and 50ng/mL zearalenone, T-2toxin and aflatoxin B1 standard substances into the sample to be detected, and detecting the content of zearalenone, T-2toxin and aflatoxin B1 again by the method to obtain a detection value shown in figure 2. Recovery (%) (value detected-background)/addition amount × 100%. From the results in the table I, the recovery rate is 87.0-98.4%, which shows that the method is stable, sensitive and accurate, and is suitable for detecting zearalenone, T-2toxin and aflatoxin B1 in a corn sample.
Table one: detection and standard recovery rate of zearalenone, T-2toxin and aflatoxin B1 in corn sample
The zearalenone aptamer AptZENThe sequence is 5 '-biotin-TCA TCT ATC TAT GGT ACA TTA CTA TCT GTA ATG TGA TAT G-3'; t-2toxin aptamer AptT-2The sequence is 5 '-biotin-CAG CTC AGA AGC TTG ATC CTG TAT ATC AAG CAT CGC GTG TTT ACA CAT GCG AGA GGT GAA GAC TCG AAG TCG TGC ATC TG-3'; aflatoxin B1Aptamer AptAFB1The sequence is 5 '-biotin AGC AGC ACA GAG GTC AGA TGG TGC TAT CAT GCG CTC AAT GGG AGA CTT TAG CTG CCC CCA CCT ATG CGT GCT ACC GTG AA-3'.
Claims (5)
1. A method for simultaneously detecting three mycotoxins based on time-resolved fluorescence labeling-aptamer recognition, the detection method comprising: respectively using biotinylated zearalenone aptamer Apt to time-resolved fluorescent nanoparticlesZENT-2toxin aptamer AptT-2And aflatoxin B1Aptamer AptAFB1Modification to obtain KYF4:Dy3+-AptZEN、KYF4:Tb3+-AptT-2And KYF4:Eu3+-AptAFB1Detecting the fluorescence signal intensity of the mixture in the presence of mycotoxin by using a fluorescent probe and using tungsten disulfide nanosheets as a fluorescence quencher;
the three mycotoxins are zearalenone, T-2toxin and aflatoxin B1；
The time-resolved fluorescent nanoparticle is KYF4:Dy3+、KYF4:Tb3+And KYF4:Eu3+。
2. The method for the simultaneous detection of three mycotoxins based on time-resolved fluorescence label-aptamer recognition of claim 1, wherein said KYF4:Dy3+-AptZEN、KYF4:Tb3+-AptT-2And KYF4:Eu3+-AptAFB1The preparation method of the fluorescent probe comprises the following steps:
step 1, time-resolved fluorescence nanoparticlesPerforming avidin modification: resolving the time fluorescence of the nano material KYF with the concentration of 1mg/mL4:Dy3+、KYF4:Tb3+And KYF4:Eu3+Respectively activating the solution and glutaraldehyde by light-shielding slight oscillation at room temperature for 2h, removing unreacted glutaraldehyde, washing with PBS at least three times, adding avidin with the concentration of 1mg/mL, oscillating at 37 ℃ overnight, washing after the reaction is finished, taking a solid phase, and obtaining the avidin-modified time fluorescence resolution nano material AV-KYF4:Dy3+、AV-KYF4:Tb3+And AV-KYF4:Eu3+；
Step 2, combining the time-resolved fluorescent nanoparticles with the aptamers: preparing the avidin modified time fluorescence resolution nano material AV-KYF prepared in the step 14:Dy3+、AV-KYF4:Tb3+And AV-KYF4:Eu3+Dispersed in PBS buffer and biotinylated aptamer Apt addedZEN、AptT-2And AptAFB1Incubating in a shaker at 37 ℃ for 12h, collecting the solid phase and washing with PBS at least three times to finally obtain the aptamer-modified time-resolved fluorescent nanoparticle KYF4:Dy3+-AptZEN、KYF4:Tb3 +-AptT-2And KYF4:Eu3+-AptAFB1A fluorescent probe.
3. The method for simultaneously detecting three mycotoxins based on time-resolved fluorescence labeling-aptamer recognition according to claim 1, wherein the zearalenone aptamer AptZENThe sequence is 5 '-biotin-TCA TCT ATC TAT GGT ACA TTA CTA TCT GTA ATG TGA TAT G-3'; t-2toxin aptamer AptT-2The sequence is 5 '-biotin-CAG CTC AGA AGC TTG ATC CTG TAT ATC AAG CAT CGC GTG TTT ACA CAT GCG AGA GGT GAA GAC TCG AAG TCG TGC ATC TG-3'; aflatoxin B1Aptamer AptAFB1The sequence is 5 '-biotin AGC AGC ACA GAG GTC AGA TGG TGC TAT CAT GCG CTC AAT GGG AGA CTT TAG CTG CCC CCA CCT ATG CGT GCT ACC GTG AA-3'.
4. The method for simultaneously detecting three mycotoxins based on time-resolved fluorescence labeling-aptamer recognition according to claim 1, wherein the detection method specifically comprises:
s1, establishing a standard curve: KYF with final concentration of 1mg/mL4:Dy3+-AptZEN、KYF4:Tb3+-AptT-2And KYF4:Eu3+-AptAFB1Mixing a fluorescent probe in a PBS (phosphate buffer solution), adding a tungsten disulfide nanosheet solution of 0.3mg/mL, incubating for 20min at room temperature, adding three mycotoxin solutions of different concentrations of 0.001-100 ng/mL into the mixture to enable the final volume to be 200 mu L, incubating for 40min at 37 ℃, detecting time-resolved fluorescence signal values of the solutions at 488nm, 544nm and 619nm after the incubation is finished, wherein the time-resolved fluorescence signal values respectively correspond to zearalenone, T-2toxin and aflatoxin B1The time-resolved fluorescence signal is gradually enhanced along with the increase of the toxin concentration, and a standard curve is established according to the fluorescence value and the corresponding standard substance concentration:
s2, sample detection: adding the sample to be detected to KYF identical to that in step S14:Dy3+-AptZEN、KYF4:Tb3+-AptT-2And KYF4:Eu3+-AptAFB1Incubating the mixed solution of the fluorescent probe and the tungsten disulfide nanosheet for 40min at 37 ℃, detecting time-resolved fluorescence signal values of the solution at 488nm, 544nm and 619nm after the incubation is finished, and obtaining zearalenone, T-2toxin and aflatoxin B according to a standard curve1The concentration of (c).
5. The method for simultaneously detecting three mycotoxins based on time-resolved fluorescence labeling-aptamer recognition according to claim 4, wherein the solution fluorescence detection is performed by using a BioTek Synergy H1 enzyme-labeled instrument, and the parameters are set to 273nm of excitation wavelength, 100 μ s of delay time and 1000 μ s of detection time.
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