Method for rapid detection of ZEN toxins

Abstract

The present invention relates to the field of detection technology, and discloses a colorimetric aptamer sensor for rapid detection of ZEN toxin, comprising a zearalenone nucleic acid aptamer and nanogold. The colorimetric aptamer sensor performs ZEN detection as follows: a nanogold solution and a zearalenone nucleic acid aptamer solution react at room temperature, then a zearalenone toxin standard sample of different concentrations is added for incubation, and then a sodium chloride solution is added and incubated at room temperature, and a standard curve is established with the absorbance value as the vertical coordinate and the zearalenone concentration as the horizontal coordinate. The present invention constructs a simple, rapid, visual, label-free colorimetric biosensor, which only takes 15 minutes to complete the ZEN detection process, and can easily detect ZEN in grains. It is low-cost and provides a new approach for on-site detection of ZEN in grains.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a novel colorimetric aptamer sensor based on aptamers and gold nanoparticles for rapid detection of ZEN toxins and a detection method thereof. Background Art

[0002] Zearalenone (ZEN), also known as F-2 toxin, is a secondary metabolite primarily produced by fungi such as Fusarium and Fusarium luteum. It is widely distributed in corn, wheat, barley, sorghum, rye, and other cereals. Previous studies have shown that ZEN can damage the immune, nervous, and reproductive systems of humans and animals, posing a serious threat to food security and human health. Many countries and organizations have established maximum residue limits (MRLs) for ZEN in agricultural products. For example, my country stipulates a maximum residue limit of 60 μg / kg for wheat, corn, and their powdered products; 500 μg / kg for corn raw materials and processed products; and 100 μg / kg for mixed feed for piglets and sows. The European Union stipulates a maximum residue limit of 75 μg / kg for cereals and products; France stipulates a maximum residue limit of 200 μg / kg for cereals and rapeseed oil; and Austria stipulates a maximum residue limit of 60 μg / kg for wheat. Therefore, establishing effective and accurate methods for detecting ZEN in various foods is crucial to protecting public health.

[0003] Currently, there are many conventional methods for detecting zearalenone, primarily GC-MS for identifying zearalenol used in food production and zearalenone contamination caused by Fusarium spp. LC-MS / MS can detect the content and related structures of hundreds of mycotoxins. While these methods offer high sensitivity and accuracy, they often require tedious sample pretreatment, sophisticated instrumentation, and skilled personnel. Immunosorbent assays are also simple, rapid, and effective methods for detecting mycotoxins, including enzyme-linked immunosorbent assays, electrochemical immunoassays, fluorescence immunoassays, microchip-based assays, electromigration-based assays, and surface plasmon resonance immunoassays. However, these immunoassays rely heavily on the use of antibodies. Antibody production through animal immunization is typically time-consuming, expensive, and sensitive. Furthermore, the kits are susceptible to inactivation during storage and transportation, making them difficult to preserve.

[0004] Aptamers are single-stranded oligonucleotide (ssDNA or ssRNA) sequences with high affinity and specificity for target molecules. Aptamers are considered the most promising alternative to antibodies due to their simple in vitro synthesis, lack of animal-required methods, broad target range, high affinity and specificity, ease of storage and modification, low cost, high stability, and reusability. Due to their unique properties, aptamers can be combined with a variety of technologies and widely used in targeted therapy, disease detection, food safety testing, and biomedicine. Biosensors developed using aptamers as recognition elements have advantages such as increased sensitivity and specificity, shorter detection times, and on-site testing, and are therefore increasingly popular among researchers. Currently, a series of aptamer-based biosensors have been developed for the detection of mycotoxins, primarily for ochratoxin A and aflatoxin B1. However, research on the rapid detection of ZEN using aptamer-based biosensors is limited. In recent years, aptamer-based colorimetric sensing methods have become an alternative method for detecting various target molecules due to their simplicity, low cost, high sensitivity, and ability to specifically detect without complex instruments. In this detection method, aptamers (single-stranded DNA or RNA oligonucleotides) are used as recognition elements because they can selectively bind to target molecules, which helps to improve the specificity of the sensor. In colorimetric sensors, gold nanoparticles (AuNPs) are widely used as colorimetric indicators due to their high color correspondence and controllable shape. Dispersed AuNP solutions are wine red. However, the aggregation of AuNPs increases the size of the particles, causing the solution to change significantly to purple or blue. In addition, AuNPs have been used in combination with other nanomaterials (e.g., reduced graphene oxide) or combined with aptamers to improve sensor performance or increase detection sensitivity. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a colorimetric aptamer sensor and a detection method for rapid detection of ZEN toxins.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions:

[0007] A colorimetric aptasensor for rapid detection of ZEN toxins, comprising zearalenone nucleic acid aptamer and gold nanoparticles.

[0008] Preferably, the zearalenone nucleic acid aptamer is a zearalenone nucleic acid aptamer solution with a concentration of 5 umol / L.

[0009] Preferably, the gold nanoparticles are in the form of a solution, and the gold nanoparticle solution is prepared by stirring and heating ultrapure water and chloroauric acid solution with a volume ratio of 100:1 to boiling, adding a sodium citrate solution with a mass fraction of 1%, wherein the volume ratio of the sodium citrate solution to ultrapure water is 2.5:100, and continuously stirring. When the color of the solution no longer changes, stop heating and continue stirring for 10 minutes, cool to room temperature, and refrigerate for later use.

[0010] Furthermore, the colorimetric aptamer sensor for rapid detection of ZEN toxin performs the following ZEN detection method: the nanogold solution and the zearalenone nucleic acid aptamer solution are reacted at room temperature, and then different concentrations of zearalenone toxin standard samples are added for incubation, and then sodium chloride solution is added and incubated at room temperature, and a standard curve is established with the absorbance value as the vertical coordinate and the zearalenone concentration as the horizontal coordinate; the product to be tested is processed and a solution containing ZEN is extracted, and the obtained solution is added to the solution incubated with the nanogold solution and the ZEN nucleic acid aptamer, and after further incubation, sodium chloride solution is added for continued incubation, the absorbance value is detected, and substituted into the standard curve to calculate the ZEN content in the product.

[0011] Preferably, the concentration of the sodium chloride solution is 2 mol / L.

[0012] Preferably, the volume ratio of the nanogold solution, the zearalenone nucleic acid aptamer solution and the sodium chloride solution is 100:10:10.

[0013] Preferably, the reaction time of the nanogold solution and the zearalenone nucleic acid aptamer solution at room temperature is 5 minutes.

[0014] Preferably, different concentrations of zearalenone toxin standard samples are added and incubated for 5 minutes.

[0015] Preferably, the sodium chloride solution is added and incubated at room temperature for 5 minutes.

[0016] Furthermore, 100 uL of AuNPs solution was added to the microplate, followed by 10 uL of 5 umol / L ZEN aptamer solution, and the mixture was incubated at room temperature for 5 min. Then, 10 uL of extraction solution was added to obtain a ZEN-containing solution. After incubation at room temperature for 5 min, 10 uL of 2 mol / L sodium chloride solution was added and incubated at room temperature for 5 min. The absorbance was then detected using a multimode plate reader.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention constructs a simple, rapid, visual, label-free colorimetric biosensor using nucleic acid aptamers as the recognition element and gold nanoparticles as the indicator. By adding a certain amount of sodium chloride and observing the color change of the gold nanoparticles, ZEN can be sensitively detected. This colorimetric aptamer sensor can detect ZEN in just 15 minutes and easily detects ZEN in grain. It requires no expensive instrumentation or aptamer modification, resulting in a low-cost, novel approach for on-site ZEN detection in grain. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0020] Figure 1 This is a schematic diagram of the detection principle of the present invention;

[0021] Figure 2 This is the UV-visible absorption spectrum of the nano-gold solution;

[0022] Figure 3 This is a transmission electron microscopy image of a nano-gold solution, where scale A is 100 nm and scale B is 5 nm;

[0023] Figure 4 (A) UV spectra of AuNPs under different NaCl concentrations; 4(B) Values of AuNPs at A650nm under different NaCl concentrations;

[0024] Figure 5 is the absorbance value at different aptamer concentrations;

[0025] Figure 6 is the absorbance value of the nanogold solution and aptamer at different reaction times;

[0026] Figure 7 is the linear relationship between different concentrations of ZEN and A650; where A is the change in the absorbance of the AuNPs solution at A650nm with increasing ZEN concentration, and B is the linear relationship between the absorbance at A650nm and the ZEN concentration in the ZEN concentration range of 5-300 ng / mL;

[0027] Figure 8 The absorbance value diagram measured under different interferences; DETAILED DESCRIPTION

[0028] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0029] Example 1

[0030] Main Materials:

[0031] Zearalenone aptamer (5′-GAT GGG GAA AGG GTC CCC CTG GGT TGG AGCATC GGA CA-3′): Zearalenone standard (ZEN) synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0032] Aflatoxin B1 standard (AFB1), deoxynivalenol (DON), and T-2 toxin: Qingdao Puruibang Bioengineering Co., Ltd.

[0033] Chloroauric acid (HAuCl4): Sinopharm Chemical Reagent Co., Ltd., ZEN enzyme-linked immunosorbent assay kit: Shanghai Kuailing Biotechnology Co., Ltd.;

[0034] Corn kernels: purchased at the supermarket;

[0035] Sodium citrate, potassium dichromate, acetonitrile, sodium chloride, Shanghai MacLean Biochemical Technology Co., Ltd.;

[0036] The water used in the experiment was ultrapure water (resistivity of 18.2 MΩ.cm). All organic solvents in the experiment were chromatographically pure, and other experimental materials were analytically pure.

[0037] Main equipment:

[0038] VICTOR Nivo multi-mode plate reader: PerkinElmer Business Management (Shanghai) Co., Ltd.;

[0039] UV spectrophotometer: Hanyi Instrument (Shanghai) Co., Ltd.

[0040] JEM-2100F transmission electron microscope: JEOL Ltd.;

[0041] KQ-500DE ultrasonic cleaner: Kunshan Ultrasonic Instrument Co., Ltd.

[0042] Vortex-Genie 2 vortex mixer: Scientific Industries, USA;

[0043] DFY-500 high-speed crusher: Jinsui Machinery Manufacturing Plant, Yongkang City, Zhejiang Province;

[0044] TH2-82T water bath constant temperature oscillator: Changzhou Ronghua Instrument Co., Ltd.

[0045] A colorimetric aptamer sensor for rapid detection of ZEN toxins comprises a zearalenone nucleic acid aptamer and gold nanoparticles. The zearalenone nucleic acid aptamer in the present invention is prior art and can be obtained by those skilled in the art through commercial purchase or other methods.

[0046] The zearalenone nucleic acid aptamer is a zearalenone nucleic acid aptamer solution with a concentration of 5 umol / L.

[0047] The gold nanoparticles (AuNPs) solution was prepared as follows:

[0048] The sodium citrate reduction method was used to prepare an AuNP solution. Specifically, 100 mL of ultrapure water and 1.00 mL of 1% chloroauric acid solution were added to a 250 mL beaker that had been pre-soaked in chromic acid solution. Stir and heat to boiling. Then, 2.5 mL of 1% sodium citrate solution was added and rapidly stirred for 10 minutes. During continued stirring, the solution color changed from light yellow to black, then from black to purple-red, and finally from purple-red to wine-red, becoming transparent. When the solution color stopped changing, heating was stopped and stirring continued for 10 minutes. The solution was then cooled to room temperature, aliquoted, and stored at 4°C until further use. The resulting gold nanoparticle solution was characterized by UV spectrophotometry to determine characteristic peaks and absorbance values, and quality was assessed. Transmission electron microscopy was used to observe the prepared gold nanoparticles.

[0049] The prepared AuNPs solution was wine red with uniform color distribution and no precipitation. The size, shape and uniformity of AuNPs particles were characterized by scanning 300-700nm UV-visible absorption spectrum and transmission electron microscopy. Figure 2 and Figure 3 The prepared gold nanoparticles solution has a single absorption peak at 520 nm, with no other peaks. Transmission electron microscopy shows that the prepared gold nanoparticles solution is relatively uniform and dispersed, and the prepared AuNPs have a relatively consistent particle size of 15 nm and are evenly dispersed spherical.

[0050] Gold nanoparticles exhibit different colors in their aggregated and dispersed states. Synthesized gold nanoparticles appear red in a dispersed state, but blue when added to a high-concentration salt solution due to aggregation. In the absence of ZEN toxins in the sample solution, ZEN aptamers electrostatically adsorb onto the surface of AuNPs, preventing aggregation and maintaining a stable dispersion in the presence of high-concentration salt solutions, resulting in a red appearance. When the target ZEN is present in the solution, ZEN specifically binds to the aptamers, causing the gold nanoparticles, now in a salty environment without the protection of the aptamers, to aggregate, accompanied by a color change from red to blue. The greater the amount of ZEN, the more pronounced the aggregation, color change, and absorbance shift. Therefore, the color changes and absorbance shifts of the sample solution under different conditions can be used to determine the state of the gold nanoparticles, enabling qualitative and quantitative detection of ZEN in corn samples.

[0051] Sodium chloride concentration optimization:

[0052] 100 μL of AuNP solution was added to a microplate. Then, 10 μL of sodium chloride solution of varying concentrations (0 mol / L, 0.5 mol / L, 1 mol / L, 1.5 mol / L, 2 mol / L, 2.5 mol / L, and 3 mol / L) was added. After reacting at room temperature for 5 minutes, the color change of the gold nanoparticles was observed visually. The absorbance of the AuNPs was measured using a multimode plate reader to determine the degree of aggregation and to determine the optimal NaCl concentration.

[0053] Appropriate sodium chloride concentration can reduce background interference and improve detection sensitivity. However, AuNPs are particularly sensitive to sodium chloride solutions. In a certain concentration of sodium chloride solution, they can aggregate and their color changes from wine red to blue. In other words, the concentration of sodium chloride directly affects the degree of aggregation of AuNPs. Figure 4 A It can be seen that with the addition of sodium chloride, the peak of AuNPs appears at A650nm, and the absorbance value at this point also increases with the increase of sodium chloride concentration. On the contrary, the peak at A520nm gradually decreases. Therefore, the present invention selects the absorbance value at A650nm to measure the aggregation degree of AuNPs. Figure 4 B shows that AuNPs hardly aggregate in sodium chloride solution with a concentration of 0-1 mol / L, and the difference cannot be seen with the naked eye, and they still maintain a stable red color. However, when the sodium chloride concentration is 1.5-3 mol / L, AuNPs solution begins to aggregate. As the sodium chloride concentration increases, the absorbance at A650nm continues to increase. When the sodium chloride concentration reaches 2 mol / L, the absorbance at A650nm tends to be stable. In order to avoid the influence of excessive sodium chloride, 2 mol / L was finally selected as the optimal sodium chloride concentration based on the UV spectrum and color changes.

[0054] Aptamer concentration optimization:

[0055] 100 μL of AuNPs solution was added to each microplate, followed by 10 μL of ZEN aptamer solution at different concentrations (0 μmol / L, 1 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 7 μmol / L, and 9 μmol / L). The mixture was incubated at room temperature for 5 minutes. 10 μL of 2 mol / L sodium chloride solution was added to the reaction solution and allowed to react at room temperature for 5 minutes. The color change was observed visually and the absorbance was measured using a multimode plate reader to determine the optimal aptamer concentration. Three parallel control groups were set up in the experiment.

[0056] Aptamers can protect AuNPs from agglomerating under high salt conditions to a certain extent, but the more aptamers there are, the better. If the amount of aptamers adsorbed on AuNPs is too much, the AuNPs will not agglomerate and change color in high salt concentration solutions, affecting the color change of the entire system and increasing costs. A small amount of nucleic acid aptamers will not play a protective role. Therefore, exploring the optimal concentration of nucleic acid aptamers is crucial to the entire invention. The results are as follows: Figure 5 As shown, when the aptamer concentration is 0-4umol / L, AuNPs are in an aggregated state and the aptamer cannot prevent salt-induced AuNPs aggregation. When the aptamer concentration is 5umol / L, AuNPs turn red, indicating that the nucleic acid aptamer has a good salt-induced protection effect on AuNPs. Correspondingly, the absorbance value at A650nm shows an increasing trend, and after the nucleic acid aptamer concentration reaches 5umol / L, the absorbance value at A650nm tends to be stable, indicating that the aptamer binding to AuNPs has been saturated.

[0057] Optimization of incubation time of AuNPs and aptamers:

[0058] To ensure full binding of the aptamer to the AuNPs, the incubation time between AuNPs and the aptamer was optimized. 100 μL of AuNP solution was added to each microplate, followed by 10 μL of 5 μmol / L ZEN aptamer and incubation at room temperature for 5 minutes. 10 μL of 30 ng / mL ZEN toxin standard was added and incubated at room temperature for 5 minutes. After that, 10 μL of 2 mol / L sodium chloride solution was added and incubated for 0, 5, 10, 15, 20, 25, and 30 minutes. The absorbance was measured using a multimode plate reader. Three parallel control groups were set up in the experiment.

[0059] like Figure 6As shown, the absorbance at A650nm is maximum when the reaction time is 5 minutes. If the reaction time is less than 5 minutes, the reaction is not complete and the color of the reaction system remains red. As the reaction time increases, the absorbance at A650nm decreases, and the color of the reaction system gradually degrades and becomes lighter. Therefore, the optimal incubation time is 5 minutes.

[0060] Linear relationship:

[0061] The colorimetric aptamer sensor for rapid detection of ZEN toxins was used for ZEN detection as follows: 100 μL of AuNPs solution was added to the microplate, followed by 10 μL of 5 μmol / L ZEN aptamer solution, incubated at room temperature for 5 minutes, followed by 10 μL of ZEN toxin standard samples with different concentrations (0 ng / mL, 5 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL, 350 ng / mL, 400 ng / mL) and incubated for 5 minutes, followed by 10 μL of 2 mol / L sodium chloride solution, incubated at room temperature for 5 minutes. Three parallel control groups were set up in the experiment, and a standard curve was established with the absorbance value as the vertical axis and the ZEN concentration as the horizontal axis.

[0062] like Figure 7 As shown in A, the absorbance of AuNPs solution at A650nm increases with the increase of ZEN concentration. However, when the ZEN concentration reaches 300 ng / mL, the absorbance of AuNPs at A650nm decreases with the increase of ZEN concentration. Figure 7 As shown in B, within the concentration range of 5-300 ng / mL, the absorbance at A650nm showed a good linear relationship with the ZEN concentration. The linear equation was y = 0.0003x + 0.5128, and the correlation coefficient was R = 0.9989. The blank control was measured 9 times under the optimized conditions, and the detection limit of this method was determined to be 5 ng / ml.

[0063] Comparing the method of the present invention with the existing reported detection methods (Table 1), the present invention has the advantages of being rapid, low cost, intuitive and visual, having a low detection limit, and a wide linear range, and does not require expensive instruments or modification of the aptamer.

[0064] Table 1 Comparison of detection methods for the determination of zearalenone

[0065]

[0066] Specificity test:

[0067] In order to verify whether the method of the present invention can specifically identify ZEN, specific detection was performed.

[0068] Add 100 μL of AuNPs solution to each microplate, followed by 10 μL of 5 μmol / L ZEN aptamer solution and incubation at room temperature for 5 minutes. Then, add 10 μL of 30 ng / mL DON, ZEN, T-2, or AFB1 toxin standards and incubate at room temperature for 5 minutes. Then, add 10 μL of 2 mol / L sodium chloride solution and incubate at room temperature for 5 minutes. Color changes were observed visually and absorbance was measured using a multimode plate reader. Three parallel control groups were included in the experiment.

[0069] like Figure 8 As shown, when ZEN and other toxins were tested under the optimal conditions, only the system solution with ZEN added changed from red to blue, and its absorbance at A650nm was significantly higher than that of other toxins, indicating that the detection method of the present invention has good specificity.

[0070] Corn sample testing and verification

[0071] Corn kernels were crushed and passed through an 80-mesh sieve. 20 g of the extract was placed in a conical flask and added to 100 mL of an extract solution (acetonitrile:water = 90:10). The extract was shaken in a 45°C waterbath for 1 hour. The extract was first filtered through qualitative filter paper and then through a 0.45 μm microporous membrane filter. The extract was then transferred to a brown volumetric flask for storage. Different concentrations of ZEN standard solutions (10.0, 50.0, and 100 ng / mL) were added to the corn extract to obtain ZEN-containing solutions. Subsequently, 100 μL of the AuNPs solution was added to a microplate, followed by 10 μL of a 5 μmol / L ZEN aptamer solution. The mixture was incubated at room temperature for 5 minutes. Finally, 10 μL of the extracted solution was added to obtain the ZEN-containing solution. After incubation at room temperature for 5 minutes, 10 μL of a 2 mol / L sodium chloride solution was added. After incubation at room temperature for 5 minutes, the absorbance was measured using a multimode plate reader. The spike recovery was performed and the recovery rate was calculated. The results are shown in Table 2. It can be seen that the recovery rate of the actual samples determined by the method of the present invention is 81.3%-96.4%, and the relative standard deviation (RSD) is 1.0%-5.7%. The results show that the aptamer-based colorimetric method of the present invention is feasible for the rapid and specific detection of ZEN levels in actual food samples.

[0072] Table 2 Recovery of ZEN in actual samples (n=5)

[0073]

[0074] As can be seen, the present invention constructs a simple, rapid, visual, and label-free colorimetric aptasensor using ZEN aptamers as recognition elements and gold nanoparticles (AuNPs) as indicators, enabling visual detection of zearalenone in grains. The sensor of the present invention directly binds the ZEN aptamer to an AuNPs solution, then adds the target ZEN and a high-concentration salt solution for detection. No modification is required, making it easy to operate and enabling visual observation of the color change of the AuNPs solution. The sensor of the present invention has a minimum detection limit of 5 ng / mL and a linear range of 5-300 ng / mL. The resulting ZEN standard curve equation is y = 0.0003x + 0.5128 (R = 0.9989). Specificity tests have demonstrated that this method can specifically detect ZEN. The method was used to determine the specificity of ZEN in corn samples, with recoveries ranging from 81.3% to 96.4%. The entire detection process takes only 15 minutes, indicating that the present method can be applied to the detection of real-world samples.

[0075] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for rapid detection of ZEN toxins, characterized in that: The following steps are involved: 1) The gold nanoparticle solution and 5 μmol / L zearalenone (ZEN) aptamer solution were reacted at room temperature for 5 min. 2) The product to be tested is processed to extract a test sample solution containing zearalenone, and the obtained test sample solution is added to the solution incubated with the nanogold solution and the ZEN nucleic acid aptamer and further incubated for 5 minutes; 3) After further incubation, add 2 mol / L sodium chloride solution and continue incubation for 5 minutes; 4) Detect the absorbance value at a wavelength of 650 nm, substitute it into the standard curve, and calculate the content of ZEN in the product. Wherein, the method for establishing the standard curve is: Replace the sample solution to be tested with ZEN standard sample solutions of different concentrations and repeat steps 1)-4). Draw a standard curve with the absorbance at 650 nm as the ordinate and the ZEN concentration as the abscissa. The nanogold solution is prepared by stirring and heating ultrapure water and a 1% chloroauric acid solution in a volume ratio of 100:1 to boiling, adding a 1% sodium citrate solution in a volume ratio of 2.5:100, and continuously stirring. When the color of the solution no longer changes, heating is stopped and stirring is continued, followed by cooling to room temperature and refrigeration.

2. The method for rapid detection of ZEN toxin according to claim 1, characterized in that: The volume ratio of the gold nanoparticle solution, the zearalenone nucleic acid aptamer solution and the sodium chloride solution is 100:10:

10.

3. The method for rapid detection of ZEN toxin according to claim 1, characterized in that: 100 μL of AuNPs solution was added to the microplate, followed by 10 μL of 5 μmol / L ZEN aptamer solution. The plates were incubated at room temperature for 5 min, and then 10 μL of the extracted ZEN solution was added. After incubation at room temperature for 5 min, 10 μL of 2 mol / L sodium chloride solution was added and incubated at room temperature for 5 min. The absorbance was then detected using a multimode plate reader.

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