Preparation method and application of manganese nanoflower-aptamer probe

Abstract

The invention discloses a preparation method and application of a manganese nano flower aptamer probe, and the preparation method comprises the following steps: adding oleic acid to a potassium permanganate solution, performing an oxidation-reduction reaction, and purifying to obtain a manganese nano flower; the manganese nano flower is dispersed in water, an ochratoxin A aptamer solution, a sodium chloride solution, a phosphate buffer solution and water are added, and incubated after mixing to obtain a manganese nano flower aptamer probe. The probe proposed in the present invention has a strong catalytic activity to a chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB), can rapidly catalyze the oxidation of the chromogenic substrate and produce a color change, therefore, the chromogenic substrate can be used to detect ochratoxin A by colorimetry or ultraviolet absorption spectrophotometry, and the detection accuracy and detection efficiency of ochratoxin A can be further improved by using the method.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and in particular to a preparation method of a manganese nanoflower-aptamer probe and application thereof. Background Art

[0002] Ochratoxin A (OTA) is a mycotoxin that is mainly found in foods such as grains, nuts, soybeans, milk, juice, wine, beer, and cooking oil, and has a wide range of contamination. Due to the carcinogenic, teratogenic, mutagenic, and immunosuppressive properties of OTA, excessive intake of OTA may lead to acute or chronic disease attacks, or even death. Therefore, it is necessary to strictly control and detect the content of ochratoxin A in agricultural products and foods.

[0003] At present, common OTA detection technologies include high performance liquid chromatography, mass spectrometry, fluorescence spectroscopy, electrochemical method and Raman enhanced spectroscopy, etc. Most of these methods have problems such as cumbersome operation, long time consumption and high price, and cannot be widely used in rapid on-site detection of OTA. Therefore, the development of fast, simple, sensitive and low-cost detection technology has attracted more and more attention from researchers. Summary of the invention

[0004] The main purpose of the present invention is to propose a preparation method and application of a manganese nanoflower-aptamer probe, aiming to provide a manganese nanoflower-aptamer probe, which is helpful for the rapid detection of ochratoxin A.

[0005] To achieve the above object, the present invention proposes a method for preparing a manganese nanoflower-aptamer probe, comprising the following steps:

[0006] Add oleic acid to potassium permanganate solution to carry out redox reaction and purify manganese nanoflowers;

[0007] The manganese nanoflowers are dispersed in water, and ochratoxin A aptamer solution, sodium chloride solution, phosphate buffer solution and water are added, mixed and incubated to obtain a manganese nanoflower-aptamer probe.

[0008] Optionally, oleic acid is added to a potassium permanganate solution to carry out a redox reaction, and in the step of purifying to obtain manganese nanoflowers, 8 to 15 ml of oleic acid is added for every gram of potassium permanganate.

[0009] Optionally, in the step of adding oleic acid to a potassium permanganate solution to carry out a redox reaction and purifying to obtain manganese nanoflowers, the redox reaction time is 40 to 55 hours.

[0010] Optionally, the step of adding oleic acid to a potassium permanganate solution to perform a redox reaction and purifying to obtain manganese nanoflowers comprises:

[0011] adding oleic acid to a potassium permanganate solution to carry out an oxidation-reduction reaction to obtain a reaction solution;

[0012] The reaction solution is centrifuged, the precipitate is collected, and washed with ultrapure water and ethanol to obtain manganese nanoflowers.

[0013] Optionally, in the step of dispersing the manganese nanoflowers in water, adding ochratoxin A aptamer solution, sodium chloride solution, phosphate buffer and water, mixing and incubating to obtain a manganese nanoflower-aptamer probe, the concentration of the ochratoxin A aptamer solution is 0.8~1.2μmol / L.

[0014] Optionally, 2.2-2.8 L of the ochratoxin A aptamer solution is added per gram of potassium permanganate.

[0015] Optionally, the manganese nanoflowers are dispersed in water, ochratoxin A aptamer solution, sodium chloride solution, phosphate buffer and water are added, mixed and incubated to obtain a manganese nanoflower-aptamer probe, and the incubation time is 0.8~1.2h.

[0016] In addition, the present invention also proposes an application of a manganese nanoflower-aptamer probe prepared by the preparation method of the manganese nanoflower-aptamer probe as described above in the detection of ochratoxin A.

[0017] In the technical solution provided by the present invention, a honeycomb manganese nanoflower material is prepared using potassium permanganate as a manganese precursor, and then an ochratoxin A aptamer is adsorbed on the surface of the manganese nanoflower to prepare a probe with high specificity. The probe has a strong catalytic activity to the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB), and can quickly catalyze the oxidation of the chromogenic substrate and produce a color change. Therefore, the chromogenic substrate can be used to detect ochratoxin A by colorimetry or ultraviolet absorption spectrophotometry. The use of this method can further improve the detection accuracy and efficiency of ochratoxin A. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 TEM image of the Mn-NFs material prepared in Example 1;

[0020] Figure 2This is the element mapping analysis diagram of the Mn-OTA-apt probe prepared in Example 1;

[0021] Figure 3 The figure is a comparison of the ultraviolet spectra of the catalytic color development substrate of the Mn-NFs material prepared in Example 1 at different times;

[0022] Figure 4 The UV spectra of the Mn-NFs material prepared in Example 1 catalyzed the color development of the substrate at different temperatures are compared;

[0023] Figure 5 The figure is a comparison of the ultraviolet spectra of the catalytic color-developing substrates of the Mn-NFs material prepared in Example 1 at different concentrations;

[0024] Figure 6 is a particle size distribution diagram of the Mn-NFs material prepared in Example 1;

[0025] Figure 7 is the particle size distribution diagram of the Mn-OTA-apt probe prepared in Example 1;

[0026] Figure 8 It is a potential comparison diagram of OTA-apt, the Mn-NFs material prepared in Example 1, and the Mn-OTA-apt probe;

[0027] Fig. 9 The UV spectra of OTA-apt, the Mn-NFs material prepared in Example 1, and the Mn-OTA-apt probe;

[0028] Fig.10 This is a colorimetric analysis comparison chart of detecting OTA in six samples using the Mn-OTA-apt probe prepared in Example 1.

[0029] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0030] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0031] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is mutually contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0032] In view of this, in order to achieve the above-mentioned purpose, the present invention proposes a method for preparing a manganese nanoflower-aptamer probe, comprising the following steps:

[0033] Step S10, adding oleic acid to the potassium permanganate solution to carry out redox reaction and purify to obtain manganese nanoflowers.

[0034] The present invention uses potassium permanganate (KMnO4) as a manganese precursor to prepare manganese nanoflowers (Manganese Nano Flower, Mn-NFs). The prepared manganese nanoflowers are honeycomb-shaped, have good biocompatibility, are easy to degrade, have high nanozyme activity and high specific surface area, and can quickly catalyze the oxidation of the chromogenic substrate and produce color changes. Taking the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) as an example, the Mn-NFs prepared by the present invention can catalyze TMB to undergo a color reaction, turning blue, and the solution after color development has an obvious ultraviolet absorption peak in the 600-700 nm band. Based on this, the absorbance of this band can be detected by ultraviolet absorption spectrophotometry, and then the TMB content therein can be calculated according to the standard curve. The ultraviolet quantitative detection method used can specifically refer to the conventional ultraviolet measurement method in the art, which will not be described in detail here.

[0035] Specifically, potassium permanganate solution can be prepared by itself. In some embodiments, a certain amount of potassium permanganate is weighed, dissolved in water (which can be distilled water), and stirred rapidly to fully dissolve to obtain a potassium permanganate solution. In this embodiment, a manganese dioxide core is generated at the oil / water interface through a redox reaction between KMnO4 and oleic acid (OA), and then the MnO2 core continuously grows into layered MnO2 lamellae, and finally forms Mn-NFs.

[0036] In this embodiment, the addition amount of the two raw materials follows the following ratio: 8-15 ml of oleic acid is added for every gram of potassium permanganate; the redox reaction time is 40-55 hours. In this way, the reaction can be fully carried out, and the potassium permanganate can be fully reduced to form Mn-NFs of uniform size and honeycomb shape.

[0037] Furthermore, in actual operation, the above step S10 can be performed as follows:

[0038] Step S11, adding oleic acid to a potassium permanganate solution to perform an oxidation-reduction reaction to obtain a reaction solution;

[0039] Step S12, centrifuging the reaction solution, collecting the precipitate, and washing it with ultrapure water and ethanol to obtain manganese nanoflowers.

[0040] Step S20, dispersing the manganese nanoflowers in water, adding ochratoxin A aptamer solution, sodium chloride solution, phosphate buffer and water, mixing and incubating to obtain a manganese nanoflower-aptamer probe.

[0041] The present invention uses a simple incubation method to adsorb ochratoxin A aptamer (OTA-apt) on the surface of manganese nanoflowers to obtain manganese nanoflowers (Mn-OTA-apt) with aptamers adsorbed on the surface. The material not only has stronger catalytic activity than manganese nanoflowers, accelerates and enhances the catalytic color development of TMB, but also when it contacts OTA, the aptamer (OTA-apt) adsorbed thereon can combine with OTA and detach from the probe material, so that the catalytic activity of the material for TMB decreases, thereby causing the color development to become lighter. In this way, by observing the color change of the sample solution, it can be judged whether the sample contains OTA. Specifically, compared with a blank control without OTA, if the sample color is lighter, it means that the sample contains OTA, and if the color is consistent, it means that the sample does not contain OTA; further, when the color of the sample containing OTA is compared, the sample with a lighter color contains a higher content of OTA. In this way, qualitative detection of OTA can be achieved. Based on this, combined with ultraviolet spectrophotometry, the content of OTA can be calculated by detecting the absorbance of the color development solution, so as to achieve quantitative detection of OTA. The detection limit of OTA by the Mn-OTA-apt prepared in the present invention is 0.0102 ng / mL, and the detection range is 0.079-37.62 ng / mL.

[0042] Specifically, in the above step S20, the ochratoxin A aptamer refers to the nucleic acid aptamer of ochratoxin A, which can be purchased on the market. The concentration of the ochratoxin A aptamer solution is 0.8~1.2μmol / L. Based on the ochratoxin A aptamer solution in this concentration range, the amount of addition should be: 2.2~2.8L of the ochratoxin A aptamer solution is added for each gram of potassium permanganate. Considering that the amount of ochratoxin A aptamer solution to be added is large, in actual operation, only a small amount of manganese nanoflower dispersion can be taken for subsequent adsorption steps. For example, based on 0.5g of manganese nanoflowers prepared with potassium permanganate, 250ml of dispersion can be obtained. In order to reduce the consumption of ochratoxin A aptamer solution, only 2μL of dispersion can be taken and incubated with 10μL of ochratoxin A aptamer solution.

[0043] The incubation temperature is preferably 37°C. A suitable system environment needs to be prepared during incubation. In this embodiment, 50 μL of NaCl (1 M) solution, 10 μL of PBS (500 mM, pH 7.5) solution, and 28 μL of ultrapure water are added for incubation. The incubation time is 0.8-1.2 h.

[0044] In the technical solution provided by the present invention, a honeycomb manganese nanoflower material is prepared using potassium permanganate as a manganese precursor, and then an ochratoxin A aptamer is adsorbed on the surface of the manganese nanoflower to prepare a probe with high specificity. The probe has a strong catalytic activity to the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB), and can quickly catalyze the oxidation of the chromogenic substrate and produce a color change. Therefore, the chromogenic substrate can be used to detect ochratoxin A by colorimetry or ultraviolet absorption spectrophotometry. The use of this method can further improve the detection accuracy and efficiency of ochratoxin A.

[0045] Based on the above advantages of Mn-OTA-apt, the present invention also proposes an application of a manganese nanoflower-aptamer probe prepared by the preparation method of the manganese nanoflower-aptamer probe as described above in the detection of ochratoxin A.

[0046] Specifically, Mn-OTA-apt not only has stronger catalytic activity than manganese nanoflowers (Mn-NFs), accelerates and enhances the catalytic color development of TMB, but also, when it comes into contact with OTA, the adsorbed aptamer (OTA-apt) on it can bind to OTA and detach from the probe material, which reduces the catalytic activity of the material for TMB, and then causes the color development to become lighter. Based on this, when testing the sample, the sample can be prepared into a liquid sample, and then Mn-OTA-apt is added. After incubation at 37°C for 2h, TMB is added for color development, and at the same time, a blank control (i.e., a pure sample without OTA) is prepared. In this way, by observing the color change of the sample solution, it can be determined whether the sample contains OTA. Specifically, compared with the blank control without OTA, a lighter color of the sample indicates that the sample contains OTA, and the same color indicates that the sample does not contain OTA; further, when the color of the sample containing OTA is compared, the lighter color sample contains a higher content of OTA. In this way, qualitative detection of OTA can be achieved.

[0047] Furthermore, in combination with ultraviolet spectrophotometry, the content of OTA can be calculated by detecting the absorbance of the color-developing solution, thereby realizing quantitative detection of OTA. Specifically, a standard solution group of different concentrations of OTA can be prepared first, and after adding Mn-OTA-apt for incubation, TMB is added for color development, and the absorbance is detected to establish a standard curve of OTA concentration-absorbance. Then, the absorbance of the color-developing solution of the sample is detected, and the content of OTA in the sample is calculated by substituting it into the standard curve.

[0048] The detection limit of OTA by the Mn-OTA-apt prepared in the present invention is 0.0102 ng / mL, and the detection range is 0.079-37.62 ng / mL.

[0049] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0050] Example 1

[0051] Weigh 0.5 g of KMnO4 and dissolve it in 250 mL of distilled water, and stir the mixture rapidly for about 0.5 h to prepare a potassium permanganate solution. Then add 5.0 mL of OA and react for 48 h. Collect the crude brown-black product by centrifugation, wash it several times with ultrapure water and ethanol to remove the residual reactants, and obtain honeycomb manganese nanoflower materials (Mn-NFs). Figure 1 ,Observation under electron microscope showed that Mn-NFs were uniform in size and high specific surface area, indicating that they have high nanozyme activity and high specific surface area.

[0052] The purified Mn-NFs were redispersed in 250 mL of ultrapure water to obtain a manganese nanoflower dispersion.

[0053] The ochratoxin A aptamer (OTA-apt) was centrifuged, ultrapure water was added and vortexed for about 1 min to prepare a 1 μmol / L ochratoxin A aptamer solution for later use.

[0054] Take 2 μL of the manganese nanoflower dispersion prepared in the above steps into a centrifuge tube, add 10 μL of 1 μmol / L ochratoxin A aptamer solution, then add 50 μL of 1 mol / L NaCl solution, 10 μL of PBS solution (pH 7.5) and 28 μL of ultrapure water. Vortex the resulting mixture and incubate it in a constant temperature water bath at 37 °C for 1 h to obtain the manganese nanoflower-aptamer probe (Mn-OTA-apt).

[0055] (1) Identification

[0056] Element mapping analysis was performed on Mn-OTA-apt. The results are as follows: Figure 2 As shown in the figure, it can be seen that phosphorus element appears in the probe, and the distribution of detected Mn, C, N, O, and P elements is highly consistent with the distribution of Mn-NFs, indicating that the adsorption of OTA-apt by manganese nanoflowers is achieved.

[0057] At the same time, the particle size distribution of Mn-NFs and Mn-OTA-apt was further tested. Figure 6 and Figure 7 As shown in the figure, it can be seen that the particle size of the former Mn-NFs is about 122nm, and the particle size of the latter Mn-OTA-apt is about 142nm, which is much larger than the particle size of Mn-NFs, indicating that OTA-apt is uniformly adsorbed on Mn-NFs.

[0058] Furthermore, the potentials of OTA-apt, Mn-NFs and Mn-OTA-apt were tested. Figure 8 As shown, through the comparison of the three potentials, it can be seen that the aptamer is successfully adsorbed onto the surface of the manganese nanoflower.

[0059] OTA-apt, Mn-NFs and Mn-OTA-apt were tested using a UV spectrophotometer. The results are as follows: Fig. 9 As shown, by comparing the three curves, it can be seen that the aptamer is successfully adsorbed onto the surface of the manganese nanoflower.

[0060] In summary, the method of the present invention successfully prepared the Mn-OTA-apt probe.

[0061] (2) Detection of catalytic activity of Mn-NFs

[0062] Dissolve 0.024 g of TMB in 1 mL of N,N-dimethylformamide (DMF) to obtain TMB solution.

[0063] The TMB solution was added into the dispersion of Mn-NFs, and the UV spectra of TMB catalyzed by Mn-NFs were measured at different time conditions (0.5, 2, 5, 10, 15, 20, 25 min). The results are shown in Figure 3 As shown in the figure, it can be seen that Mn-NFs can catalyze the color development of TMB, and the catalytic activity continues to increase with time.

[0064] The TMB solution was added into the dispersion of Mn-NFs, and the UV spectra of TMB catalyzed by Mn-NFs were measured at different temperatures (30, 40, 50, 60, 70, 80°C). The results are shown in Figure 4 As shown in the figure, it can be seen that Mn-NFs can catalyze TMB color development and is still active at 70°C, indicating that its catalytic activity is highly stable.

[0065] Different concentrations of manganese nanoflower dispersions (5, 10, 15, 20, 25, 30 μg / mL) were prepared, TMB solution was added to the Mn-NFs dispersion, and the UV spectra of TMB catalyzed by Mn-NFs of different concentrations were measured. The results are shown in Figure 5 As shown in the figure, it can be seen that Mn-NFs can catalyze the color development of TMB.

[0066] OTA detection: Take 2 μL of different samples (blank, wheat flour, wine, OTA, wheat flour-OTA, wine-OTA) and add the same amount of Mn-OTA-apt, mix well, and incubate in a constant temperature water bath for 2 hours to obtain a mixed solution. Take 20 μL of each mixed solution, dilute with 70 μL ultrapure water, and then add 2.5 μL 100 mM TMB for color reaction. The results are shown in the figure. Fig.10 As shown in the figure, the three bottles on the left are darker blue, and among the three bottles on the right, the OTA bottle and the wheat flour-OTA bottle solution are very light blue, almost colorless, and the wine-OTA solution is colorless. Through colorimetry, it can be seen that after adding Mn-OTA-apt, the sample containing OTA is lighter in color, indicating that Mn-OTA-apt reacts with OTA in the sample. Through this color comparison, it is possible to quickly determine whether the sample contains OTA.

[0067] Example 2

[0068] Weigh 0.5 g of KMnO4 and dissolve it in 250 mL of distilled water, and stir the mixture rapidly for about 0.5 h to prepare a potassium permanganate solution. Then add 4.0 mL of OA and react for 40 h. The crude brown-black product was collected by centrifugation and washed several times with ultrapure water and ethanol to remove the residual reactants to obtain honeycomb manganese nanoflower materials (Mn-NFs). The purified Mn-NFs were redispersed in 250 mL of ultrapure water to obtain a manganese nanoflower dispersion.

[0069] The ochratoxin A aptamer (OTA-apt) was centrifuged, ultrapure water was added and vortexed for about 1 min to prepare a 1 μmol / L ochratoxin A aptamer solution for later use.

[0070] Take 2 μL of the manganese nanoflower dispersion prepared in the above steps into a centrifuge tube, add 10 μL of ochratoxin A aptamer solution, then add 50 μL of 1 mol / L NaCl solution, 10 μL of PBS solution (pH 7.5) and 28 μL of ultrapure water. Vortex the resulting mixture and incubate it in a constant temperature water bath at 37 °C for 0.8 h to obtain a manganese nanoflower-aptamer probe (Mn-OTA-apt).

[0071] Example 3

[0072] Weigh 0.5 g of KMnO4 and dissolve it in 250 mL of distilled water, and stir the mixture rapidly for about 0.5 h to prepare a potassium permanganate solution. Then add 6.0 mL of OA and react for 45 h. The crude brown-black product was collected by centrifugation and washed several times with ultrapure water and ethanol to remove the residual reactants to obtain honeycomb manganese nanoflower materials (Mn-NFs). The purified Mn-NFs were redispersed in 250 mL of ultrapure water to obtain a manganese nanoflower dispersion.

[0073] The ochratoxin A aptamer (OTA-apt) was centrifuged, ultrapure water was added and vortexed for about 1 min to prepare a 0.8 μmol / L ochratoxin A aptamer solution for later use.

[0074] Take 2 μL of the manganese nanoflower dispersion prepared in the above steps into a centrifuge tube, add 11.2 μL of ochratoxin A aptamer solution, then add 50 μL of 1 mol / L NaCl solution, 10 μL of PBS solution (pH 7.5) and 28 μL of ultrapure water. Vortex the resulting mixture and incubate it in a constant temperature water bath at 37 °C for 1.2 h to obtain the manganese nanoflower-aptamer probe (Mn-OTA-apt).

[0075] Example 4

[0076] Weigh 0.5 g of KMnO4 and dissolve it in 250 mL of distilled water, and stir the mixture rapidly for about 0.5 h to prepare a potassium permanganate solution. Then add 7.5 mL of OA and react for 55 h. The crude brown-black product was collected by centrifugation and washed several times with ultrapure water and ethanol to remove the residual reactants to obtain honeycomb manganese nanoflower materials (Mn-NFs). The purified Mn-NFs were redispersed in 250 mL of ultrapure water to obtain a manganese nanoflower dispersion.

[0077] The ochratoxin A aptamer (OTA-apt) was centrifuged, ultrapure water was added and vortexed for about 1 min to prepare a 1.2 μmol / L ochratoxin A aptamer solution for later use.

[0078] Take 2 μL of the manganese nanoflower dispersion prepared in the above steps into a centrifuge tube, add 8.8 μL of ochratoxin A aptamer solution, then add 50 μL of 1 mol / L NaCl solution, 10 μL of PBS solution (pH 7.5) and 28 μL of ultrapure water. Vortex the resulting mixture and incubate it in a constant temperature water bath at 37 °C for 1 h to obtain the manganese nanoflower-aptamer probe (Mn-OTA-apt).

[0079] In summary, it can be seen that the method of the present invention can prepare manganese nanoflowers with uniform size, high specific surface area and high nanozyme activity, and can successfully adsorb OTA-apt on its surface, further improving the catalytic activity of manganese nanoflowers, so that it can quickly catalyze the oxidation of TMB and change its color; in addition, the Mn-OTA-apt prepared by the method of the present invention has high specificity for OTA. When added to a sample containing OTA, the OTA in the sample can specifically bind to the OTA-apt in the probe, causing it to fall off the Mn-NFs material, thereby reducing the activity of the Mn-NFs material in catalyzing the chromogenic substrate (TMB) into a blue substance (oxTMB). The lighter the color, the higher the detected OTA concentration, so that the detection personnel can quickly qualitatively or quantitatively detect OTA in the sample by colorimetry or ultraviolet spectrophotometry, with high detection efficiency and accuracy.

[0080] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. Application of a manganese nanoflower-aptamer probe in detecting ochratoxin A, characterized in that: The method for preparing the manganese nanoflower-aptamer probe comprises the following steps: Add oleic acid to the potassium permanganate solution to carry out redox reaction and purify the manganese nanoflowers, wherein 8-15 ml of oleic acid is added for each gram of potassium permanganate, and the redox reaction time is 40-55 hours; The manganese nanoflowers are dispersed in water, and ochratoxin A aptamer solution, sodium chloride solution, phosphate buffer solution and water are added, mixed and incubated to obtain a manganese nanoflower-aptamer probe; The detection of ochratoxin A comprises the following steps: The sample was prepared into a liquid sample, and then the manganese nanoflower-aptamer probe was added, incubated at 37° C. for 2 h, and TMB was added for color development; By observing the color change of the sample solution, it can be determined whether the sample contains ochratoxin A; By detecting the absorbance of the color-developing solution, the content of ochratoxin A is calculated, thereby achieving quantitative detection of ochratoxin A.

2. The use of the manganese nanoflower-aptamer probe in detecting ochratoxin A according to claim 1, characterized in that: The steps of adding oleic acid to a potassium permanganate solution to perform a redox reaction and purifying to obtain manganese nanoflowers include: adding oleic acid to a potassium permanganate solution to carry out an oxidation-reduction reaction to obtain a reaction solution; The reaction solution is centrifuged, the precipitate is collected, and washed with ultrapure water and ethanol to obtain manganese nanoflowers.

3. The use of the manganese nanoflower-aptamer probe in detecting ochratoxin A according to claim 1, characterized in that: In the step of dispersing the manganese nanoflowers in water, adding ochratoxin A aptamer solution, sodium chloride solution, phosphate buffer and water, mixing and incubating to obtain a manganese nanoflower-aptamer probe, the concentration of the ochratoxin A aptamer solution is 0.8~1.2 μmol / L.

4. The use of the manganese nanoflower-aptamer probe in detecting ochratoxin A as claimed in claim 3, characterized in that: For each gram of potassium permanganate, 2.2-2.8 L of the ochratoxin A aptamer solution is added.

5. The use of the manganese nanoflower-aptamer probe in detecting ochratoxin A according to claim 1, characterized in that: The manganese nanoflowers are dispersed in water, ochratoxin A aptamer solution, sodium chloride solution, phosphate buffer and water are added, mixed and then incubated to obtain the manganese nanoflower-aptamer probe, and the incubation time is 0.8~1.2h.

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