A bichromatic detection probe is used for naked-eye visual detection of zearalenone

Abstract

The application discloses a kind of bicolour detection probe for zearalenone naked eye visual detection, comprising the following steps: step one: configuration solution 1;Step two: configuration solution 2;Step three: configuration solution 3;Step four: 3 μL of solution 2 is added to 37 μL of solution 1, then 10 μL of solution 3 is added to carry out reaction, and the absorption value of solution at 418 nm and 480 nm within 1-30 min is recorded using enzyme label instrument, the color change of solution is recorded by mobile phone photographing every minute, and the picture is digitized using RGB image conversion technology to improve the discernibility of naked eye.The application belongs to the technical field of ZEN detection, and is a kind of bicolour detection probe for zearalenone naked eye visual detection, which has high specificity, high sensitivity, good stability, simple operation, easy to carry, low cost, does not need professional technical personnel, fast detection time (10 minutes can be naked eye to see color change), and can be used as a useful tool for on-site analysis of ZEN in food and drug samples.

Description

Technical Field

[0001] This invention belongs to the field of ZEN detection technology, specifically referring to a dual-color detection probe for naked-eye visual detection of zearalenone. Background Technology

[0002] Zearalenone (ZEN), also known as F-2 toxin, is a class of organic compounds produced by Fusarium graminearum. Zearalenone and its derivatives are mainly found in moldy grains and traditional Chinese medicinal materials, and are characterized by their heat resistance, difficulty in decomposition, and challenges in removal. Zearalenone has estrogenic effects, causing damage to the reproductive system of female animals. It is also toxic to the nervous system, heart, kidneys, liver, and lungs of male animals. Short-term, high-dose exposure to ZEN can easily lead to acute poisoning, manifesting as restlessness, muscle tremors, lethargy, loss of appetite, nausea, vomiting, and other systemic discomfort. Long-term exposure leads to chronic poisoning, resulting in ovarian cysts, breast swelling, etc. Due to its strong carcinogenicity, neurotoxicity, immunotoxicity, reproductive toxicity, and teratogenicity, ZEN has been classified as a Group 3 carcinogen by the International Agency for Research on Cancer.

[0003] Currently, the main methods available for ZEN detection include chromatography (high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (HPLC-MS), and enzyme-linked immunosorbent assay (ELISA). However, HPLC and HPLC / MS suffer from high instrument costs, complex sample pretreatment and data analysis, and the need for specialized technical personnel. ELISA, on the other hand, is limited by the inherent difficulties in antibody preservation, easy inactivation, and high cost, making it unsuitable for routine use. While aptamer-based biosensors can detect ZEN, methods include electrochemical, colorimetric, fluorescence detection, and elliptic polarization assays. Electrochemical, colorimetric, and fluorescence detection methods are complex in their preparation and cannot achieve rapid detection, while elliptic polarization assays are not only complex in their preparation but also require specialized equipment, making them unsuitable for routine use. Therefore, existing technologies struggle to achieve highly sensitive, visually perceptible detection of ZEN, and are even less suitable for immediate ZEN screening. Thus, there is an urgent need to develop a simple, rapid, and highly sensitive method for ZEN detection. Summary of the Invention

[0004] In response to the above situation and to overcome the shortcomings of the existing technology, this invention provides a dual-color detection probe for naked-eye visual detection of zearalenone. It has the advantages of high specificity, high sensitivity, good stability, simple operation, portability, low cost, and can be operated without professional technicians. The detection time is fast (the color change can be seen with the naked eye in 10 minutes). It can be used as a useful tool for on-site analysis of ZEN in food and drug samples.

[0005] The technical solution adopted by this invention is as follows: This invention provides a dual-color detection probe for naked-eye visual detection of zearalenone, comprising the following steps:

[0006] Step 1: Prepare solution 1:

[0007] Solution 1 was prepared using a pH 7.0 buffer containing 40 mM HEPES, 1 μM probe, 400 mM NaCl, 20 mM KCl, 0.05% Triton-X-100, and 1 μM Hemin.

[0008] Step 2: Prepare solution 2:

[0009] Prepare solutions containing ZEN at different concentrations, ranging from 0 to 100 μM;

[0010] Step 3: Prepare solution 3:

[0011] Solution 3 was prepared using a pH 7.0 buffer containing 40 mM HEPES, 2 mM H2O2, and 1.5 mM MABTS.

[0012] Step Four:

[0013] 3 μL of solution 2 was added to 37 μL of solution 1, and then 10 μL of solution 3 was added to carry out the reaction. The absorbance values ​​of the solution at 418 nm and 480 nm were recorded using an ELISA reader within 1-30 minutes, and the color change of the solution was recorded every minute using a mobile phone.

[0014] Further, in the step one probe is 5'-XGGGYXZGGGTACAGATGTCATCTATCTATGGTACATTATT-3' and 5'-AATAATGTGATCTGAAGGGHXWGGGX-3', where A, T, G, and C are adenine, thymine, guanine, and cytosine, respectively, which are essential base components of DNA. When preparing the solution, X, Y, Z, H, and W in the probe can be any one of the three bases A, T, and C mentioned above, and the bases Y and W, and Z and H are complementary.

[0015] Furthermore, a small amount of ZEN is present in the solution during step four, and the solution is dark green.

[0016] Furthermore, in step four, if the ZEN concentration in the solution is too high, the solution will be either yellow or yellowish-brown.

[0017] Furthermore, in step four, the solution does not contain ZEN and is colorless.

[0018] The beneficial effects of this invention using the above structure are as follows: This solution provides a dual-color detection probe for naked-eye visual detection of zearalenone. When a small amount of ZEN is present in the solution, the probe is activated and forms a DNAzyme structure. Under the action of H2O2, the DNAzyme can catalyze ABTS to ABTS· + At this point, the solution appears dark green, and its absorbance at 418 nm will increase; when the ZEN concentration in the solution is too high, the excess ZEN will further react with ABTS· + The reaction produces new substances, at which point the solution is yellow or yellowish-brown. Due to the formation of new substances, the absorbance of the solution at 418 nm will decrease, while the absorbance at 480 nm will increase. When ZEN is not present in the solution, the probe will not be activated, and DNAzyme cannot be formed. The corresponding reaction will not occur, and the solution will be colorless. At this point, the absorbance of the solution at either 418 nm or 480 nm will not change significantly.

[0019] ZEN can be detected by recording the changes in absorbance at 418nm or 480nm using an ELISA reader. Alternatively, the color change of the solution can be observed directly with the naked eye, or the color change of the image can be observed after G→RGB conversion of the photograph.

[0020] This method is label-free, rapid, sensitive, and can be monitored in real time with the naked eye. This invention is expected to enable rapid on-site screening of ZEN, laying an experimental foundation for ensuring the safety of food and traditional Chinese medicine products, and expanding the design ideas for ZEN detection probes. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the principle of a dual-color detection probe for naked-eye visual detection of zearalenone in zearalenone according to the present invention.

[0022] Figure 2 This is a detection result diagram of Example 1 of the present invention, which uses a dual-color detection probe for naked-eye visual detection of zearalenone.

[0023] Figure 3 This is a low-concentration probe detection result diagram of Example 2 of the present invention, which uses a dual-color detection probe for naked-eye visual detection of zearalenone in zearalenone.

[0024] Figure 4 This is a high-concentration probe detection result image from Example 2 of the present invention, which uses a dual-color detection probe for naked-eye visual detection of zearalenone in zearalenone.

[0025] Figure 5 This is a specificity diagram of the probe for detecting ZEN in Example 3 of the present invention, which uses a dual-color detection probe for naked-eye visual detection of zearalenone.

[0026] Figure 6 This is a probe detection result diagram from Example 4 of the present invention, which uses a dual-color detection probe for naked-eye visual detection of zearalenone.

[0027] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0029] Example 1:

[0030] Naked-eye visual probes are used for ZEN detection:

[0031] Step 1: Prepare Solution 1 using pH 7.0 containing 40 mM HEPES buffer, 1 μM probe, 400 mM NaCl, 20 mM KCl, 0.05% Triton-X-100, and 1 μM Hemin;

[0032] Step 2: Take 37 μL of the above solution 1 and add it to 3 μL of ZEN test solutions containing 0 μM, 30 μM and 100 μM respectively to obtain solution A;

[0033] Step 3: Prepare the substrate solution as Solution 3 using pH 7.0 containing 40 mM HEPES buffer, 2 mM H2O2 and 1.5 mM MABTS;

[0034] Step Four:

[0035] Add 10 μL of solution to solution A above, carry out the reaction, and record the absorbance values ​​of the solution at 418 nm and 480 nm at 1-30 min using a microplate reader. Record the color change of the solution every minute using a mobile phone. The results are as follows: Figure 2 .

[0036] Example 2:

[0037] Establishing the ZEN concentration dependence curve:

[0038] Using the detection steps in Example 1, probes were used for detection. Low concentrations of ZEN test solutions were used at concentrations of 0, 0.1, 0.5, 1, 2, 3, 5, 10, 20, and 30 μM. The absorbance at 418 nm was recorded after 15 minutes, and the data was fitted. Samples were photographed, and the images taken at 15 minutes were converted from G to RGB. The probe detection results are shown below. Figure 3 ;

[0039] Similarly, high concentrations of ZEN were detected at concentrations of 30, 35, 40, 50, 60, 70, 80, 90, and 100 μM. The absorption peak at 480 nm was recorded after 15 minutes, fitted, and the samples were photographed. Finally, the images taken at 15 minutes were converted from G to RGB. The probe detection results are as follows: Figure 4 .

[0040] Example 3: Specificity detection of ZEN using naked-eye visual probes:

[0041] The detection steps in Example 1 were used, with the ZEN solution concentration being 10 μM. Other substances included AFB1, AFB2, AFG1, AFM1, T-2, OTA, PAT, and DON. AFB1 represents aflatoxin B1, AFB2 represents aflatoxin B2, AFG1 represents aflatoxin G1, AFM1 represents aflatoxin M1, T-2 represents T-2 toxin, OTA represents ochratoxin, PAT represents patulin, and DON represents vomitoxin. The solution concentration was 100 μM, and the absorbance at 418 nm was recorded after 15 min.

[0042] Example 4: Probe detection of known concentrations of ZEN in oats:

[0043] The detection steps in Example 1 were used, with ZEN concentrations of 0, 0.1, 0.5, 1, 2, 3, 5, 10, 20, and 30 μM. The absorbance at 418 nm was recorded after 15 minutes, and G→RGB conversion was performed. The probe detection results are as follows. Figure 6 .

[0044] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0045] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0046] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A dual-color detection probe for naked-eye visual detection of zearalenone, characterized in that, Includes the following steps: Step 1: Prepare solution 1: Solution 1 was prepared using a pH 7.0 buffer containing 40 mM HEPES, 1 μM probe, 400 mM NaCl, 20 mM KCl, 0.05% Triton-X-100, and 1 μM Hemin. Step 2: Prepare solution 2: Prepare solutions containing ZEN at different concentrations, ranging from 0 to 100 μM; Step 3: Prepare solution 3 Solution 3 was prepared using a pH 7.0 buffer containing 40 mM HEPES, 2 mM H2O2, and 1.5 mM MABTS. Step Four: Add 3 μL of solution 2 to 37 μL of solution 1, then add 10 μL of solution 3 to react. Use an ELISA reader to record the absorbance values ​​of the solution at 418 nm and 480 nm within 1-30 min, and use a mobile phone to record the color change of the solution every minute. In step one, the probes are 5'-XGGGYXZGGGTACAGATGTCATCTATCTATGGTACATTATT-3' and 5'-AATAATGTGATCTGAAGGGHXWGGGX-3', where A, T, G, and C are adenine, thymine, guanine, and cytosine, respectively, which are essential bases for DNA. When preparing the solution, X, Y, Z, H, and W in the probe can be any one of the three bases A, T, and C mentioned above, and Y and W, and Z and H are complementary bases.

2. The dual-color detection probe according to claim 1 for naked-eye visual detection of zearalenone, characterized in that: In step four, a small amount of ZEN is present in the solution, which is dark green in color.

3. The dual-color detection probe according to claim 1 for naked-eye visual detection of zearalenone, characterized in that: In step four, if the ZEN concentration in the solution is too high, the solution will be either yellow or yellowish-brown.

4. The dual-color detection probe according to claim 1 for naked-eye visual detection of zearalenone, characterized in that: In step four, the solution does not contain ZEN and is colorless.

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