Method for measuring content of aflatoxin b1 by fluorescence analysis based on photocleavage

Abstract

A method for measuring the content of aflatoxin B1 by fluorescence analysis based on photocleavage, relating to the technical field of biological detection. The method comprises the step of using a fluorescent probe combination to measure the content of aflatoxin B1. The fluorescent probe combination comprises fluorescent probes Aptamer and PC-strand; the nucleotide sequence of Aptamer is as shown in SEQ ID NO. 1, and an FAM group is marked at the 3' terminus of the nucleotide sequence as shown in SEQ ID NO. 1; the nucleotide sequence of PC-strand is as shown in SEQ ID NO. 2, a TAMRA group is marked at the 5' terminus of the nucleotide sequence as shown in SEQ ID NO. 2, and a PC group is marked between bases at 10 bp and 11 bp of the nucleotide sequence as shown in SEQ ID NO. 2. The method can implement photosensitive detection of aflatoxin B1, and has high detection sensitivity and high specificity.

Description

A method for detecting aflatoxin B1 content by fluorescence analysis based on photocleavage Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for detecting aflatoxin B1 content by fluorescence analysis based on photo-cleavage. Background Art

[0002] With the advancement of science and technology, people's living standards are constantly improving, and their demand for food safety is becoming increasingly stringent. Improper food storage during production and storage can easily produce aflatoxins, especially in mold-prone crops such as corn, soybeans, and their products. Aflatoxins are extremely toxic and carcinogenic, especially aflatoxin B1 (AFB1), which has been classified as a Class I carcinogen by the World Health Organization. To ensure food safety, the use of relevant detection technologies to detect the food toxin AFB1 has long been a hot topic.

[0003] Existing methods for detecting AFB1 include colorimetry, fluorescence, electrochemistry, high-performance liquid chromatography (HPLC), surface plasmon resonance, and novel fluorescent aptamer sensors. However, most current biosensors with target recognition units are passive, meaning the target probe is always active and outputs a specific signal upon reacting with the target molecule AFB1, making it difficult to achieve controllable and precise control of this process. Due to the complexity of actual test samples, such as their high number of components, low abundance, and complex structure, it is necessary to achieve real-time, in-situ controllable activation while pursuing high sensitivity and specificity to achieve precise detection of the target molecule AFB1.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting the content of aflatoxin B1 based on fluorescence analysis of photosection to solve the problems existing in the above-mentioned prior art. The method provided by the present invention can realize the photosensitivity detection of aflatoxin B1 with high detection sensitivity and strong specificity.

[0006] This invention utilizes the instantaneous operation and remote triggering capabilities of light to develop an "activity-inhibited" biosensor, avoiding the shortcomings of existing technologies. During the biosensing process, the target recognition unit in this "activity-inhibited" state remains inactive even in the presence of the target molecule AFB1. It remains inactive until a specific time, when it is selectively activated and switched to the "on" state, binding to the target molecule AFB1 and generating a fluorescent signal output.

[0007] Based on this, the present invention provides the following solutions:

[0008] The present invention provides a fluorescent probe combination for detecting aflatoxin B1 content based on a fluorescence analysis method of light cleavage, comprising a fluorescent probe Aptamer and a PC-strand;

[0009] The nucleotide sequence of the Aptamer is shown in SEQ ID NO.1, and the FAM group is labeled at the 3' end of the nucleotide sequence shown in SEQ ID NO.1;

[0010] The nucleotide sequence of the PC-strand is shown in SEQ ID NO.2, the TAMRA group is labeled at the 5' end of the nucleotide sequence shown in SEQ ID NO.2, and the PC group is labeled between the bases at 10 bp and 11 bp of the nucleotide sequence shown in SEQ ID NO.2;

[0011] The structural formula of the PC group is as follows:

[0012] The present invention also provides the use of the fluorescent probe combination in preparing a kit for detecting the content of aflatoxin B1 by a fluorescence analysis method based on photocleavage.

[0013] The present invention also provides a kit for detecting the content of aflatoxin B1 by fluorescence analysis based on photocleavage, wherein the kit comprises the above-mentioned fluorescent probe combination.

[0014] The present invention also provides the use of the fluorescent probe combination or kit in detecting the content of aflatoxin B1.

[0015] The present invention also provides a method for detecting aflatoxin B1 content based on fluorescence analysis of light cutting, comprising the following steps:

[0016] (1) The Aptamer solution, PC-strand solution, and Tris buffer solution are mixed and reacted, followed by ultraviolet irradiation to obtain a mixed reaction system solution;

[0017] (2) taking the same volume of the mixed reaction system solution obtained in step (1), adding the same volume of aflatoxin B1 standard solution but with different concentrations, respectively, detecting the fluorescence intensity after the reaction, and then drawing a standard curve of aflatoxin B1 concentration and fluorescence intensity;

[0018] (3) taking the mixed reaction system solution obtained in step (1), adding the sample solution to be tested, detecting the fluorescence intensity after the reaction, and then calculating the aflatoxin B1 content in the sample solution to be tested based on the standard curve;

[0019] In step (1), the nucleotide sequence of the Aptamer is as shown in SEQ ID NO.1, and the FAM group is labeled at the 3' end of the nucleotide sequence shown in SEQ ID NO.1; the nucleotide sequence of the PC-strand is as shown in SEQ ID NO.2, the TAMRA group is labeled at the 5' end of the nucleotide sequence shown in SEQ ID NO.2, and the PC group is labeled between the bases at 10 bp and 11 bp of the nucleotide sequence shown in SEQ ID NO.2;

[0020] The structural formula of the PC group is as follows:

[0021] In step (3), the volume ratio of the mixed reaction system solution to the sample solution to be tested is the same as the volume ratio of the mixed reaction system solution to the aflatoxin B1 standard solution in step (2).

[0022] Furthermore, in step (1), in the mixed solution of the Aptamer solution, the PC-strand solution and the Tris buffer solution, the concentration of the Aptamer is 0.15 μmol / L; the concentration of the PC-strand is 0.24 μmol / L.

[0023] Furthermore, in step (1), the temperature of the mixing reaction is 4°C.

[0024] Furthermore, in step (1), the irradiation intensity of the ultraviolet light is 5mW / cm 2 .

[0025] Furthermore, the irradiation time of the ultraviolet light is 8 minutes.

[0026] Furthermore, in step (2) and step (3), the reaction temperature is 37°C.

[0027] The present invention discloses the following technical effects:

[0028] The present invention uses a fluorescently labeled single-stranded DNA Aptamer as a fluorescent probe and a PC-strand labeled with an optically active group to partially complement the fluorescent probe Aptamer, forming a double strand. At this point, the quenching group TAMRA and the fluorescent group FAM are close together, quenching the fluorescence. Under illumination, the PC-strand opens at the position of the photosensitive group, forming two segments, which each form a double strand with the fluorescent probe Aptamer. At this point, the binding force between the PC-strand and Aptamer is weak. When the target molecule AFB1 is present, AFB1 specifically binds to the fluorescent probe Aptamer, forming a secondary structure. This disrupts the double-stranded structure formed by the PC-strand and the fluorescent probe Aptamer, causing the quenching group TAMRA to move away from the fluorescent group FAM, and restoring fluorescence. Based on the specific recognition effect of Aptamer on AFB1, when ochratoxin (OTA) or zearalenone (ZEN) with a similar structure to AFB1 is added, the fluorescence does not recover. By monitoring the changes in the fluorescence signal, the specific detection of the target molecule AFB1 is achieved, and it is applied to the detection of AFB1 in food (such as rice, corn, soybeans, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] FIG1 is a schematic diagram showing the principle of the detection method of the present invention;

[0031] Figure 2 is a schematic diagram of AD double chain, A-PD double chain and A-nPD double chain;

[0032] FIG3 shows the fluorescence detection results of the concentration optimization experiment of the Aptamer solution;

[0033] FIG4 shows the experimental results of optimizing the reaction temperature of Aptamer and PC-strand; wherein (A) is a fluorescence curve graph; (B) is a bar graph;

[0034] Figure 5 shows the results of the PC-strand concentration screening experiment; (A) is a fluorescence curve graph; (B) is a dot plot;

[0035] Figure 6 shows the results of the base effect experiment; wherein A is the fluorescence intensity detection result before and after the addition of AFB1 standard solution to test tube No. 1; B is the fluorescence intensity detection result before and after the addition of AFB1 standard solution to test tube No. 2;

[0036] FIG7 shows the results of the PC health effect experiment; wherein A is the fluorescence intensity detection result of test tubes 1-4; B is the fluorescence intensity detection result of test tubes 5-8;

[0037] FIG8 shows the results of the light effect experiment; wherein A is the fluorescence intensity test result of test tubes 1-2; B is the fluorescence intensity test result of test tubes 3-4;

[0038] Figure 9 is a gel electrophoresis diagram of the detection of AFB1 by illumination; M represents a 20 bp DNA leader marker, 1 represents Aptamer, 2 represents PC-strand, 3 represents Aptamer+PC-strand+AFB1 in the absence of illumination, 4 represents Aptamer+PC-strand in the presence of illumination, 5 represents Aptamer+PC-strand+AFB1 in the presence of illumination; 6 represents Aptamer+AFB1; the boxed portion represents the product fragment resulting from the cleavage of the PC bond in the PC-strand chain after illumination;

[0039] Figure 10 shows the results of the experiment on the effect of illumination time; A is a fluorescence curve graph; B is a dot graph;

[0040] Figure 11 is a circular dichroism spectrum of the effect of light on the structure of Aptamer; wherein A is the structure diagram of Aptamer, Aptamer+PC-strand, and Aptamer+PC-strand+AFB1 under light conditions; B is the structure diagram of Aptamer, Aptamer+PC-strand, and Aptamer+PC-strand+AFB1 under no light conditions;

[0041] FIG12 shows the effect of reaction temperature on AFB1 detection; wherein A is a fluorescence curve graph; B is a broken line graph;

[0042] FIG13 shows the detection results of AFB1 sensitivity; wherein (A) is a fluorescence curve graph; (B) is a broken line graph;

[0043] FIG14 shows the specificity experiment results; wherein A is a fluorescence curve graph; B is a bar graph;

[0044] Figure 15 shows the results of the corn water application exploration experiment; A is the fluorescence curve graph; B is the dot graph;

[0045] Figure 16 shows the results of the rice water application exploration experiment; wherein A is a fluorescence curve graph; B is a dot graph;

[0046] Figure 17 shows the results of the soybean water application exploration experiment; A is the fluorescence curve graph; B is the dot graph. DETAILED DESCRIPTION

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0049] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0050] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0051] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0052] The present invention is based on experimental research conducted under photocleavage, as shown in Figure 1. The basic principle is: the fluorescent probe Aptamer is partially complementary to the PC-strand labeled with an optically active group to form a double chain (A-PD in Figure 2). Since the quenching group TAMRA is close to the fluorescent group FAM, the fluorescence is quenched; under light conditions, the PC-strand opens at the position of the photosensitive group, and the PC-strand breaks into two segments, which respectively form a double chain with the fluorescent probe Aptamer (AD and A-nPD in Figure 2). At this time, Aptamer and PC-strand form two double-stranded structures with weak binding force. When AFB1 is introduced, Aptamer has a specific recognition effect on AFB1 to form a secondary structure. At this time, the double-stranded structure formed by Aptamer and PC-strand is destroyed, the quenching group TAMRA is far away from the fluorescent group FAM, and the fluorescence is restored. Based on the specific recognition effect of Aptamer on AFB1, when ochratoxin (OTA) or zearalenone (ZEN) with a similar structure to AFB1 is added, the fluorescence does not recover. By detecting the changes in the fluorescence signal, the specific detection of the target molecule AFB1 is achieved, and it is applied to the detection of AFB1 in food (such as rice, corn, soybeans, etc.).

[0053] In the following examples, Aptamer, PC-strand, None-PC-strand and DNA were synthesized by Shanghai Bioengineering Co., Ltd., wherein:

[0054] Aptamer:

[0055] 5'-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCC-FAM-3' (SEQ ID NO. 1);

[0056] PC-strand: 5'-TAMRA-GGGCCTAGCG-PC-AAGGGCAC-3' (SEQ ID NO. 2);

[0057] None-PC-strand: 5'-TAMRA-GGGCCTAGCGAAGGGCAC-3' (SEQ ID NO. 3);

[0058] DNA: 5'-TAMRA-GGGCCTAGCG-3' (SEQ ID NO. 4).

[0059] The chemical structure of PC is as follows:

[0060] Example 1

[0061] 1 Method

[0062] 1.1 Optimization of Aptamer Solution Concentration

[0063] Take 7 centrifuge tubes, take different volumes of Aptamer (1 μmol / L) solution into the centrifuge tubes, then add Tris buffer solution (10mM Tris, 120mM NaCl, 5mM KCl, pH=7.2) respectively, mix thoroughly, and the total reaction volume is 80μL. The final concentrations of Aptamer in the 7 centrifuge tubes are 0.5μmol / L, 0.25μmol / L, 0.2μmol / L, 0.15μmol / L, 0.1μmol / L, 0.07μmol / L, and 0.03μmol / L. Mix thoroughly and detect its fluorescence intensity. Fluorescence intensity detection conditions: excitation wavelength 480nm, scanning range 490-700nm, voltage 700V, slit 5 / 5.

[0064] 1.2 Screening of reaction temperature between Aptamer and PC-strand

[0065] To four centrifuge tubes, add Aptamer, PC-strand, and Tris buffer (10mM Tris, 120mM NaCl, 5mM KCl, pH 7.2) to each tube and mix thoroughly. The total reaction volume is 80μL. The final concentration of Aptamer is 0.15μmol / L, and the final concentration of PC-strand is 0.18μmol / L. Incubate the solutions in the four centrifuge tubes at 4°C, 15°C, 25°C, and 37°C for 1 hour, and measure the fluorescence intensity.

[0066] Fluorescence intensity detection conditions: excitation wavelength of 480 nm, scanning range of 490-700 nm, voltage of 700 V, slit of 5 / 5.

[0067] 1.3 Screening of PC-strand concentration

[0068] Take 12 centrifuge tubes, add Aptamer, PC-strand, and Tris (10mM Tris, 120mM NaCl, 5mM KCl, pH=7.2) buffer solution to each tube, mix thoroughly, and the total reaction volume is 80μL. After reacting at 4°C for 1 hour, the fluorescence intensity is measured. The final concentration of Aptamer is 0.15μmol / L, and the final concentrations of PC-strand are 0, 0.03μmol / L, 0.06μmol / L, 0.09μmol / L, 0.12μmol / L, 0.15μmol / L, 0.18μmol / L, 0.21μmol / L, 0.24μmol / L, 0.27μmol / L, 0.3μmol / L, and 0.33μmol / L, respectively.

[0069] Fluorescence intensity detection conditions: excitation wavelength of 480 nm, scanning range of 490-700 nm, voltage of 700 V, slit of 5 / 5.

[0070] 1.4 Effect of bases

[0071] Take two centrifuge tubes and, as shown in Table 1, add Aptamer, DNA / PC-strand, and Tris buffer (10mM Tris, 120mM NaCl, 5mM KCl, pH 7.2), respectively. Mix thoroughly to a total reaction volume of 80μL. Incubate at 4°C for 1 hour. Then, add AFB1 standard solution and incubate at 37°C for 1 hour before measuring fluorescence intensity. The final concentrations of Aptamer, PC-strand, and DNA were 0.24μmol / L, respectively, and the final concentration of AFB1 standard solution was 150ng / mL.

[0072] Table 1

[0073] Fluorescence intensity detection conditions: excitation wavelength of 480 nm, scanning range of 490-700 nm, voltage of 700 V, slit of 5 / 5.

[0074] 1.5 Impact of PC Health

[0075] Take 8 centrifuge tubes and number them 1-8 as shown in Table 2, and perform the following steps:

[0076] Aptamer, DNA and Tris buffer solution were added to centrifuge tubes 1-4 respectively, and Aptamer, none-PC-strand and Tris buffer solution were added to centrifuge tubes 5-8 respectively and mixed thoroughly. No. 1-8 were reacted at 4°C for 1 hour. Afterwards, no light treatment was performed on centrifuge tubes 1-2 and 5-6. AFB1 standard solution was added to centrifuge tubes 2 and 6. After reacting at 37°C for 1 hour, the fluorescence intensity was detected. The total reaction volume was 80 μL. No. 1 and No. 5 centrifuge tubes were used as the control group for experiments No. 2 and No. 6, and no AFB1 standard solution was added. No. 3-4, 7-8 centrifuge tubes were treated with ultraviolet light (irradiation intensity 5 mW / cm 2 ) for 20 minutes. AFB1 standard solution was added to centrifuge tubes 4 and 8. After incubation at 37°C for 1 hour, fluorescence intensity was measured. The total reaction volume was 80 μL. Tubes 3 and 7 served as controls for experiments 4 and 8, without the addition of AFB1 standard solution.

[0077] The final concentration of Aptamer was 0.15 μmol / L, the final concentration of none-PC-strand was 0.24 μmol / L, the final concentration of DNA was 0.24 μmol / L, and the final concentration of AFB1 standard solution was 150 ng / mL.

[0078] Fluorescence intensity detection conditions: excitation wavelength of 480 nm, scanning range of 490-700 nm, voltage of 700 V, slit of 5 / 5.

[0079] Table 2

[0080] 1.6 Effect of Light

[0081] Take 4 centrifuge tubes numbered 1-4, add Aptamer, PC-strand and Tris buffer solution respectively, react at 4℃ for 1h, and irradiate tubes 1-2 with ultraviolet light (irradiation intensity 5mW / cm 2 ) After 20 minutes of UV irradiation, AFB1 standard solution was added to centrifuge tube No. 2. After incubation at 37°C for 1 hour, fluorescence intensity was measured. The total reaction volume was 80 μL. Centrifuge tube No. 1 served as a control group for centrifuge tube No. 2 and did not introduce AFB1 standard solution. Centrifuge tubes No. 3-4 were not treated with UV irradiation. AFB1 standard solution was added to centrifuge tube No. 4. After incubation at 37°C for 1 hour, fluorescence intensity was measured. The total reaction volume was 80 μL. Centrifuge tube No. 1 served as a control group for centrifuge tube No. 2 and did not introduce AFB1 standard solution.

[0082] The final concentration of Aptamer is 0.15 μmol / L, the final concentration of PC-strand is 0.24 μmol / L, and the final concentration of AFB1 standard solution is 150 ng / mL.

[0083] Fluorescence intensity detection conditions: excitation wavelength of 480 nm, scanning range of 490-700 nm, voltage of 700 V, slit of 5 / 5.

[0084] 30% acrylamide (5 mL), 5×TBE buffer solution (2 mL), formamide (2 mL), urea (4.2 g), 10% ammonium persulfate (90 μL) and tetramethylethylenediamine (90 μL) were mixed evenly, loaded into a gel electrophoresis plate, and left at room temperature until the gel solidified. DNA sample (10 μL), 6× loading buffer (2 μL) and Gel red dye (1 μL) were added to each well of the plate. The voltage was 90 V and the electrophoresis experiment was stopped for 1.5 hours. The gel was taken out and the DNA sample bands were observed under a gel electrophoresis imager.

[0085] 1.7 Effect of photoperiod

[0086] Take 10 centrifuge tubes numbered 1-10, add Aptamer, PC-strand and Tris buffer solution, react at 4℃ for 1h, and then irradiate with ultraviolet light (irradiation intensity 5mW / cm 2 ) were irradiated for 0 min, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, and 9 min, and then AFB1 standard solution was added to 10 centrifuge tubes. After reacting at 37 ° C for 1 h, the fluorescence intensity was detected. The total reaction volume was 80 μL.

[0087] The final concentration of Aptamer is 0.15 μmol / L, the final concentration of PC-strand is 0.24 μmol / L, and the final concentration of AFB1 standard solution is 150 ng / mL.

[0088] Fluorescence intensity detection conditions: excitation wavelength of 480 nm, scanning range of 490-700 nm, voltage of 700 V, slit of 5 / 5.

[0089] Take 4 centrifuge tubes numbered 1-4, add Aptamer, PC-strand and Tris buffer solution to each tube and react at 4°C for 1 hour. After irradiating centrifuge tube No. 4 with ultraviolet light (irradiation intensity 5 mW) for 8 minutes, add AFB1 standard solution to centrifuge tube No. 3 and centrifuge tube No. 4 and react at 37°C for 1 hour. The circular dichroism spectrum is measured. The total reaction volume is 80 μL.

[0090] The final concentration of Aptamer was 0.15 μmol / L, the final concentration of PC-strand was 0.24 μmol / L, and the concentration of AFB1 standard solution was 150 ng / mL.

[0091] Circular dichroism spectrometer test conditions: the spectral scanning range is 200-340 nm, the interval is 0.5 nm, and the spectral background is subtracted with Tris buffer solution.

[0092] 1.8 Screening of AFB1 reaction temperature

[0093] Take 4 centrifuge tubes numbered 1-4, add Aptamer, PC-strand and Tris buffer solution respectively, react at 4℃ for 1h, and then irradiate with ultraviolet light (irradiation intensity 5mW / cm 2 ) for 8 min, then add AFB1 standard solution, react at different temperatures (4℃, 15℃, 25℃, 37℃) for 1 h, and then detect the fluorescence intensity. The total reaction volume is 80 μL.

[0094] The final concentration of Aptamer is 0.15 μmol / L, the final concentration of PC-strand is 0.24 μmol / L, and the final concentration of AFB1 standard solution is 150 ng / mL.

[0095] Fluorescence intensity detection conditions: excitation wavelength of 480 nm, scanning range of 490-700 nm, voltage of 700 V, slit of 5 / 5.

[0096] 1.9 AFB1 Detection Sensitivity

[0097] Take 13 centrifuge tubes numbered 1-13, add Aptamer, PC-strand and Tris buffer solution respectively, react at 4℃ for 1h, then irradiate with ultraviolet light (irradiation intensity 5mW / cm 2 ) for 8 min, then different concentrations of AFB1 standard solution were added, and the reaction was carried out at 37°C for 1 h. The fluorescence intensity was detected. The total reaction volume was 80 μL.

[0098] Among them, the final concentration of Aptamer is 0.15 μmol / L, the final concentration of PC-strand is 0.24 μmol / L, and the final concentrations of AFB1 standard solution are 0 ng / mL, 0.00625 ng / mL, 0.0875 ng / mL, 0.0125 ng / mL, 0.025 ng / mL, 0.0625 ng / mL, 0.625 ng / mL, 6.25 ng / mL, 62.5 ng / mL, and 625 ng / mL.

[0099] Fluorescence intensity detection conditions: excitation wavelength of 480 nm, scanning range of 490-700 nm, voltage of 700 V, slit of 5 / 5.

[0100] 1.10 AFB1 Detection Specificity

[0101] Take 6 centrifuge tubes numbered 1-6, add Aptamer, PC-strand and Tris buffer solution respectively, react at 4℃ for 1h, then irradiate with ultraviolet light (irradiation intensity 5mW / cm 2 ) for 8 min, take centrifuge tube No. 1 as the control sample without adding fungal toxins, add fungal toxin AFB1 standard solution, OTA standard solution and ZEN standard solution to centrifuge tubes 2-4, respectively, add AFB1 standard solution and OTA standard solution to centrifuge tube No. 5, and add AFB1 standard solution and ZEN standard solution to centrifuge tube No. 6. After reacting at 37°C for 1 h, the fluorescence intensity was detected. The total reaction volume was 80 μL.

[0102] Among them, the final concentration of Aptamer is 0.15 μmol / L, the final concentration of PC-strand is 0.24 μmol / L, and the final concentrations of AFB1 standard solution, OTA standard solution, and ZEN standard solution are all 150 ng / mL.

[0103] Fluorescence intensity detection conditions: excitation wavelength of 480 nm, scanning range of 490-700 nm, voltage of 700 V, slit of 5 / 5.

[0104] 1.11 AFB1 Detection Application Experiment

[0105] AFB1 Extraction from Food Samples: Food samples (corn, rice, and soybeans) were ground into powder using a grinder, and aflatoxin AFB1 was extracted from the food samples according to the method specified in the Chinese National Food Safety Standard (GB5009.22-2016). Briefly, 3 g of food sample was added to 10 mL of a 7:3 methanol / water solution, shaken at 30°C for 1 hour, filtered, and the filtrate was centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was collected, filtered through a 0.45 μm filter, and 3 mL of Tris solution was added. The mixture was mixed thoroughly and stored at 4°C until use.

[0106] Detection of AFB1 in food samples:

[0107] Take 90 μL of food sample (corn, rice, soybean) extract, add different volumes of AFB1 standard solution, mix well, and prepare food sample solutions with different AFB1 concentrations (AFB1 concentrations are 0 ng / mL, 0.075 ng / mL, 0.125 ng / mL, 0.75 ng / mL and 1.25 ng / mL, respectively), and store at 4°C for use.

[0108] Take 5 centrifuge tubes numbered 1-5, add Aptamer, PC-strand and Tris buffer solution, the final concentration of Aptamer is 0.15μmol / L, the final concentration of PC-strand is 0.24μmol / L, react at 4℃ for 1h, and ultraviolet light (irradiation intensity 5mW / cm 2 ) for 8 min, and then the above-mentioned food sample solutions with different AFB1 concentrations were added respectively, and reacted at 37°C for 1 h. The total reaction volume was 80 μL.

[0109] Detection conditions in AFB1 solution: excitation wavelength 480 nm, scanning range 490-700 nm, voltage 700 V, slit 5 / 5.

[0110] 2 Experimental results

[0111] 2.1 Aptamer concentration screening results

[0112] Aptamer is a fluorescent probe with excellent fluorescence properties, and TAMRA can effectively quench FAM fluorescence. Therefore, before using PC-strand, it is necessary to screen the Aptamer concentration to select one with a good luminescence reaction effect, while also saving stock solution and achieving economic principles. As shown in Figure 3, as the concentration of the fluorescent probe Aptamer increases, the fluorescence intensity gradually increases. At a concentration of 0.15 μmol / L, the fluorescence value is 225.78633. It can be determined that the Aptamer solution with a concentration of 0.15 μmol / L should be used for subsequent experiments.

[0113] 2.2 Reaction temperature screening results of Aptamer and PC-strand

[0114] Temperature will affect the effectiveness of fluorescence quenching to a certain extent. In order to restore fluorescence to its optimal effect when AFB1 is added in subsequent experiments, it is necessary to optimize the reaction temperature and select the optimal temperature for fluorescence quenching. According to the optimized fluorescence curve and histogram of the reaction temperature of Aptamer and PC-strand (as shown in Figure 4), it can be seen that the fluorescence value increases with increasing reaction temperature, indicating that low temperature is conducive to the hybridization of Aptamer and PC-strand. The fluorescence quenching effect is best when the reaction temperature is 4°C, so the temperature used in subsequent experiments is 4°C.

[0115] 2.3 Screening results of PC-strand concentration

[0116] As shown in Figure 5, with the increase of PC-strand concentration, the fluorescence intensity of Aptamer gradually decreased. When the concentration was 0.24 μmol / L (C Aptamer :CPC-strand =1:1.6), the fluorescence quenching tended to be stable. Therefore, in subsequent experiments, the concentration of PC-strand was selected to be 0.24 μmol / L.

[0117] 2.4 Effect of bases

[0118] As shown in Figure 6, in the absence of light, at 150 ng / mL of AFB1, the fluorescence change of A-PD was weak due to the FRET effect, with a 2.1-fold increase in fluorescence signal (Figure 6B). However, AD, which does not contain the eight bases following the PC bond, produced a significant fluorescence signal change, with a 7.37-fold increase in fluorescence signal (Figure 6A). This suggests that increasing the number of bases strengthens the binding of AFB1's aptamer to DNA, reducing sensing performance.

[0119] 2.5 Impact of PC Health

[0120] As shown in Figure 7, in the absence of illumination and in the presence of 150 ng / mL AFB1, the fluorescence signal of A-nPD, lacking a PC bond, increased by 210%, while the fluorescence signal of AD, lacking a PC bond and containing eight bases, increased by 601%. In the presence of illumination and 150 ng / mL AFB1, the fluorescence signal of A-nPD increased by 223% and that of AD by 613%. Illumination had no effect on the fluorescence changes of A-nPD and AD. This demonstrates that the introduction of a PC bond into A-PD is crucial for the time-gated design of AFB1 detection.

[0121] 2.6 Effect of Light

[0122] As shown in Figure 8, when the irradiation intensity is 5mW / cm 2 After irradiation under the conditions of 5 mW / cm, the fluorescence intensity increased by 430% when AFB1 was 100 ng / mL, while without irradiation, the fluorescence intensity increased by 430%. 2 Under irradiation conditions, the fluorescence intensity increased by 178% at 100 ng / mL of AFB1. This result shows that light can activate the sensing function and broaden the detection range.

[0123] As can be seen from Figure 9, under the light condition, the PC bond of the PC-strand is disconnected.

[0124] 2.7 Effect of photoperiod

[0125] As shown in Figure 10, when the irradiation intensity is 5mW / cm 2 Under the same conditions, at an AFB1 concentration of 100 ng / mL, the fluorescence intensity of A-PD gradually increased. Prolonged illumination caused more PC bonds to break, generating more photoactivated probes, which in turn bound to AFB1 and increased fluorescence. However, after 8 minutes, the fluorescence gradually leveled off. These results indicate that illumination can activate the sensing function, and the irradiation time is 8 minutes.

[0126] As shown in Figure 11, in the circular dichroism spectrum, CD 280nm The positive peak is the base complementary pairing peak, CD 240nm The negative peak is the DNA helical structure peak. When the irradiation intensity of A-PD is 5mW / cm 2 After irradiation under the conditions of 100ng / mL of AFB1, CD 280nm Positive peak increased by 39.1%, CD 240nm The negative peak is reduced by 565%. 2 Under the condition of irradiation, when AFB1 was 100ng / mL, CD 280nm Positive peak decreased by 20.0%, CD 240nm The negative peak decreased by 163.8%. The results showed that light was more conducive to the binding of AFB1 to the aptamer and the change of its conformation.

[0127] 2.8 Effect of reaction temperature on AFB1 detection

[0128] As shown in Figure 12, since temperature has a great influence on the specific recognition of target molecules by aptamers, the irradiation intensity is 5mW / cm 2 Under the conditions of 100 ng / mL AFB1, the change in reaction temperature had a significant impact on the fluorescence intensity of the reaction system. When the reaction temperature was 37°C, the fluorescence value was the highest. Therefore, 37°C was selected as the reaction temperature in subsequent reactions.

[0129] 2.9 AFB1 detection sensitivity experimental results

[0130] As shown in Figure 13, after 8 minutes of illumination, the fluorescence value increased with the increase of AFB1 concentration (0-625 ng / mL). According to the fitting straight line and standard deviation calculation in the line graph (B) in Figure 13, the detection limit LOD was

[0131] 2.10 AFB1 Detection Specificity Experimental Results

[0132] As shown in Figure 14, after 8 minutes of illumination, the fluorescence intensity increased 4.4-fold in the presence of AFB1 (100 ng / mL). When OTA or ZEN (100 ng / mL) were present at the same concentration, the fluorescence intensity remained essentially unchanged. However, the addition of AFB1 at the same concentration significantly increased the fluorescence intensity. These results demonstrate that this system exhibits good selectivity for AFB1.

[0133] 2.11 AFB1 Detection Applicability

[0134] As shown in Figure 15, after 8 minutes of illumination, when AFB1 was present in the system, the fluorescence intensity gradually increased with the increase in the concentration of AFB1. The results show that the sensor has good applicability for AFB1 in the rice system.

[0135] As shown in Figure 16, after 8 minutes of illumination, when AFB1 was present in the system, the fluorescence intensity gradually increased with the increase in AFB1 concentration. The results show that the sensor has good applicability for AFB1 in the corn system.

[0136] As shown in Figure 17, after 8 minutes of illumination, when AFB1 was present in the system, the fluorescence intensity gradually increased with the increase in the concentration of AFB1. The results show that the sensor has good applicability for AFB1 in the soybean system.

[0137] In summary, the present invention conducted a series of investigations and selected optimal values ​​from the experimental results. Specifically, the optimal aptamer solution concentration was selected to be 0.15 μmol / L, and the optimal tris buffer solution (10 mM Tris, 120 mM NaCl, 5 mM KCl, pH = 7.2) was selected. Photocleavage was demonstrated to be effective in quenching fluorescence in the reaction between aptamer and PC strands. The optimal reaction temperature and time for aptamer and PC strands were 4°C, 20 minutes, and a 1:1.2 ratio of aptamer to PC strand concentrations. The feasibility of experiments demonstrating fluorescence recovery after photocleavage by adding AFB1 was also demonstrated, with the optimal photocleavage time being 20 minutes. The optimal reaction temperature for AFB1 was selected to be 37°C, and the sensitivity of AFB1 was determined to be 0.07018 ng / mL. This experimental method was then applied to detect AFB1 content in food samples such as corn, rice, and soybeans.

[0138] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A fluorescent probe combination for detecting aflatoxin B1 content based on photo-cleavage fluorescence analysis, characterized in that: Includes fluorescent probes Aptamer and PC-strand; The nucleotide sequence of the Aptamer is shown in SEQ ID NO.1, and the FAM group is labeled at the 3' end of the nucleotide sequence shown in SEQ ID NO.1; The nucleotide sequence of the PC-strand is as shown in SEQ ID NO.2, the TAMRA group is labeled at the 5' end of the nucleotide sequence shown in SEQ ID NO.2, and the PC group is labeled between the bases at 10bp and 11bp of the nucleotide sequence shown in SEQ ID NO.2; The structural formula of the PC group is as follows:

2. Use of the fluorescent probe combination as claimed in claim 1 in preparing a kit for detecting aflatoxin B1 content by fluorescence analysis based on photocleavage.

3. A kit for detecting aflatoxin B1 content based on fluorescence analysis of light cutting, characterized in that: The kit comprises the fluorescent probe combination according to claim 1.

4. Use of the fluorescent probe combination according to claim 1 or the kit according to claim 3 in detecting the content of aflatoxin B1.

5. A method for detecting aflatoxin B1 content based on fluorescence analysis of light cutting, characterized in that: The following steps are involved: (1) After the Aptamer solution, the PC-strand solution and the Tris buffer solution are mixed and reacted, the mixture is irradiated with ultraviolet light to obtain a mixed reaction system solution; (2) taking the same volume of the mixed reaction system solution obtained in step (1), respectively adding the same volume of aflatoxin B1 standard solution but with different concentrations, respectively detecting the fluorescence intensity after the reaction, and then drawing a standard curve of aflatoxin B1 concentration and fluorescence intensity; (3) taking the mixed reaction system solution obtained in step (1), adding the sample solution to be tested, detecting the fluorescence intensity after the reaction, and then calculating the aflatoxin B1 content in the sample solution to be tested according to the standard curve; In step (1), the nucleotide sequence of the Aptamer is as shown in SEQ ID NO.1, and the FAM group is labeled at the 3' end of the nucleotide sequence shown in SEQ ID NO.1; the nucleotide sequence of the PC-strand is as shown in SEQ ID NO.2, and the TAMRA group is labeled at the 5' end of the nucleotide sequence shown in SEQ ID NO.2, and the PC group is labeled between the bases at 10 bp and 11 bp of the nucleotide sequence shown in SEQ ID NO.2; The structural formula of the PC group is as follows: In step (3), the volume ratio of the mixed reaction system solution to the sample solution to be tested is the same as the volume ratio of the mixed reaction system solution to the aflatoxin B1 standard solution in step (2).

6. The method according to claim 5, characterized in that In step (1), in the mixed solution of the Aptamer solution, the PC-strand solution and the Tris buffer solution, the concentration of the Aptamer is 0.15 μmol / L; the concentration of the PC-strand is 0.24 μmol / L.

7. The method according to claim 5, characterized in that In step (1), the temperature of the mixed reaction is 4°C.

8. The method according to claim 5, characterized in that In step (1), the irradiation intensity of the ultraviolet light is 5 mW / cm 2 .

9. The method according to claim 8, characterized in that The irradiation time of the ultraviolet light is 8 minutes.

10. The method according to claim 5, characterized in that In step (2) and step (3), the reaction temperature is 37°C.

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