An aptamer sensor and method for detecting aflatoxin B1 based on aggregation-induced emission effect

Abstract

The present invention discloses an aptamer sensor and method for detecting aflatoxin B1 based on the aggregation-induced emission effect, and belongs to the field of biological detection technology. The aptamer sensor comprises a single-stranded DNA aptamer that specifically binds to aflatoxin B1, a small molecule fluorescent dye that can bind to the single-stranded DNA aptamer and produce the aggregation-induced emission effect, and a carbon nanomaterial that undergoes fluorescence resonance energy transfer with the small molecule fluorescent dye. The aptamer sensor is used to detect aflatoxin B1, and aflatoxin B1 can be quantitatively detected by changes in the intensity of the fluorescent signal. The method of the present invention can achieve high-sensitivity detection of aflatoxin B1, with a low detection limit and good specificity, and provides an effective method for detecting trace amounts of aflatoxin B1 and other harmful substances.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to an aptamer sensor and a method for detecting aflatoxin B1 based on aggregation-induced luminescence effect. Background Art

[0002] Aflatoxins are secondary metabolites secreted by Aspergillus flavus and Aspergillus parasiticus in nature. They are the most stable mycotoxins found in nature with strong toxicity, carcinogenicity, mutagenicity and teratogenicity. They mainly include aflatoxin B1 (aflatoxin B1, AFB1), aflatoxin B2 (aflatoxin B2, AFB2), aflatoxin G1 (aflatoxin G1, AFG1), aflatoxin G2 (aflatoxin G2, AFG2) and their metabolites aflatoxin M1 (aflatoxin M1, AFM1) and aflatoxin M2 (aflatoxin M2, AFM2). Among them, aflatoxin B1 is the most toxic and has been identified as a Class I carcinogen by the International Agency for Research on Cancer.

[0003] Traditional AFB1 detection methods include thin layer chromatography, high performance thin layer chromatography, HPLC, enzyme-linked immunosorbent assay and fluorescence colorimetry. Traditional detection methods often have disadvantages such as long detection cycle, expensive instruments, cumbersome operation and the need for professionals, which limits the application of these methods in actual production and life. Therefore, it is necessary to develop a new detection method that is fast, convenient, low in detection cost and high in detection sensitivity.

[0004] With the development of biotechnology, biosensor technology is gradually being applied to the detection of various harmful factors in food due to its characteristics of short time consumption and simple sample pretreatment, becoming a rapidly emerging new detection technology. Recognition molecules, as important components of biosensors, are key factors affecting the specificity, stability and scope of application of biosensors. Antibodies are currently the most commonly used recognition molecules in biosensors, with high sensitivity and strong specificity, but they are high in cost and easy to inactivate, which greatly limits their application and development.

[0005] Aptamer is an oligonucleotide sequence screened from a synthetic DNA or RNA library by using the systematic evolution of ligands by exponential enrichment (SELEX). Under certain conditions, it can efficiently and specifically recognize and bind to the target molecule. Therefore, aptamer is also called "chemical antibody". Compared with traditional antibodies, aptamers have many advantages, such as easy preparation, wide range of target molecules, strong specificity, high affinity, stable properties, etc. In view of these advantages, as a new type of recognition molecule, aptamer has received extensive attention from multidisciplinary researchers in molecular biology, nanomaterials, molecular recognition and analysis, biomedicine, etc. at home and abroad. Linking aptamers with biological detection is of great significance for the efficient detection of aflatoxin. Summary of the invention

[0006] The purpose of the present invention is to provide an aptamer sensor and method for detecting aflatoxin B1 based on aggregation-induced emission effect, so as to solve the problems existing in the above-mentioned prior art. This method greatly improves the sensitivity of detection, reduces the detection cost, does not require complicated detection means and expensive instruments, and is a new detection method that is fast, convenient, low in detection cost and high in detection sensitivity.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides an aptamer sensor for detecting aflatoxin B1 based on the aggregation-induced emission effect. The aptamer sensor comprises: a single-stranded DNA aptamer that specifically binds to aflatoxin B1, a small molecule fluorescent dye that can bind to the single-stranded DNA aptamer and produce the aggregation-induced emission effect, and a carbon nanomaterial that undergoes fluorescence resonance energy transfer with the small molecule fluorescent dye.

[0009] Preferably, the single-stranded DNA adaptor sequence is 5'-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3'.

[0010] Preferably, the small molecule fluorescent dye includes 4,4'-(1E, 1'E)-2,2'-(anthracene-9,10-diyl)bis(ethylene-2,1-diyl)bis(N,N-dimethylaniline).

[0011] Preferably, the carbon nanomaterial comprises single-walled carbon nanohorns.

[0012] The present invention also provides a method for detecting aflatoxin B1 using the aptamer sensor, comprising the following steps:

[0013] (1) using Tris buffer to prepare aflatoxin B1 standard solution, aptamer solution, small molecule fluorescent dye solution and carbon nanomaterial solution of different concentrations;

[0014] (2) allowing the aptamer solution and the aflatoxin B1 standard solution to react at room temperature, then adding a small molecule fluorescent dye solution and mixing, and allowing the mixture to react at room temperature in the dark;

[0015] (3) adding the carbon nanomaterial solution to the mixed solution obtained after the reaction in step (2), reacting at room temperature in the dark, scanning with a fluorescence spectrophotometer, and establishing a standard curve based on the relationship between the aflatoxin B1 standard solution and the fluorescence intensity;

[0016] (4) According to the obtained standard curve, aflatoxin B1 in the sample to be tested is detected to obtain the content of aflatoxin B1 in the sample to be tested.

[0017] Preferably, the concentration of the aptamer solution is 200 nM; the concentration of the small molecule fluorescent dye solution is 12 μM.

[0018] Preferably, in step (2), the aptamer solution and the aflatoxin B1 standard solution are mixed in equal volumes, left to stand at room temperature for 15-20 minutes, and then the small molecule fluorescent dye solution is added and mixed, and left to stand at room temperature in the dark for 15-20 minutes; wherein the volume ratio of the small molecule fluorescent dye solution to the aflatoxin B1 standard solution is 1:1.

[0019] Preferably, in step (3), the volume ratio of the carbon nanomaterial solution to the aflatoxin B1 standard solution is 1:1, the concentration of the carbon nanomaterial solution is 20 μg / mL; and the reaction time at room temperature and in the dark is 20-30 min.

[0020] Preferably, in step (3), the scanning wavelength of the fluorescence spectrophotometer is 460 to 650 nm.

[0021] The present invention also provides the application of the aptamer sensor or the method in detecting harmful components in food or food raw materials. More preferably, the food or food raw material is soy sauce.

[0022] The present invention discloses the following technical effects:

[0023] The present invention uses a nucleic acid aptamer that can specifically bind to aflatoxin B1 as a recognition element to construct a fluorescent biosensor based on aggregation-induced emission effect for the detection of aflatoxin B1, thereby improving the sensitivity of detecting aflatoxin B1 and being able to detect aflatoxin B1 at a minimum of 1.83 ng / mL. The method of the present invention provides an effective method for the detection of trace amounts of aflatoxin B1 and other harmful substances.

[0024] As a method for detecting aflatoxin B1 based on the aggregation-induced luminescence effect, the present invention greatly reduces the cost of detecting the target toxin and is simple and convenient to operate;

[0025] The present invention solves the problems of high cost, complicated detection scheme and long detection time of aflatoxin B1 detection. The present invention adopts nucleic acid aptamers that can specifically identify aflatoxin B1, combines the aggregation-induced emission effect of small molecule fluorescent dyes and the fluorescence resonance energy transfer effect between dye molecules and carbon nanomaterials, greatly improves the sensitivity of detection, reduces the detection cost, does not require complex detection means and expensive instruments, and is a new detection method that is fast, convenient, low in detection cost and high in detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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 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 drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 The schematic diagram of the detection of aflatoxin B1 based on the aggregation-induced emission effect;

[0028] Figure 2 is the fluorescence quenching efficiency of single-walled carbon nanohorns at different concentrations;

[0029] Figure 3 The figure is a standard curve obtained by detecting aflatoxin B1 standard solution; wherein A is the fluorescence spectrum curve of aflatoxin B1 at different concentrations; and B is the standard curve of different aflatoxin B1. DETAILED DESCRIPTION

[0030] 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The technical principle of the present invention (such as Figure 1The aptamer specifically recognizes and binds to aflatoxin B1 to form a stable folded secondary structure when aflatoxin B1 is present in the system. A small molecule fluorescent dye DSAI with aggregation-induced emission effect is added. The dye aggregates with the aptamer DNA chain (its design and synthesis refer to "Fereshte Sadat Sabet, Morteza Hosseini, Hossein Khabbaz, Mehdi Dadmehr, Mohammad Reza Ganjali. FRET-based aptamer biosensor for selective and sensitive detection of aflflatoxinB1in peanut and rice. Food Chemistry, 220 (2017) 527–532."), and the aggregation-induced emission effect occurs, generating a significantly enhanced fluorescence signal. After adding single-walled carbon nanohorns, the folded secondary structure formed by the aptamer and aflatoxin B1 can resist the adsorption of single-walled carbon nanohorns, so that the small molecule fluorescent dye is far away from the single-walled carbon nanohorns and cannot produce fluorescence resonance energy transfer effect. Therefore, the aggregation-induced fluorescence signal generated by the small molecule fluorescent dye binding to the aptamer DNA cannot be quenched, so that the fluorescent signal is retained, and the fluorescent signal in the system is strong. On the contrary, when AFB1 is not present in the system, the aptamer is in a free single-stranded DNA state, and the single-walled carbon nanohorn has a strong adsorption effect on the aptamer DNA chain in the free single-stranded state, so that the free single-stranded DNA is adsorbed to its surface, and the small molecule fluorescent dye bound to the single-stranded DNA produces a fluorescence resonance energy transfer effect due to the close distance to the single-walled carbon nanohorn, so that the fluorescent signal is quenched, and the fluorescent signal in the system is weak. Therefore, the present invention can realize the quantitative detection of AFB1 according to the difference in the detected fluorescent signals.

[0036] Example 1 A method for constructing an aptamer sensor for detecting aflatoxin B1 based on aggregation-induced emission effect

[0037] 1) Prepare aflatoxin B1 standard solutions of different concentrations

[0038] Aflatoxin B1 standard solutions of different concentrations (20, 40, 80, 200, 400, 2000, 4000, 8000 ng / mL) were prepared using 10 mM Tris buffer (pH 8.0, 120 mM NaCl, 5 mM KCl, 10 mM MgCl2, 20 mM CaCl2).

[0039] 2) The above-mentioned Tris buffer was used to prepare a 200 nM aptamer solution, a 12 μM small molecule fluorescent dye with aggregation-induced emission effect (4,4'-(1E, 1'E)-2,2'-(anthracene-9,10-diyl)bis(ethylene-2,1-diyl)bis(N,N-dimethylaniline), DSAI) solution, and a 20 μg / mL single-walled carbon nanohorn solution (purchased from Nanjing Jicang Nanotechnology Co., Ltd.).

[0040] 3) 100 μL of the aptamer solution obtained in step 2) and 100 μL of the standard solution obtained in step 1) were mixed, and the mixture was allowed to react at room temperature for 15 min. Then, 100 μL of the DSAI solution obtained in step 2) was added, and the mixture was allowed to react at room temperature in the dark for 15 min.

[0041] 4) Add 100 μL of the single-walled carbon nanohorn solution to the mixed solution obtained in step 3), react at room temperature in the dark for 30 minutes, measure the fluorescence three times under the conditions of a scanning wavelength range of 460 to 650 nm, an excitation wavelength of 430 nm, and a slit width of 5 nm to obtain an average value, scan using a fluorescence spectrophotometer, and establish a standard curve based on the relationship between the concentration and the corresponding fluorescence intensity value.

[0042] 5) According to the standard curve obtained in step 4), the negative actual sample added with a known concentration of aflatoxin B1 is tested to determine the content of aflatoxin B1.

[0043] Example 2

[0044] 2.1 Optimization of carbon nanohorn concentration

[0045] Since there is a strong non-covalent binding force between the single-stranded aptamer DNA and the single-walled carbon nanohorn, the single-stranded DNA molecule chain can be adsorbed (entangled) on the surface of the single-walled carbon nanohorn, resulting in the fluorescence resonance energy transfer effect of the fluorescent dye molecule (DSAI) bound to the single-stranded DNA due to the close distance to the single-walled carbon nanohorn, resulting in fluorescence quenching. Therefore, the concentration of single-walled carbon nanohorns in the reaction system is a very important factor. If the proportion of carbon nanohorns is too low, the fluorescence background will be too high. On the contrary, if the proportion of carbon nanohorns is too high, the adsorption force on the aptamer molecules will be too strong, resulting in a decrease in sensitivity.

[0046] The concentration of fixed aptamer DNA was 50 nM, and the concentration of DSAI was 3 μM. On this basis, single-walled carbon nanohorn solutions with concentrations of 0, 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL, 12.5 μg / mL, and 15 μg / mL were prepared, respectively. The solutions were allowed to stand at room temperature for 30 minutes, and the fluorescence spectrum was measured.

[0047] The experimental results are as follows Figure 2As shown in the figure, the maximum fluorescence absorption intensity decreases with the increase of the single-walled carbon nanohorn concentration. When the single-walled carbon nanohorn concentration reaches 5μg / mL, the fluorescence intensity decreases by 90%. Considering that when the single-walled carbon nanotube concentration is too high, it will provide too much force restriction and adsorb the single-stranded DNA aptamer, which will inevitably affect the sensitivity of the aptamer to the target molecule aflatoxin B1 and affect the experimental effect. Therefore, the experiment finally chose the single-walled carbon nanohorn concentration of 5μg / mL, which not only ensures a good fluorescence quenching efficiency, but also does not produce too much force on the aptamer DNA to reduce the detection sensitivity.

[0048] 2.2 Testing process and testing conditions

[0049] 100 μL of aptamer DNA solution (concentration of 200 nM) was mixed with 100 μL of aflatoxin B1 solution of different concentrations, and allowed to react at room temperature for 15 min. Then, 100 μL of DSAI solution (concentration of 12 μM) was added, and allowed to react at room temperature in the dark for 15 min. Then, 100 μL of single-walled carbon nanohorn solution (concentration of 20 μg / mL) was added to the system, and the total volume was 400 μL. After equilibration at room temperature in the dark for 30 min, fluorescence measurement was performed. The wavelength range was 460-650 nm, the excitation wavelength was 430 nm, the slit width was 5 nm, and the average value was taken after 3 scans.

[0050] Example 3

[0051] 3.1 Sensitivity determination

[0052] The detection process was carried out according to the detection conditions in 2.2 (i.e. the optimal conditions).

[0053] According to the fluorescence spectrum curves corresponding to different aflatoxin B1 solutions ( Figure 3 A), select the fluorescence intensity value corresponding to the wavelength of 522nm, use the logarithm of the concentration of aflatoxin B1 as the horizontal axis and the fluorescence intensity value corresponding to 522nm as the vertical axis to draw a standard curve ( Figure 3 B), the analysis showed that the system had a good fluorescence response to AFB1 molecules in the range of 5-500 ng / mL, with a good linear relationship, the regression equation was y=33.2963x+22.9573, and the correlation coefficient was R 2 =0.9905. According to the 3σ / slope calculation rule, the detection limit of this method was calculated to be 1.83 ng / mL.

[0054] 3.2 Specificity determination

[0055] Two structural analogs of aflatoxin B2, aflatoxin M1 and three other common mycotoxins zearalenone (ZEA), fumonisin B1 (FB1) and ochratoxin A (OTA) were used as detection targets instead of aflatoxin B1 for specificity study. The concentrations of the two structural analogs and the other three mycotoxins added were the same as the concentration of aflatoxin B1 mentioned above, and the operation method was consistent with that of aflatoxin B1 detection. The fluorescence intensity values ​​of the above five toxins at 522nm showed no obvious change compared with the blank samples without any target added. It was concluded that the aptamer did not recognize other structural analogs and mycotoxins except aflatoxin B1, and this method had good specificity.

[0056] 3.3 Sample addition and recovery

[0057] The negative soy sauce sample was diluted 50 times with Tris buffer, and aflatoxin B1 standards with concentrations of 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL were added respectively. This method was used to determine the recovery rate of the added standard. The final recovery rate ranged from 91.3% to 109.6%, which can be used for the determination of actual samples.

[0058] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An aptamer sensor for detecting aflatoxin B1 based on aggregation-induced emission effect, characterized in that: The aptamer sensor comprises: a single-stranded DNA aptamer that specifically binds to aflatoxin B1, a small molecule fluorescent dye that can bind to the single-stranded DNA aptamer and produce an aggregation-induced luminescence effect, and a carbon nanomaterial that undergoes fluorescence resonance energy transfer with the small molecule fluorescent dye; The single-stranded DNA adaptor sequence is 5'-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3'; The carbon nanomaterial includes single-walled carbon nanohorns.

2. The aptamer sensor according to claim 1, characterized in that: The small molecule fluorescent dye includes 4,4'-(1E, 1'E)-2,2'-(anthracene-9,10-diyl)bis(ethylene-2,1-diyl)bis(N,N-dimethylaniline).

3. A method for detecting aflatoxin B1 using the aptamer sensor according to any one of claims 1 to 2, characterized in that: The steps include: (1) using Tris buffer to prepare aflatoxin B1 standard solution, aptamer solution, small molecule fluorescent dye solution and carbon nanomaterial solution of different concentrations; (2) allowing the aptamer solution and the aflatoxin B1 standard solution to react at room temperature, then adding a small molecule fluorescent dye solution and mixing, and allowing the mixture to react at room temperature in the dark; (3) adding the carbon nanomaterial solution to the mixed solution obtained after the reaction in step (2), reacting at room temperature in the dark, scanning with a fluorescence spectrophotometer, and establishing a standard curve based on the relationship between the aflatoxin B1 standard solution and the fluorescence intensity; (4) According to the obtained standard curve, aflatoxin B1 in the sample to be tested is detected to obtain the content of aflatoxin B1 in the sample to be tested.

4. The method according to claim 3, characterized in that The concentration of the aptamer solution is 200 nM; the concentration of the small molecule fluorescent dye solution is 12 μM.

5. The method according to claim 3, characterized in that In step (2), the aptamer solution and the aflatoxin B1 standard solution are mixed in equal volumes, left to stand at room temperature for 15-20 minutes, and then the small molecule fluorescent dye solution is added and mixed, and left to stand at room temperature in the dark for 15-20 minutes; wherein the volume ratio of the small molecule fluorescent dye solution to the aflatoxin B1 standard solution is 1:

1.

6. The method according to claim 3, characterized in that In step (3), the volume ratio of the carbon nanomaterial solution to the aflatoxin B1 standard solution is 1:1, the concentration of the carbon nanomaterial solution is 20 μg / mL; and the reaction time at room temperature and in the dark is 20-30 min.

7. The method according to claim 3, characterized in that In step (3), the scanning wavelength of the fluorescence spectrophotometer is 460 to 650 nm.

8. Use of the aptamer sensor according to any one of claims 1 to 2, or the method according to any one of claims 3 to 7, in detecting harmful components in food and food raw materials.

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