A preparation method of a fluorescent aptamer sensor based on H-type DNA nanostructure

Abstract

The application belongs to the technical field of biosensing detection, and discloses a preparation method of a fluorescence aptamer sensor based on H-type DNA nanostructure and application thereof, wherein a certain amount of cDNA1, cDNA2 and a displacement strand ZH are mixed and incubated, then an aflatoxin B1 aptamer strand (AFB1-Apt) is added and incubated to obtain an H-type DNA nanostructure, and hollow carbon spheres are added to the above-mentioned mixed system and mixed and incubated; when AFB1 is absent, AFB1-Apt and ZH undergo strand displacement and are free in the system, at this time, ZH is adsorbed by a quenching material, so that the fluorescence thereof is quenched; when AFB1 exists, AFB1 preferentially combines with AFB1-Apt, at this time, ZH participating in the formation of the H-type structure is free in the system in a double-stranded structure, so that the fluorescence signal is maintained, and thus the preparation of the fluorescence aptamer sensor is completed; AFB1 is quantitatively analyzed according to the response signal change. According to the above-mentioned steps, a real sample with a standard addition is detected, and a recovery rate is calculated. The prepared fluorescence aptamer sensor has high sensitivity, good selectivity and excellent practical application ability.

Description

Technical Field

[0001] This invention belongs to the field of biosensor detection technology, specifically relating to a method for preparing a fluorescent aptamer sensor based on an H-type DNA nanostructure. Background Technology

[0002] In recent years, many agricultural products and animal feeds have been susceptible to aflatoxin contamination, posing a potential threat to human and animal health due to its high toxicity. Aflatoxin B1 (AFB1) is a toxin produced by certain strains of Aspergillus flavus and Aspergillus parasiticus, and is known to have genotoxic, immunotoxic, teratogenic, carcinogenic, and mutagenic effects. Therefore, it has been classified as a Group 1 naturally occurring carcinogen by the International Agency for Research on Cancer (IARC) of the World Health Organization. According to the Codex Alimentarius Commission, the maximum limit for total aflatoxin contamination in food is 10-15 μg / kg, while the national standard (GB2761-2005) specifies a maximum allowable content of 5.0 μg / kg. AFB1 can enter the food chain through contaminated food and animal feed, posing a threat to human health. Due to its high heat resistance, conventional food processing methods are insufficient to eliminate the hazards of AFB1. Therefore, it is necessary to establish a simple and accurate method for detecting AFB1 in agricultural products.

[0003] Currently, conventional methods for AFB1 detection are based on high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). These methods can achieve high sensitivity and selectivity for AFB1 detection. However, HPLC and LC-MS methods are limited by expensive equipment, cumbersome sample pretreatment, and the need for skilled operators. Immunoassay methods, such as enzyme-linked immunosorbent assay (ELISA), are simple to operate and highly sensitive. However, ELISA requires high purity of both antigen and antibody, purification is difficult, antibody preparation is complex, and antibody stability issues arise during transportation and storage.

[0004] Nucleic acid aptamers (aptamers for short) are specific target-binding molecules screened using the Systematic Evolution of Ligands Exponential Enrichment (SELEX) technique. They can bind tightly to and selectively target specific analytes. Existing techniques, such as the method for detecting aflatoxin B1 in feed based on nucleic acid aptamers (by Peng Zhenfei et al., "Method for Detecting Aflatoxin B1 in Feed Based on Nucleic Acid Aptamers"), utilize the specific binding of FAM-labeled nucleic acid aptamers to aflatoxin B1, while failing to pair with the complementary sequence of coupled BHQ1, resulting in a change in fluorescence value and thus achieving the detection of AFB1. This method demonstrates the high affinity of nucleic acid aptamers for their targets. Compared to antibodies, nucleic acid aptamers have advantages such as higher thermal stability, lower synthesis cost, and ease of long-term storage and transportation, and have been widely used in many fields such as cell imaging, disease treatment, and microbial detection. Detection methods based on nucleic acid aptamers can avoid some limitations of chromatographic and immunoassay methods. Fluorescent aptamer sensors have been widely used in the field of chemistry due to their simple operation and high sensitivity.

[0005] In summary, the fluorescence sensor method based on nucleic acid aptamers has relatively significant advantages, including high specificity, simple operation, and good stability, and has good application prospects. Summary of the Invention

[0006] A method for preparing a fluorescent aptamer sensor based on an H-type DNA nanostructure comprises the following steps:

[0007] (1) Formation of H-type DNA nanostructure: A certain amount of complementary strand cDNA1 and cDNA2 and substitution strand ZH were placed in a constant temperature shaker and incubated for a certain time. Then, a certain amount of aflatoxin B1 aptamer strand (AFB1-Apt) and different concentrations of aflatoxin B1 (AFB1) were added to the above mixture and incubated for a certain time to finally obtain H-type DNA nanostructure.

[0008] (2) Construction of the fluorescent aptamer sensor: A certain amount of quenching material was added to the system in step (1) above and incubated in a constant temperature oscillator for a certain time. When AFB1 was not present, AFB1-Apt produced a chain substitution reaction to displace ZH and was adsorbed by the quenching material, resulting in fluorescence resonance energy transfer (FRET) effect, which quenched its fluorescence and produced a lower background signal. When AFB1 was added, AFB1 preferentially bound to AFB1-Apt. At this time, ZH, which participated in the formation of H-type DNA nanostructure, maintained the fluorescence signal in the solution due to the free double-stranded structure. Thus, the fluorescent aptamer sensor was prepared. Finally, the volume was adjusted with buffer and thoroughly mixed by vortex mixer. The excitation wavelength, measurement range and measurement voltage were set, and the fluorescence intensity of the emission wavelength was recorded by fluorescence spectrophotometer. The quantitative analysis of AFB1 was achieved by the change in response signal before and after the addition of AFB1.

[0009] Further specifying, in steps (1) and (2), the sequence of ZH is 5'-AATGTGTGTTGGTTCTCTCTGTC-3', wherein the fluorescent group labeled at the 3' end is one or more of Alexa Fluor 594, FAM, ROX, Cy3, and Cy5, with FAM being preferred.

[0010] Further, the temperature conditions for the isothermal oscillator treatment in steps (1) and (2) are 25~50℃ and the time is 0.5~2h.

[0011] Further specifying, in step (1), the sequence of cDNA1 is 5'-GAGACACAGAGAGACTGAGAAGCGCATCACAACACGTCCA-3'.

[0012] Further specifying, in step (1), the sequence of cDNA2 is 5'-GAGACACAGAGAGATGATGCGCTTCTCGGCAACACGTCCA-3'.

[0013] Further specifying, in step (2), the quenching material is one of graphene oxide, three-dimensional nitrogen-doped carbon nanosheets, nitrogen-doped carbon nanorods, and hollow carbon spheres, with hollow carbon spheres being preferred.

[0014] Further specifying, in step (2), the concentration of the quenching material is 0.1~0.5 mg / mL.

[0015] Further specifying, in step (2), the buffer solution is one or two of Tris-HCl, PBS, and HEPES, with Tris-HCl preferred and a pH of 7.4.

[0016] Compared with the prior art, the present invention has the following significant features:

[0017] (1) The spherical carbon nanomaterials with hollow structure prepared by the present invention have high quenching efficiency.

[0018] (2) This invention utilizes the strong specificity, high affinity and good stability of nucleic acid aptamers to invent two complementary H-type backbone strands that can be combined with ZH for complementary hybridization. The aptamer strands can replace ZH through a strand substitution reaction, thereby constructing a novel fluorescent aptamer sensor for detecting AFB1.

[0019] (3) The fluorescent aptamer sensor prepared in this invention has the characteristics of high accuracy, good selectivity, fast response speed, wide linear range and low detection limit for the detection of AFB1. At the same time, the detection results of actual samples show that the prepared sensor has good practical application value.

[0020] The above description is only an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the detection principle of the fluorescent aptamer sensor of the present invention.

[0022] Figure 2 The fluorescence intensity of FAM was measured for the sensor prepared in Example 1 at 0 ng / mL AFB1 (dotted line), 50 ng / mL AFB1 (dashed line), and 500 ng / mL AFB1 (solid line).

[0023] Figure 3 This is a comparison chart of the selectivity of the sensor constructed in Example 1 for AFB1 in the presence of other interfering toxins.

[0024] Figure 4 This demonstrates the application of the fluorescent aptamer sensor constructed in Example 1 in a real sample. Implementation

[0025] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments. Example

[0026] A method for fabricating a fluorescent aptamer sensor based on an H-type DNA nanostructure and its application are described below. Figure 1 As shown.

[0027] A method for preparing a fluorescent aptamer sensor based on an H-type DNA nanostructure includes the following steps:

[0028] (1) Take 10 μL of 1 μmol / L cDNA1 and cDNA2 and 10 μL of 2 μmol / L ZH respectively, mix them, shake them thoroughly with a vortex mixer, and incubate them in a 37℃ constant temperature shaker for 30 min to obtain H-type DNA nanostructures.

[0029] (2) Add 10 μL of 2 μmol / L AFB1-Apt and AFB1 test solution to the solution in step (1) above and mix thoroughly. Incubate in a 37 ℃ constant temperature shaker for 30 min. Due to the high specificity of nucleic acid aptamers, AFB1-Apt preferentially binds to AFB1 and no strand displacement reaction occurs, so that ZH in the H-type DNA nanostructure remains in double strand state. Then add 10 μL of 0.25 mg / mL hollow carbon spheres to the above system and incubate in a 37 ℃ constant temperature shaker for 30 min to obtain the fluorescent aptamer sensor. Finally, take Tris-HCl buffer to make up the volume and mix thoroughly with a vortex mixer. Detect the fluorescence intensity of FAM by a fluorescence spectrophotometer.

[0030] The specific steps for detecting fluorescence intensity are as follows: use a micro-volume cuvette to hold the sample to be tested, set the excitation wavelength of the fluorescence spectrophotometer to 490 nm, the emission wavelength measurement range to 510~610 nm, the slit to 10 nm, and the measurement voltage to 600 V.

[0031] Figure 2 The fluorescence intensity of FAM was measured for the sensor prepared in Example 1 at 0 ng / mL AFB1 (dotted line), 50 ng / mL AFB1 (dashed line), and 500 ng / mL AFB1 (solid line).

[0032] Comparative Example 1

[0033] A method for preparing a fluorescent aptamer sensor based on an H-type DNA nanostructure is described. Unless otherwise specified, the steps are the same as in Example 1, except that in step (2), AFB1-Apt is first added to the solution from step (1), thoroughly mixed, and incubated for 30 min. Then, the AFB1 test solution is added to the mixed solution and incubated for another 30 min before measuring the fluorescence intensity of FAM. Compared to Example 1, the relative fluorescence intensity value when measuring the same concentration of AFB1 (50 ng / mL) is significantly lower, indicating that adjusting the order of adding the AFB1 test solution can increase the relative fluorescence intensity between the blank and spiked solutions, thereby improving the sensor's sensitivity.

[0034] Comparative Example 2

[0035] A method for preparing a fluorescent aptamer sensor based on H-type DNA nanostructures is described. Unless otherwise specified, the steps are the same as in Example 1, except that in step (2), the quenching material used is nitrogen-doped carbon nanorods, which are added to a mixed solution and incubated for 30 min before measuring the fluorescence intensity of FAM. Compared to Example 1, the relative fluorescence intensity value when measuring the same concentration of AFB1 (50 ng / mL) is significantly lower, indicating that the hollow carbon spheres used in Example 1 have a higher efficiency in fluorescence quenching. Example

[0036] A method for preparing a fluorescent aptamer sensor based on an H-type DNA nanostructure and its application, the practical application of which includes the following steps:

[0037] To verify the specific recognition of AFB1 by the novel fluorescent biosensor, AFB1 standard was added to Tris buffer solution to achieve a concentration of 50 ng / mL in the sample. Nine other standard solutions of interfering mycotoxins were prepared using Tris buffer solution, each with a concentration of 500 ng / mL, ten times the concentration of AFB1. The detection system constructed in Example 1 was used to detect the above nine different interfering mycotoxin standard solutions and their mixtures with AFB1. The detection results are as follows: Figure 3 As shown, this demonstrates that the method of the present invention has good selectivity for AFB1. Example

[0038] A method for preparing a fluorescent aptamer sensor based on an H-type DNA nanostructure and its application, the practical application of which includes the following steps:

[0039] (1) Actual sample processing: Take 0.5 g of spiked sample (corn flour, wheat flour), add it to 10 mL of methanol / water solution (ratio 7:3), mix thoroughly and shake for 20 minutes, then take it out and centrifuge for 10 min at a speed of 10000 rpm. After centrifugation, take out the supernatant to obtain the sample extract.

[0040] (2) Sample detection: After operating the sample extract according to steps (1) to (3) of Example 1, the fluorescence intensity signal was measured and substituted into the standard curve to obtain the concentration of AFB1 in the sample. Each sample was measured three times and the average value was taken. The average recovery rate is shown in the table below:

[0041]

[0042] The prepared fluorescent aptamer sensor has been verified to exhibit high sensitivity, wide linear range, and low detection limit for AFB1 detection. Furthermore, detection results on actual samples (such as corn flour and wheat flour) demonstrate the reliable practical application value of the prepared sensor.

[0043] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0044] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a fluorescent aptamer sensor based on an H-type DNA nanostructure, characterized in that, Includes the following steps: (1) Formation of H-type DNA nanostructure: A certain amount of complementary strand cDNA1 and cDNA2 and substitution strand ZH were placed in a constant temperature shaker and incubated for a certain time. Then, a certain amount of aflatoxin B1 aptamer strand AFB1-Apt and different concentrations of aflatoxin B1 (AFB1) were added to the above mixture and incubated for a certain time to finally obtain H-type DNA nanostructure. (2) Construction of fluorescent aptamer sensor: Add a certain concentration of quenching material to the system in step (1) above, and incubate in a constant temperature shaker for a certain time. When AFB1 is not present, AFB1-Apt will displace ZH and be adsorbed by the quenching material, thus quenching its fluorescence. When AFB1 is added, AFB1 preferentially binds to AFB1-Apt. At this time, ZH, which participates in the formation of H-type DNA nanostructure, remains free in the solution due to the double-stranded structure, maintaining the fluorescence signal. Thus, the fluorescent aptamer sensor is prepared. Finally, adjust the volume with buffer and mix thoroughly with a vortex mixer. Set a fixed excitation wavelength, measurement range and measurement voltage, and record the fluorescence intensity of the emission wavelength with a fluorescence spectrophotometer. Quantitative analysis of AFB1 is achieved by the change in response signal before and after the addition of AFB1. In step (1), the sequence of ZH is 5'-AATGTGTGTTGGTTCTCTCTGTC-3'; the sequence of cDNA1 is 5'-GAGACACAGAGAGACTGAGAAGCGCATCACAACACGTCCA-3'; and the sequence of cDNA2 is 5'-GAGACACAGAGAGATGATGCGCTTCTCGGCAACACGTCCA-3'.

2. The method for preparing a fluorescent aptamer sensor based on an H-type DNA nanostructure according to claim 1, characterized in that, The 3' end is labeled with one of the following fluorescent groups: Alexa Fluor 594, FAM, ROX, Cy3, or Cy5.

3. The method for preparing a fluorescent aptamer sensor based on an H-type DNA nanostructure according to claim 1, characterized in that, In step (1), the conditions for the constant temperature oscillator treatment are: temperature 25~50℃, time ≤3h.

4. The method for preparing a fluorescent aptamer sensor based on an H-type DNA nanostructure according to claim 1, characterized in that, In step (2), the quenching material is one of graphene oxide, three-dimensional nitrogen-doped carbon nanosheets, nitrogen-doped carbon nanorods, and hollow carbon spheres.

5. The method for preparing a fluorescent aptamer sensor based on an H-type DNA nanostructure according to claim 1, characterized in that, In step (2), the concentration of the quenching material is 0.1~0.5 mg / mL.

6. The method for preparing a fluorescent aptamer sensor based on an H-type DNA nanostructure according to claim 1, characterized in that, In step (2), the buffer solution is one or two of Tris-HCl, PBS, and HEPES, with a volume range of 10 to 200 μL and a pH range of 6.0 to 8.0.

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