Preparation method of fluorescent aptamer sensor based on boron nitride doped porous carbon composite material

Abstract

The invention belongs to the technical field of biosensing detection, and discloses a preparation method and application of a fluorescent aptamer sensor based on a boron nitride doped porous carbon composite material. The boron nitride doped porous carbon composite material with the fluorescence quenching capability is prepared. The method comprises the following steps: by utilizing three accurately designed hairpin chain structures, when target acrylamide AA exists, after a hairpin H1 is combined with AA, the structure of the hairpin H1 is opened and is combined with a hairpin H2, exonuclease (EXO III) is used for shearing the hairpin H1, a single chain S1 obtained after shearing is combined with a hairpin H3 marked with carboxyl fluorescein FAM to form double chains S1-H3, EXO III continues to play a role, and S1-H3 is sheared to obtain a lot of free FAM, so that the FAM can be used for detecting the activity of the FAM. Therefore, the fluorescent powder cannot be adsorbed by the boron nitride doped porous carbon composite material, and the fluorescent signal is strong. Quantitative detection of AA is realized by responding to the change of the signal. Compared with other fluorescent sensors for acrylamide detection, the prepared fluorescent aptamer sensor has the advantages of high sensitivity, good repeatability and high accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and particularly relates to a preparation method and application of a fluorescence aptamer sensor based on a boron nitride-doped porous carbon composite material. Background Art

[0002] Since acrylamide (AA) contamination in foods rich in carbohydrates such as fried or baked foods was first discovered and reported in 2002, as a food safety and public health issue, it has become the focus of public attention. Due to its wide application in various industries, residual acrylamide monomers are discharged into environmental media, resulting in environmental pollution. At present, acrylamide has become a global health problem and is listed as a Group 2A carcinogen by the International Agency for Research on Cancer (IARC). Due to its genotoxicity, carcinogenicity, developmental and reproductive effects, many countries have established limit standards for AA in foods to ensure food safety and protect human health. Therefore, in order to meet current regulations and protect the rights and interests of consumers, it is necessary to develop a simple, efficient, rapid, accurate, highly sensitive and specific method for detecting acrylamide.

[0003] Currently, the methods used for acrylamide detection mainly include gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay, etc. These methods can achieve highly sensitive detection. However, the equipment and technical operation requirements of these methods are relatively high, and the sample treatment is complex, time-consuming and costly, which is not suitable for large-scale detection. Therefore, it is necessary to establish a method with high sensitivity, low cost and suitable for rapid detection to detect acrylamide in foods. Fluorescence analysis technology has been widely used in biosensor analysis due to its mature basic theory, simple operation and rapid detection process. Nucleic acid aptamer, screened by systematic evolution of ligands by exponential enrichment technology, is a molecular probe that can specifically bind to the target substance, and has the advantages of simple operation, low cost and good stability, which can improve the specificity of detection. Therefore, by combining a fluorescence biosensor and an aptamer, the anti-interference ability of the fluorescence sensor can be improved through the high specificity of the aptamer. In addition, developing a method with high sensitivity and low background noise is of great significance for the preparation of biosensors. Based on this, the present invention first prepares a boron nitride-doped carbon composite material as a quenching material to reduce the background signal of the sensor, and combines exonuclease-assisted signal amplification technology to effectively improve the sensitivity of the sensor.

[0004] In view of the problems existing in the existing detection technologies, such as poor selectivity and low sensitivity, the present invention constructs a novel fluorescence biosensor. First, nucleic acid aptamers are used to specifically recognize the target AA to enhance the detection specificity. Secondly, an exonuclease-assisted cascade signal amplification strategy is adopted to achieve signal amplification and improve the detection sensitivity. Finally, boron nitride-doped carbon composite materials are introduced as quenching materials to reduce the interference of background signals. Based on the fluorescence sensing strategy of the present invention, while overcoming the problems existing in the above-mentioned existing technologies, it has the characteristics of being more sensitive, economical, simple and stable, which is conducive to the popularization and application of the invention. Summary of the Invention

[0005] The present invention designs a preparation method of a fluorescence aptasensor based on boron nitride-doped porous carbon composite materials A preparation method of a fluorescence aptasensor based on boron nitride-doped porous carbon composite materials is carried out according to the following steps: (1) Preparation of boron nitride-doped porous carbon composite materials: Dissolve soluble zinc salts and imidazole compounds in methanol, add boric acid to this solution, stir evenly and then place it in an oven for reaction. Cool to room temperature, wash and then dry. Calcine the obtained powder under argon to obtain boron nitride-doped porous carbon composite materials.

[0006] (2) Pretreatment of DNA strands H1, H2 and H3: After H1, H2 and H3 are activated and configured into a mother liquor of 100 μM, they should be annealed before use to form a hairpin structure. The specific steps are as follows: Add 100 μM of H1, H2 and H3 to two sterilized centrifuge tubes respectively, then add 50 mM Tric-HCl buffer solution to each centrifuge tube, perform high-temperature annealing on the centrifuge tubes, and then let it stand to slowly cool to room temperature to obtain a reaction product, and store the reaction product in a low-temperature environment.

[0007] (3)Preparation of Fluorescent Aptamer Sensor: When the target acrylamide AA exists in the system, the aptamer sequence of H1 binds to the target, causing the hairpin structure to open. H2 is introduced and incubated. The opened H1 will expose a DNA sequence that binds to H2, forming a double-stranded H1-AA-H2 structure. Exonuclease is added and incubated. It can shear the ends of the H1-AA-H2 structure. After shearing, H1-AA and single-stranded S1 are obtained. H1-AA continues to cycle and bind to the next H2, and S1 binds to H3 labeled with carboxyfluorescein FAM to form a DNA double-stranded S1-H3 structure. The exonuclease continues to function and shear the ends of the S1-H3 structure, resulting in a lot of free FAM. Boron nitride-doped carbon composite is introduced and incubated. The free FAM will not be adsorbed onto the boron nitride-doped carbon composite, and the fluorescence signal is strong. The change in fluorescence intensity is measured by a fluorometer, and a relationship curve between the fluorescence signal response value and the concentration of the target is established.

[0008] Further defined, in step (1), the soluble zinc salt is one or more of zinc chloride, zinc sulfate, zinc carbonate, zinc nitrate hexahydrate, etc.; the imidazole compound is one or more of 5-bromo-1-methyl-1H-imidazole, 4-methyl-5-cyanoimidazole, 2-ethylimidazole, 2-methylimidazole, etc.; the reaction temperature and time are 150 °C and 12 h; the calcination temperature and time are 900 °C and 6 h.

[0009] Further defined, in step (2), the annealing temperature is 95 °C, the duration is 10 min, and the low-temperature storage environment of the reaction product is 4 °C.

[0010] Further defined, in step (3), the exonuclease is one or more of Exo I, Exo III, RecJf Exo, the dosage is 1 - 10 U, and the incubation time is 30 - 150 min; the concentrations of DNA strands H1, H2, and H3 are 1 - 2 μM, the volume used is 5 - 10 μL, and the incubation time is 0.5 - 2 h; the concentration of the boron nitride-doped carbon composite is 0.8 - 1.2 mg / mL, the volume used is 10 μL, and the incubation time is 10 - 50 min.

[0011] Further defined, in steps (2) and (3), the sequence of H1 is 5'-GAT TTG CCG TTT CGG GTAGTT ACG GCA ACA TGC CGA AGG AAC TAC CGG AAA CGG CAA ATC CTC G-3'; the sequence of H2 is 5'-TTC GAC CTT CCC ACT TGT TCA CTC AAG TGT GAA TGA GTG AAC AAG TGG GAA GGTCG AA CCC GAA ACG GCA AAT C-3'; the sequence of H3 is 5'-CAC TTG AGT GAA CAA GTG GGAAGG TCG AA-3', where the 5' end is modified with carboxyfluorescein FAM.

[0012] Compared with the prior art, the present invention has the following remarkable advantages: 1. The present invention utilizes three precisely designed hairpin chain structures and introduces an exonuclease-assisted cascade signal amplification strategy to achieve signal amplification and enhance the sensitivity of the fluorescence biosensor.

[0013] 2. The present invention successfully prepares a novel boron nitride-doped porous carbon composite material as a quenching material, which has a large specific surface area and strong adsorption capacity, etc., effectively quenches the fluorescence of FAM, and reduces the interference of background signals.

[0014] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it in accordance with the content of the specification, the following is a detailed description of the preferred embodiments of the present invention. Brief Description of the Drawings

[0015] Figure 1 It is a preparation schematic diagram of a preparation method of a fluorescence aptasensor based on a boron nitride-doped porous carbon composite material.

[0016] Figure 2 It is the fluorescence detection result of the sensor constructed in Example 1 of the present invention before (dashed line) and after (solid line) adding AA.

[0017] Figure 3 It is the standard curve of the sensor constructed in Example 1 of the present invention for detecting AA.

[0018] Figure 4 It is the selectivity of the sensor constructed in Example 1 of the present invention for AA in the presence of other interfering substances. Detailed Description of the Invention

[0019] The following will further describe in detail the specific embodiments of the present invention in conjunction with the accompanying drawings and embodiments. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention. Example

[0020] A preparation method of a fluorescence aptamer sensor based on boron nitride-doped porous carbon composite material, and its implementation method is as Figure 1 shown.

[0021] The specific steps are as follows: (1) Preparation of boron nitride-doped porous carbon composite material Synthesis of precursor: Add 1.116 g of zinc nitrate hexahydrate and 1.232 g of 2-methylimidazole to 30 mL of methanol, stir for 15 min, then add 2.7880 g of boric acid, stir again for 15 min, and then place it in an oven and react at 150 °C for 12 h. Cool to room temperature, wash with methanol and centrifuge. Finally, place it in an oven at 80 °C and dry for 24 h to obtain precursor powder.

[0022] Synthesis of boron nitride-doped porous carbon composite material: Calcinate the precursor powder at 900 °C for 6 h under argon to obtain a black powder material. Wash the black powder with absolute ethanol to remove unreacted boric acid and boron oxide impurities generated at high temperature. Finally, dry it in an oven at 80 °C for 12 h to obtain the boron nitride-doped porous carbon composite material.

[0023] (2) Pretreatment of DNA strands H1, H2 and H3: Take 100 μM of H1, H2 and H3 and add them to two sterilized centrifuge tubes respectively. Then add 50 mM Tric-HCl buffer solution to each centrifuge tube, perform high-temperature annealing on the centrifuge tubes, and then let it stand to slowly cool to room temperature to obtain a reaction product, and store the reaction product at 4 °C.

[0024] (3) Add buffer solutions without AA and with AA to the H1 (10 μL 1 μmol / L) system, and incubate at 37 °C for 45 min. Then add 10 μL 1 μmol / L of H2, incubate at 37 °C for 1 h, continue to add 5 U of Exo III to the system, and continue to incubate at 37 °C for 1.5 h. Then add 10 μL 1.4 μmol / L of H3 and 5 U of Exo III, and continue to incubate at 37 °C for 1.5 h. Finally, add 10 μL 1 mg / mL of boron nitride-doped porous carbon composite material and incubate at 37 °C for 20 min. After incubation, supplement the solution volume to 200 μL with Tris-HCl buffer solution with pH = 7.4. Set the excitation wavelength to 490 nm, the emission wavelength measurement range to 510 - 600 nm, and record the fluorescence intensity at the emission wavelength of 520 nm under a fluorescence spectrophotometer. (4) Establishment of the standard curve: Add 10 μL of AA standard solutions with different concentrations to step (3) to obtain sample detection solutions with different gradients. After incubation, different fluorescence signals are obtained. Using the logarithm of the AA concentration as the abscissa and the difference in fluorescence signals with and without the target as the ordinate for linear fitting, establish the standard curve of the sensor for AA.

[0025] As Figure 2 shown, the fluorescence detection results of the sensor constructed in Example 1 of the present invention before (dashed line) and after (solid line) adding AA are presented.

[0026] As Figure 3 shown, the standard curve of the sensor constructed in Example 1 of the present invention for detecting AA is presented. Example

[0027] A preparation method and application of a fluorescence aptamer sensor based on boron nitride-doped porous carbon composite material, its practical application includes the following steps: To verify that the newly prepared fluorescence biosensor has specific recognition for AA, add acrylamide standard to Tris-HCl buffer solution to make the AA concentration in the sample 50 nM; use Tris-HCl buffer solution to prepare standard solutions of other 7 interfering substances (acrylic acid, coffee, ascorbic acid, glycine, aspartic acid, glucose, starch) respectively, with a concentration of 50 nM. Detect the above 7 different interfering substance standard solutions and their mixed samples with AA according to the detection system constructed in Example 1. The detection results are as Figure 4 shown, indicating that the method of the present invention has good selectivity for AA. Example

[0028] A preparation method and application of a fluorescence aptamer sensor based on boron nitride-doped porous carbon composite material, its practical application includes the following steps: (1) Food sample treatment: Disperse coffee samples and ground potato chip samples in water (200 mg / mL) respectively, and perform ultrasonic treatment for 30 min. Then add 2.5 mL of n-hexane, place it in a constant temperature oscillator and shake for 15 min, centrifuge at 4000 rpm for 15 min and repeat the operation to complete the defatting process. The obtained precipitate is treated with Carrez I and II (1 mL each) to break the emulsion, precipitate proteins and carbohydrates. After centrifuging at 4000 rpm for 15 min, obtain the supernatant, filter it with a 0.45 μm microporous membrane and dilute it 500 times with Tris-HCl. Using the standard addition method, obtain the food extract.

[0029] (2)Sample detection: Take 10 μL of the food extract, measure the fluorescence signal according to step (3) of Example 1, and substitute it into the standard curve to obtain the concentration of AA in the sample.

[0030] (3)When using coffee as the food sample for determination, based on the addition amount of 5 nmol / L, add AA standard products with 0.01 times and 10 times of the reference amount to the coffee respectively. Take 10 μL of the sample solution, measure the fluorescence signal according to step (3) of Example 1, substitute it into the standard curve detected in Example 1 to obtain the concentration of AA in the sample. Each sample is measured 3 times and the average value is taken. After calculation, the average recovery rate of the sensor is 97.7% - 109.6%.

[0031] It is verified that the prepared novel fluorescence sensor has the advantages of high sensitivity, good selectivity, good reliability and stability in the detection of AA. The detection of actual samples shows that the prepared sensor has very good practical application value.

[0032] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention. Any reference signs in the claims should not be regarded as limiting the claimed rights.

[0033] In addition, it should be understood that although this specification is described according to the embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard 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 preparation method of a fluorescent aptamer sensor based on boron nitride-doped porous carbon composite material, characterized in that, It includes the following steps: (1) Preparation of boron nitride-doped porous carbon composite: Dissolve soluble zinc salt and imidazole compound in methanol, add boric acid to this solution, stir evenly and then place it in an oven for reaction. After cooling to room temperature, wash and dry it. Calcinate the obtained powder under argon to obtain the boron nitride-doped porous carbon composite, which has a better quenching effect compared with other carbon materials; (2) Preparation of fluorescent aptamer sensor: First, the aptamer sequence of DNA hairpin H1 binds to the target acrylamide AA, causing the hairpin structure to open; introduce DNA hairpin H2 for incubation. The opened H1 will expose a DNA sequence to bind to H2, forming a DNA double strand H1-AA-H2 structure; add exonuclease for incubation, which can shear the ends of the H1-AA-H2 structure. After shearing, H1-AA and DNA single strand S1 are obtained; H1-AA continues to cycle and bind to the next H2, and S1 binds to DNA hairpin H3 labeled with carboxyfluorescein FAM to form a DNA double strand S1-H3 structure; the exonuclease continues to act and shear the ends of the S1-H3 structure, thus obtaining a lot of free FAM; introduce the boron nitride-doped carbon composite for incubation. The free FAM will not be adsorbed onto the boron nitride-doped carbon composite, thus causing a change in the response signal; quantitative analysis of AA is achieved according to the change in the fluorescence intensity of FAM before and after adding the target. Three hairpin DNAs are designed and an exonuclease-assisted signal amplification strategy is introduced.

2. The preparation method of a fluorescence aptamer sensor based on boron nitride-doped porous carbon composite material according to claim 1, wherein, In step (1), the soluble zinc salt is one or more of zinc chloride, zinc sulfate, zinc carbonate, zinc nitrate hexahydrate, etc.; the imidazole compound is one or more of 5-bromo-1-methyl-1H-imidazole, 4-methyl-5-cyanoimidazole, 2-ethylimidazole, 2-methylimidazole, etc.; the reaction temperature and time are 150 °C and 12 h; the calcination temperature and time are 900 °C and 6 h.

3. The preparation method of a fluorescence aptamer sensor based on boron nitride-doped porous carbon composite material according to claim 1, wherein, In step (2), the exonuclease is one or more of Exo I, Exo III, RecJf Exo, the dosage is 1 - 10 U, and the incubation time is 30 - 150 min; the concentrations of DNA hairpins H1, H2, and H3 are 1 - 2 μM, the volume is 5 - 10 μL, and the incubation time is 45 - 120 min; the concentration of the boron nitride-doped carbon composite is 0.8 - 1.2 mg / mL, the volume is 10 μL, and the incubation time is 10 - 50 min.

4. The preparation method of a fluorescence aptamer sensor based on boron nitride-doped porous carbon composite material according to claim 1, wherein, In step (2), the sequence of the DNA hairpin H1 is 5'-GAT TTG CCG TTT CGGGTA GTT ACG GCA ACA TGC CGA AGG AAC TAC CGG AAA CGG CAA ATC CTC G-3'; the sequence of the DNA hairpin H2 is 5'-TTC GAC CTT CCC ACT TGT TCA CTC AAG TGT GAA TGA GTG AAC AAGTGG GAA GG TCG AA CCC GAA ACG GCA AAT C-3'; the sequence of the DNA hairpin H3 is 5'-CAC TTGAGT GAA CAA GTG GGA AGG TCG AA-3', where the 5' end is modified with carboxyfluorescein 6-FAM.

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