阅读说明：本技术 一种适配体dna-荧光探针传感器及其制备方法和利用其定量检测gtx1/4的方法 (Aptamer DNA-fluorescent probe sensor, preparation method thereof and method for quantitatively detecting GTX1/4 by using aptamer DNA-fluorescent probe sensor ) 是由 王广凤 戴天玥 盛祝涛 于 2021-08-30 设计创作，主要内容包括：本发明公开了一种用于GTX1/4检测的适配体DNA-荧光探针传感器及其制备方法和利用其定量检测GTX1/4的方法；所述制备方法包括以下步骤：首先制备双链S1-Spc溶液；将HKUST-1以甲醇蒸汽熏蒸7h,得到HK-7；将双链S1-Spc溶液与HK-7的分散液混合后稀释,进行震荡培养；向上述溶液中加入DNA S2溶液,震荡培养得到装载有S1-Spc双链与S2单链的HK-7溶液,即为用于GTX1/4检测的适配体DNA-荧光探针传感器；根据本发明的方法构建的适配体DNA-荧光探针传感器,仅利用适配体DNA与荧光探针的识别作用即可实现对于GTX1/4的定量检测,其操作简单,灵敏度高。(The invention discloses an aptamer DNA-fluorescent probe sensor for GTX1/4 detection, a preparation method thereof and a method for quantitatively detecting GTX1/4 by using the same; the preparation method comprises the following steps: firstly, preparing a double-chain S1-Spc solution; fumigating HKUST-1 with methanol vapor for 7h to obtain HK-7; mixing the double-chain S1-Spc solution with the dispersion liquid of HK-7, diluting, and performing shake culture; adding a DNA S2 solution into the solution, and performing shake culture to obtain an HK-7 solution loaded with an S1-Spc double chain and an S2 single chain, namely the aptamer DNA-fluorescent probe sensor for GTX1/4 detection; the aptamer DNA-fluorescent probe sensor constructed by the method can realize quantitative detection of GTX1/4 only by utilizing the recognition effect of the aptamer DNA and the fluorescent probe, and has simple operation and high sensitivity.)

1. A preparation method of an aptamer DNA-fluorescent probe sensor for GTX1/4 detection is characterized by comprising the following steps:

(1) mixing and diluting equal-volume DNA S1 solution and DNA Spc solution, heating for reaction, and cooling to obtain double-stranded S1-Spc solution, wherein the gene sequences of the DNA S1 and the DNA Spc are respectively as follows:

DNA S1:3’-CCTTAATGGATTGGAGTTCCTC-5’-6-FAM；

DNA Spc:Dabcyl-3’-GAGGATTTCCAATCC-5’；

(2) fumigating metal framework compound HKUST-1 with methanol vapor for 7h to obtain HK-7;

(3) mixing the double-chain S1-Spc solution with the dispersion liquid of HK-7, diluting, and performing shake culture;

(4) adding a DNA S2 solution into the solution obtained in the step (3), and performing shake culture to obtain an HK-7 solution loaded with S1-Spc double chains and S2 single chains, namely an aptamer DNA-fluorescent probe sensor S1-Spc/S2/HK-7 for GTX1/4 detection;

the gene sequence of the DNA S2 is as follows: 3 '-GAGGAACTCCAATCC-5'.

2. The method for preparing an aptamer DNA-fluorescent probe sensor for GTX1/4 detection according to claim 1, wherein the method specifically comprises the following steps:

(1) mixing equal-volume equal-concentration DNA S1 solution and DNA Spc solution, diluting until the concentration of DNA S1 is 3 mu M, heating at 95 ℃ for reaction for 5min, and cooling to obtain double-stranded S1-Spc solution;

(2) fumigating metal framework compound HKUST-1 with methanol vapor for 7h to obtain HK-7;

(3) mixing 20 μ L of double-stranded S1-Spc solution with 10 μ L of 20mg/mL HK-7 dispersion, diluting to total volume of 200 μ L, and shake culturing at 37 deg.C for 3 h;

(4) and (3) adding 10 mu L of 4 mu M DNA S2 solution into 200 mu L of the solution obtained in the step (3), and performing shake culture at 37 ℃ for 2h to obtain an HK-7 solution loaded with S1-Spc double chains and S2 single chains, namely the aptamer DNA-fluorescent probe sensor S1-Spc/S2/HK-7 for GTX1/4 detection.

3. The method for preparing the aptamer DNA-fluorescent probe sensor for GTX1/4 detection according to claim 1 or 2, wherein the DNA S1 solution, the DNA Spc solution, the DNA S2 solution and the HK-7 dispersion are prepared by dissolving the DNA S1, the DNA Spc, the DNA S2 and the HK-7 in TAE/Mg containing 40mM Tris, 1mM EDTA and 12.5mM magnesium acetate at pH 8.0, respectively²⁺Obtained in a buffer solution.

4. The method for preparing an aptamer DNA-fluorescent probe sensor for GTX1/4 detection according to claim 1 or 2, wherein in the steps (1) and (3), the solution used for dilution is TAE/Mg containing 40mM Tris, 1mM EDTA and 12.5mM magnesium acetate, and the pH value of the solution is 8.0²⁺And (4) buffer solution.

5. An aptamer DNA-fluorescent probe sensor for GTX1/4 detection prepared by the preparation method according to any one of claims 1 to 4.

6. The use of the aptamer DNA-fluorescent probe sensor for GTX1/4 detection according to claim 5 for quantitative detection of GTX1/4 concentration.

7. A quantitative detection method of GTX1/4 is characterized by comprising the following steps:

A. repeating steps (1) to (4) of the production method according to any one of claims 1 to 4;

B. heating and cooling the DNA apt solution, mixing the DNA apt solution with an isovolumetric DNA Inducer solution with equal concentration, and diluting to obtain a partially paired double-stranded apt-In solution;

the gene sequences of the DNA apt and the DNA inductor are respectively as follows:

DNA apt：

3’-TTGGATGGAACGGGCTGGTTTCCAA-5’；

DNA Inducer：3’-TCCAATCCATTAAGG-5’；

C. mixing the partially paired double-chain apt-In solution with GTX1/4 solutions with different concentrations, incubating, adding into HK-7 solution loaded with S1-Spc double chains and S2 single chains, and standing for reaction;

D. and (3) testing the fluorescence intensity of each reaction system under the excitation wavelength of 475nm, constructing a linear curve by taking the concentration C of the GTX1/4 solution as a horizontal coordinate and the fluorescence intensity I at 520nm as a vertical coordinate, further obtaining a linear equation, and obtaining the concentration of the GTX1/4 solution to be tested corresponding to any fluorescence intensity I according to the linear equation.

8. The quantitative detection method of GTX1/4 according to claim 7, wherein the detection method specifically comprises the steps of:

A. repeating steps (1) to (4) of the production method according to any one of claims 1 to 4;

B. heating the DNA apt solution at 95 ℃ for 5min, cooling to room temperature, mixing with the DNA Inducer solution with the same volume and concentration, and diluting until the concentration of the DNA apt solution and the DNA Inducer solution are both 4 mu M to obtain a partially-paired double-stranded apt-In solution;

C. respectively mixing 5 mu L of partially paired double-chain apt-In solution with GTX1/4 solutions with different concentrations, reacting for 1h at 37 ℃, then respectively adding the mixture into 200 mu L of HK-7 solution loaded with S1-Spc double chains and S2 single chains, and standing for 15 min;

D. and (3) testing the fluorescence intensity of each reaction system under the excitation wavelength of 475nm, constructing a linear curve by taking the concentration C of the GTX1/4 solution as a horizontal coordinate and the fluorescence intensity I at 520nm as a vertical coordinate, further obtaining a linear equation, and obtaining the concentration of the GTX1/4 solution to be tested corresponding to any fluorescence intensity I according to the linear equation.

9. The method of claim 7 or 8, wherein the final concentration of GTX1/4 in the reaction system is 100nM, 75nM, 50nM, 25nM, 20nM, 15nM, 10nM, 5nM, respectively.

10. The quantitative detection method of GTX1/4 according to claim 5 or 6, wherein the linear equation is: 31.206 XC +1769.323 with a linear correlation coefficient R²0.99766, where C is in nM.

Technical Field

The invention belongs to the technical field of environmental detection, and particularly relates to an aptamer DNA-fluorescent probe sensor, a preparation method thereof and a method for quantitatively detecting GTX1/4 by using the aptamer DNA-fluorescent probe sensor.

Background

Gonyautoxin 1/4(GTX1/4) is the most representative neurotoxin in paralytic shellfish toxins, and the toxic death events of consumers caused by the neurotoxin have wide distribution and high incidence in coastal areas of China. In recent years, quantitative detection of GTX1/4 has received increasing attention.

Paralytic shellfish poison to which GTX1/4 belongs is one of the three ocean biohazards worldwide. Misuse of foods containing such toxins can lead to reduced neurotransmission due to reduced sodium ion channel activity. The serious person with poisoning will die quickly due to its strong toxicity.

For the research of the quantitative detection of GTX1/4, the current research methods are mainly limited to isotachophoresis, capillary electrophoresis, liquid chromatography, enzyme-linked immunosorbent assay (ELISA), and the like. However, the precise operation steps of the methods for detecting GTX1/4 in equal amount have high requirements on the operation skills of experimenters and are difficult to popularize, so that the popularization of the quantitative detection of GTX1/4 is limited.

Therefore, the quantitative detection of GTX1/4 has been the research focus of food environment detection, and the quantitative detection which can realize simple operation and high sensitivity to GTX1/4 is the problem to be solved at present.

Disclosure of Invention

In order to solve the technical problems, the invention provides an aptamer DNA-fluorescent probe sensor for GTX1/4 detection, a preparation method thereof and a method for quantitatively detecting GTX1/4 by using the same. The aptamer DNA-fluorescent probe sensor constructed by the method can realize quantitative detection of GTX1/4 only by utilizing the recognition effect of the aptamer DNA and the fluorescent probe, and has simple operation and high sensitivity.

The technical scheme adopted by the invention is as follows:

a method for preparing an aptamer DNA-fluorescent probe sensor for GTX1/4 detection, the method comprising the steps of:

(1) mixing and diluting equal-volume DNA S1 solution and DNA Spc solution, heating for reaction, and cooling to obtain double-stranded S1-Spc solution, wherein the gene sequences of the DNA S1 and the DNA Spc are respectively as follows:

DNA S1:3’-CCTTAATGGATTGGAGTTCCTC-5' -6-FAM; DNA S1 has a fluorophore;

DNA Spc:Dabcyl-3’-GAGGATTTCCAATCC-5'; the DNA Spc has a quencher group, and the DNA S1 is the same as that of the DNA SpcThe gene sequences can be partially matched;

(2) fumigating metal framework compound HKUST-1 with methanol vapor for 7h to obtain HK-7;

(3) mixing the double-chain S1-Spc solution with the dispersion liquid of HK-7, diluting, and performing shake culture;

(4) adding a DNA S2 solution into the solution obtained in the step (3), and performing shake culture to obtain an HK-7 solution loaded with S1-Spc double chains and S2 single chains, namely an aptamer DNA-fluorescent probe sensor S1-Spc/S2/HK-7 for GTX1/4 detection;

the gene sequence of the DNA S2 is as follows: 3' -GAGGAACTCCAATCC-5'; the gene sequence of DNA S2 is mostly identical to the gene sequence of DNA Spc.

The preparation method of the ligand DNA-fluorescent probe sensor for GTX1/4 detection specifically comprises the following steps:

(1) mixing equal-volume equal-concentration DNA S1 solution and DNA Spc solution, diluting until the concentration of DNA S1 is 3 mu M, heating at 95 ℃ for reaction for 5min, and cooling to obtain double-stranded S1-Spc solution;

(2) fumigating metal framework compound HKUST-1 with methanol vapor for 7h to obtain HK-7;

(3) mixing 20 μ L of double-stranded S1-Spc solution with 10 μ L of 20mg/mL HK-7 dispersion, diluting to total volume of 200 μ L, and shake culturing at 37 deg.C for 3 h;

(4) and (3) adding 10 mu L of 4 mu M DNA S2 solution into 200 mu L of the solution obtained in the step (3), and performing shake culture at 37 ℃ for 2h to obtain an HK-7 solution loaded with S1-Spc double chains and S2 single chains, namely the aptamer DNA-fluorescent probe sensor S1-Spc/S2/HK-7 for GTX1/4 detection.

The dispersions of the DNA S1 solution, the DNA Spc solution, the DNA S2 solution and the HK-7 were prepared by dissolving the DNA S1, the DNA Spc, the DNA S2 and the HK-7 in TAE/Mg containing 40mM Tris, 1mM EDTA and 12.5mM magnesium acetate at pH 8.0, respectively²⁺Obtained in a buffer solution.

The concentrations of the DNA S1 solution, the DNA Spc solution and the DNA S2 solution are 10 mu M, 10 mu M and 4 mu M respectively.

In the steps (1) and (3), the solution used for dilution was 40mM Tris, 1mM Tris, pH 8.0EDTA, 12.5mM magnesium acetate TAE/Mg²⁺And (4) buffer solution.

In the step (2), the preparation method of the HK-7 comprises the following steps:

(a) dissolving 1,3, 5-benzenetricarboxylic acid (1,3,5-BTC) in 15mL of a mixture of ethanol and N, N-Dimethylformamide (DMF) which are mixed in equal volume, wherein the final concentration of the 1,3,5-BTC is 0.15862M;

(b) dissolving a solid copper nitrate trihydrate in ultrapure water to prepare a 0.5M copper nitrate solution;

(c) mixing 30mL of the solution obtained in the step (a) with 15mL of the solution obtained in the step (b), adding the mixture into a reaction kettle with the capacity of 100mL, putting the reaction kettle into an oven, and reacting for 10 hours at 100 ℃;

(d) after the reaction in step (c) is finished, removing the supernatant of the reaction, and carrying out solvent exchange on the obtained solid with 30mL of DMF and dichloromethane for 24h respectively, wherein the final product is HKUST-1 which is also called HK-0;

(e) taking 0.1g of HKUST-1, putting the HKUST-1 on a platform built in a reaction kettle with the capacity of 100mL, adding 10mL of methanol solution at the bottom of the reaction kettle, wherein the methanol solution in the reaction kettle is not in contact with the built platform and the HKUST-1, and putting the reaction kettle containing reactants into an oven to react for 7 hours at 180 ℃;

(f) and (e) after the reaction in the step (e) is finished, taking out the reaction kettle, rapidly cooling to room temperature, rapidly removing the product on the reaction platform, collecting the product, marking as HK-7, and enabling the average pore diameter of the product to be 2.0-3.0 nm.

The invention also provides a ligand DNA-fluorescent probe sensor for GTX1/4 detection, which is prepared by the preparation method.

The invention also provides application of the ligand DNA-fluorescent probe sensor for detecting GTX1/4 in quantitative detection of GTX1/4 concentration.

The invention also provides a quantitative detection method of GTX1/4, which comprises the following steps:

A. repeating the steps (1) to (4) of the preparation method;

B. heating and cooling the DNA apt solution, mixing the DNA apt solution with an isovolumetric DNA Inducer solution with equal concentration, and diluting to obtain a partially paired double-stranded apt-In solution;

the gene sequences of the DNA apt and the DNA inductor are respectively as follows:

DNA apt：

five bases In the DNA apt can pair with five bases In the DNA Inducer to form a partially paired double-stranded apt-In.

DNA Inducer：3’-TCCAATCCATTAAGG-5'; the gene sequence of the DNA apt can be partially paired with the gene sequence of the DNA inductor to form a partially paired double-stranded apt-In;

C. mixing the partially paired double-chain apt-In solution with GTX1/4 solutions with different concentrations, incubating, adding into HK-7 solution loaded with S1-Spc double chains and S2 single chains, and standing for reaction;

D. and (3) testing the fluorescence intensity of each reaction system under the excitation wavelength of 475nm, constructing a linear curve by taking the concentration C of the GTX1/4 solution as a horizontal coordinate and the fluorescence intensity I at 520nm as a vertical coordinate, further obtaining a linear equation, and obtaining the concentration of the GTX1/4 solution to be tested corresponding to any fluorescence intensity I according to the linear equation.

The detection method specifically comprises the following steps:

A. repeating the steps (1) to (4) of the preparation method;

B. heating the DNA apt solution at 95 ℃ for 5min, cooling to room temperature, mixing with the DNA Inducer solution with the same volume and concentration, diluting until the concentration of the DNA apt solution and the DNA Inducer solution are both 4 mu M, and standing for 1h at room temperature to obtain a partially-paired double-stranded apt-In solution;

C. respectively mixing 5 mu L of partially paired double-chain apt-In solution with GTX1/4 solutions with different concentrations, reacting for 1h at 37 ℃, then respectively adding the mixture into 200 mu L of HK-7 solution loaded with S1-Spc double chains and S2 single chains, and standing for 15 min;

D. and (3) testing the fluorescence intensity of each reaction system under the excitation wavelength of 475nm, constructing a linear curve by taking the concentration C of the GTX1/4 solution as a horizontal coordinate and the fluorescence intensity I at 520nm as a vertical coordinate, further obtaining a linear equation, and obtaining the concentration of the GTX1/4 solution to be tested corresponding to any fluorescence intensity I according to the linear equation.

The final concentrations of GTX1/4 in the reaction were 100nM, 75nM, 50nM, 25nM, 20nM, 15nM, 10nM, and 5nM, respectively.

The linear equation is: 31.206 XC +1769.323 with a linear correlation coefficient R²0.99766, where C is in nM.

The DNA apt solution, the DNA Inducer solution and the GTX1/4 solution are respectively prepared by dissolving DNA apt, DNA Inducer and GTX1/4 white powder in TAE/Mg containing 40mM Tris, 1mM EDTA and 12.5mM magnesium acetate and having a pH of 8.0²⁺Obtained in a buffer solution.

The concentrations of the DNA apt solution and the DNA inductor solution are both 10 mu M.

In step B, the solution used for dilution was TAE/Mg containing 40mM Tris, 1mM EDTA, 12.5mM magnesium acetate, pH 8.0²⁺And (4) buffer solution.

The invention adjusts the aperture of the metal frame compound HKUST-1 through methanol vapor to obtain the carrier HK-7 material with higher DNA loading rate, and the carrier HK-7 has better adaptability with the aptamer DNA-fluorescent probe. The method comprises the steps of firstly mixing DNA S1 solution and DNA Spc solution with equal molar concentrations in equal volumes, diluting, heating and cooling to obtain partially paired double-stranded S1-Spc solution, then modifying the double-stranded S1-Spc and DNA S2 partially coincided with the DNA Spc sequence to a carrier HK-7 with adaptive aperture, and further preparing the DNA apt-fluorescent probe sensor S1-Spc/S2/HK-7.

And mixing the DNA apt solution with equal molar concentration and the DNA inductor solution to form a partially paired double-chain apt-In solution, wherein the apt-In can identify a target object Goodpastoxin GTX1/4, and further the apt In the apt-In can be combined with the target object to form a G-quadruplex stable structure, so that double chains are forcedly disintegrated to release the DNA inductor. Because the DNA Inducer is partially paired with the DNA S1 and has stronger competitive power than the DNA Spc, the modified double strand S1-Spc on the carrier HK-7 is pulled out firstly, and then the DNA Spc with a quenching group is replaced to form the double strand S1-In, and because of the special structure of the DNA Spc, the single strand DNA Spc can spontaneously form a hairpin structure and does not participate In the competition with the DNA Inducer any more. After that, S1-In will pull DNA S2 out of the carrier, and because the competitive power of DNA S2 is stronger than that of DNA Inducer, S1-S2 structure is formed, and the released DNA Inducer will continue to pull out new double-stranded S1-Spc, so as to form a circulation amplification system. Because the DNA S1 has the fluorescent group, the strong fluorescence is generated after the separation of the fluorescent group and the quenching group, and the concentration of the DNA inductor and the concentration of the target object are in a relation of 1:1, the quantitative detection of the concentration of the gonyautoxin GTX1/4 can be realized by using a fluorescence spectrophotometer, and the detection method has the advantages of high sensitivity, low detection limit and convenient operation.

Drawings

FIG. 1 is a schematic diagram of a method for detecting the concentration of gonyautoxin GTX1/4 by using an aptamer DNA-fluorescent probe sensor constructed by the invention;

FIG. 2 is a graph showing the distribution of particle sizes of HK-0, HK-5, HK-5.5, HK-6, HK-6.5, HK-7.5, HK-8, HK-8.5, HK-9;

FIG. 3 is a graph showing fluorescence spectra of HK-5, HK-5.5, HK-6, HK-6.5, HK-7.5, HK-8, HK-8.5, and HK-9 loaded with DNA S2;

FIG. 4 is a graph showing fluorescence spectra of HK-5, HK-5.5, HK-6, HK-6.5, HK-7.5, HK-8, HK-8.5, and HK-9 after loading S1-Spc;

FIG. 5 is a graph showing the loading of HK-5, HK-5.5, HK-6, HK-6.5, HK-7.5, HK-8, HK-8.5, HK-9 to DNAs S2, S1-Spc;

FIG. 6 is a fluorescence spectrum of the environmental detection sensor a, the environmental detection sensor b and the blank control c in example 2;

FIG. 7 is a dot-matrix diagram constructed with the concentration of gonyautoxin GTX1/4 solution as abscissa and the fluorescence intensity at 520nm as ordinate.

Detailed Description

Example 1

The preparation method of HK-7 comprises the following steps:

(a) dissolving 1,3, 5-benzenetricarboxylic acid (1,3,5-BTC) in 15mL of a mixture of ethanol and N, N-Dimethylformamide (DMF) which are mixed in equal volume, wherein the final concentration of the 1,3,5-BTC is 0.15862M;

(b) dissolving a solid copper nitrate trihydrate in ultrapure water to prepare a 0.5M copper nitrate solution;

(c) mixing 30mL of the solution obtained in the step (a) with 15mL of the solution obtained in the step (b), adding the mixture into a reaction kettle with the capacity of 100mL, putting the reaction kettle into an oven, and reacting for 10 hours at 100 ℃;

(d) after the reaction in step (c) is finished, removing the supernatant of the reaction, and carrying out solvent exchange on the obtained solid with 30mL of DMF and dichloromethane for 24h respectively, wherein the final product is HKUST-1 which is also called HK-0;

(e) taking 0.1g of HKUST-1, putting the HKUST-1 on a platform built in a reaction kettle with the capacity of 100mL, adding 10mL of methanol solution at the bottom of the reaction kettle, wherein the methanol solution in the reaction kettle is not in contact with the built platform and the HKUST-1, and putting the reaction kettle containing reactants into an oven to react for 7 hours at 180 ℃;

(f) and (e) after the reaction in the step (e) is finished, taking out the reaction kettle, rapidly cooling to room temperature, rapidly removing the product on the reaction platform, collecting the product, marking as HK-7, and enabling the average pore diameter of the product to be 2.0-3.0 nm.

Respectively replacing the reaction time in the step (e) with 5.0, 5.5, 6.0, 6.5, 7.5, 8.0, 8.5 and 9.0h to respectively prepare HK-0, HK-5, HK-5.5, HK-6, HK-6.5, HK-7.5, HK-8, HK-8.5 and HK-9.

The BET of HK-5, HK-5.5, HK-6, HK-6.5, HK-7, HK-7.5, HK-8, HK-8.5, HK-9, the fluorescence spectra after loading with DNA S2, and the fluorescence spectra after loading with S1-Spc were then tested for the loading of DNA S2, S1-Spc, respectively, as shown in FIGS. 2-5. As can be seen from the figure, HK-7 has the best compatibility with DNAS2, S1-Spc, and DNAS2, S1-Spc have the highest loading on HK-7.

The method for loading the DNA S2 comprises the following steps: mixing 10 μ L of 4 μ M DNA S2 solution with 10 μ L of 20mg/mL HK-n dispersion, diluting to total volume of 200 μ L, and shake culturing at 37 deg.C for 3 h;

the method for loading the S1-Spc comprises the following steps: 20 μ L of 3 μ M double-stranded S1-Spc solution was mixed with 10 μ L of 20mg/mL HK-n dispersion, diluted to a total volume of 200 μ L, and shake-cultured at 37 ℃ for 3h

The method for testing the loading capacity of the DNA S2 and S1-Spc comprises the following steps: the loading rates of the DNAs S2 and S1-Spc in HK-n were calculated using the following formula:

η_μis the loading rate of DNA S2 or S1-Spc, I₀Is the fluorescence intensity, I, of the DNA S2 or S1-Spc solution before loading (before mixing with the HK-n dispersion)_nIs the fluorescence intensity of the solution supernatant after loading.

Example 2

A preparation method of an aptamer DNA-fluorescent probe sensor for GTX1/4 detection comprises the following steps:

(1) mixing equal-volume equal-concentration DNA S1 solution and DNA Spc solution, diluting until the concentration of DNA S1 is 3 mu M, heating at 95 ℃ for reaction for 5min, and cooling to obtain double-stranded S1-Spc solution;

(2) mixing 20 μ L of double-stranded S1-Spc solution with 10 μ L of 20mg/mL HK-7 dispersion, diluting to total volume of 200 μ L, and shake culturing at 37 deg.C for 3 h;

(3) and (3) adding 10 mu L of 4 mu M DNA S2 solution into 200 mu L of the solution obtained in the step (2), and performing shake culture at 37 ℃ for 2h to obtain an HK-7 solution loaded with S1-Spc double chains and S2 single chains, namely the aptamer DNA-fluorescent probe sensor S1-Spc/S2/HK-7 for GTX1/4 detection.

It was not verified that the aptamer DNA-fluorescent probe sensor S1-Spc/S2/HK-7 prepared in this example can be used for detecting GTX1/4, and the following experiment was performed:

heating the DNA apt solution at 95 ℃ for 5min, cooling to room temperature, mixing with the DNA Inducer solution with the same volume and concentration, diluting until the concentration of the DNA apt solution and the DNA Inducer solution are both 4 mu M, and standing for 1h at room temperature to obtain a partially-paired double-stranded apt-In solution;

the gene sequences of the DNA apt and the DNA inductor are respectively as follows:

DNA apt：

DNA Inducer：3’-TCCAATCCATTAAGG-5’；

5 mu.L of the partially paired double-stranded apt-In solution and 5 mu.L of GTX1/4 solution are mixed, the final concentration of GTX1/4 In the system is 100nM, the reaction is carried out for 1h at 37 ℃, and then the mixture is added into 200 mu.L of HK-7 solution loaded with S1-Spc double strands and S2 single strands, and the mixture is kept stand for 15min and is marked as an environmental detection sensor a.

After the reaction, 100. mu.L of the reaction solution was transferred to a micro fluorescence cuvette, and the fluorescence spectrum of the reaction solution at an excitation wavelength of 475nm was detected by a fluorescence spectrophotometer, and the results are shown in FIG. 6, using HK-7 solution loaded with S1-Spc double strand and S2 single strand obtained in step (3) as a control, and the dispersion of HK-7 used in step (2) as a blank control c. As can be seen from the figure, the HK-7 solution loaded with the S1-Spc double strand and the S2 single strand had very weak fluorescence at 520nm, which was generated from S1 not fully loaded in the carrier HK-7, as background noise, before the GTX1/4 solution of the target was not added; the dispersion of HK-7 did not produce fluorescence.

It is demonstrated that, in the aptamer DNA-fluorescent probe sensor for GTX1/4 detection constructed in this embodiment, after the target substance GTX1/4 is added, GTX1/4 will combine with the aptamer DNA apt to form a stable structure, releasing the partially linked DNA Inducer chain, the free DNA Inducer chain will continuously break away the DNA S1 with a fluorophore in the vector from the constraint of the vector, thereby generating fluorescence, and the characteristic fluorescence of the fluorophore FAM is at 520 nm.

Example 3

The quantitative detection method of GTX1/4 comprises the following steps:

A. repeating the steps (1) to (3) in example 2 to prepare HK-7 solution loaded with S1-Spc double strand and S2 single strand;

B. heating the DNA apt solution at 95 ℃ for 5min, cooling to room temperature, mixing with the DNA Inducer solution with the same volume and concentration, diluting until the concentration of the DNA apt solution and the DNA Inducer solution are both 4 mu M, and standing for 1h at room temperature to obtain a partially-paired double-stranded apt-In solution;

C. respectively mixing 5 mu L of partially paired double-chain apt-In solution with 45 mu L of GTX1/4 solution with different concentrations, reacting for 1h at 37 ℃, then respectively adding the mixture into 200 mu L of HK-7 solution loaded with S1-Spc double chains and S2 single chains, and standing for 15 min; the final concentrations of GTX1/4 in the reaction system were 100nM, 75nM, 50nM, 25nM, 20nM, 15nM, 10nM, 5nM, respectively;

D. and (3) testing the fluorescence intensity of each reaction system under the excitation wavelength of 475nm, constructing a linear curve by taking the concentration C of the GTX1/4 solution as a horizontal coordinate and the fluorescence intensity I at 520nm as a vertical coordinate, and obtaining a linear equation according to the linear equation, wherein the concentration of the GTX1/4 solution to be tested corresponding to any fluorescence intensity I can be obtained according to the linear equation. The linear equation is: 31.206 XC +1769.323 with a linear correlation coefficient R²0.99766, where C is in nM.

The above detailed description of an aptamer DNA-fluorescent probe sensor, a method for preparing the same, and a method for quantitatively detecting GTX1/4 using the same, with reference to examples, is illustrative and not restrictive, and several examples can be cited within the scope defined, and thus, variations and modifications thereof without departing from the general inventive concept should fall within the scope of the present invention.