CN111220666A - Efficient miRNA detection based on hemin-induced biocatalysis photoelectric sensitive interface - Google Patents
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Abstract
The invention discloses a micro-RNA high-efficiency detection method based on hemin induced biological catalytic precipitation reaction. After the gold nanoparticles and the cadmium sulfide quantum dots are sequentially modified on the paper chip, the capture DNA of the hairpin structure is modified on the surface of the electrode. Then the double-stranded structure formed by hybridization of the micro-RNA and the capture DNA can be sheared by the DSN, so that the micro-RNA is released, and the released micro-RNA is sheared by the DSN after the hybridization with the capture DNA, so that the DSN-assisted micro-RNA circulation is formed. Thereafter, a plurality of short DNA fragments obtained on the surface of the electrode are hybridized with the DNA marked by the hemin added later to form a double-stranded structure again. In the presence of hydrogen peroxide, hemin catalyzes 4-chloro-1-naphthol, so that insoluble precipitates are formed on the surface of an electrode, and the transfer of ascorbic acid to the surface of the electrode can be effectively inhibited, thereby reducing the photocurrent. The method has low detection limit and wider detection range, provides a thought for trace detection of RNA sequences, and has wide application prospect in early diagnosis of a plurality of diseases.
Description
Technical Field
The invention relates to the technical fields of nano material preparation, RNA detection technology and paper chips, in particular to efficient miRNA detection based on a hemin-induced biocatalysis photoelectric sensitive interface.
Background
As one of the important components in healthy cells and pathogenic cells, miRNA plays an important role in gene expression and regulation. They affect more than fifty percent of the protein-encoding genes in mammals, and their dysregulation can lead to cancer and other diseases. Therefore, it is very important to realize efficient and sensitive detection of miRNA.
Photo-electrochemical sensing has received much attention due to its advantages such as simple equipment, high sensitivity, and simple operation. In PEC sensing systems, a number of signal amplification strategies are used to achieve detection of a range of targets, such as target cyclic amplification, loop-mediated isothermal amplification, rolling circle amplification, and the like. The enzyme-assisted target cyclic amplification technology realizes the cyclic amplification function by placing a sensing electrode in a target solution containing enzyme for cultivation. The technology does not need an additional cultivation process and complex operation, and has the characteristic of cyclic amplification, thereby having wide application prospect.
In order to further improve the detection range and detection limit of a target object, enzyme biocatalytic precipitation (BCP) is introduced to realize efficient and sensitive detection of micro-RNA. BCP can cause an insoluble substance insulating layer to be formed on the surface of the electrode, so that the interface electron transfer characteristic is changed, the electron transfer process of a photoelectrode and an electron (donor) acceptor is blocked, the signal is reduced, and the sensitive detection of a target object is realized.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a miRNA high-efficiency detection method based on a hemin-induced biocatalysis photoelectric sensitive interface. The invention is realized by the following measures:
(1) firstly, designing a pattern of a microfluidic paper chip by using Adobe illustrator CS4 software, printing the designed pattern on A4 chromatographic paper by using a wax printer, then putting the printed chromatographic paper in an oven, and heating for 60 s at 100 ℃ to form a hydrophobic region and a hydrophilic working region;
(2) printing a three-electrode system (a carbon working electrode, a carbon counter electrode and an Ag/AgCl reference electrode) on the paper chip obtained in the step (1) by utilizing a screen printing technology;
(3) growing polypyrrole conductive polymer in a working area of a carbon electrode by using an in-situ chemical synthesis method, which comprises the following specific steps: firstly, measuring 20 mu L-30 mu L of pyrrole monomer, dripping the pyrrole monomer on a working area of an electrode (2), standing for 10 min at room temperature, then taking 20 mu L of newly-configured mixed solution of ferric chloride with the molar concentration of 0.49M and hydrochloric acid with the molar concentration of 0.3M, transferring the mixed solution to the surface of the electrode, standing for 2-3 h at 4 ℃, then sequentially and carefully cleaning the surface of the working electrode with 50 mu L of 0.3M hydrochloric acid solution, 0.1M sodium chloride solution and deionized water, and finally drying at room temperature;
(4) fixing 20 mu L of chitosan modified cadmium sulfide quantum dots in the working area of the electrode in the step (3), after carefully cleaning the electrode, placing 20 mu L of glutaraldehyde solution on the surface of the electrode to activate the chitosan modified cadmium sulfide quantum dots, and then cleaning the electrode three times by using Phosphate Buffered Saline (PBS) with the pH value of 7.4;
(5) mu.L of 1. mu.M capture DNA strand was immobilized on the working region of the electrode obtained in step (4), followed by blocking the non-immobilized active sites with BSA, washing with PBS pH7.4, and then 20. mu.L of the electrode containing miRNA at different concentrations and 0.03U. mu.L-1Placing the mixed solution of DSN and 5 mM magnesium chloride on a paper chip, incubating for 2 h at room temperature, and washing with PBS (pH 7.4) for three times;
(6) adding 20 μ L of 1 μ M hemin-labeled DNA onto a paper chip, carefully washing with PBS (pH 7.4), adding 50 μ L of BCP solution into the working area of the electrode obtained in step (5), standing at room temperature for 10 min, washing with PBS (pH 7.4), and drying at room temperature;
(7) and (4) obtaining a working area of the electrode in the step (6), dripping 20 mu L of PBS (phosphate buffer solution) containing 0.01M ascorbic acid and having pH of 7.4, and carrying out photoelectrochemical signal detection through a time-current curve under the assistance of a xenon lamp by using a three-electrode system.
The preparation process of the chitosan modified cadmium sulfide quantum dot comprises the following steps:
pouring 10 mL of 0.1M cadmium nitrate methanol solution into 10 mL of chitosan solution (0.5% M/v), stirring for 30min at room temperature, then adding 10 mL of 0.1M sodium sulfate methanol/water solution (1:1, v/v), stirring vigorously for 3h, centrifuging the synthesized product, washing with methanol and water, and re-dispersing in 5mL of water to prepare the chitosan modified cadmium sulfide quantum dot.
The preparation process of the BCP solution comprises the following steps:
9.79M of H2O2(namely the mass fraction is 30%) to 0.03M; dissolving 4-chloro-1-naphthol in ethanol, diluting with 0.01M PBS (pH 7.4) to volume of 1mL, wherein the volume fraction of ethanol is 2% and the molar concentration of 4-chloro-1-naphthol is 1 mM, and collecting 5 μ L of diluted H2O2Adding into the above solution, and shaking for 1 min.
The invention has the beneficial effects that:
(1) the method uses chromatographic paper as substrate, and has the advantages of low cost and portability.
(2) The method has fewer biomarker processes, is simple to operate, and has an easily controlled process.
(3) The method does not need additional material assistance and cultivation process, but has the function of biological amplification.
(4) The method has low detection limit and wider detection range, provides an idea for trace detection of RNA sequences, and has wide application prospect in early diagnosis of a plurality of diseases.
Detailed Description
In order to further illustrate the miRNA efficient detection method based on hemin-induced biocatalytic photoelectric sensitive interface, this example is implemented according to the technical scheme of the present invention, and specific embodiments are given, but the present invention is not limited to the following examples.
Example 1 efficient detection of miRNA based on hemin-induced biocatalysis photoelectric sensitive interface
(1) Firstly, designing a pattern of a microfluidic paper chip by using Adobe illustrator CS4 software, printing the designed pattern on A4 chromatographic paper by using a wax printer, then putting the printed chromatographic paper in an oven, and heating for 60 s at 100 ℃ to form a hydrophobic region and a hydrophilic working region;
(2) printing a three-electrode system (a carbon working electrode, a carbon counter electrode and an Ag/AgCl reference electrode) on the paper chip obtained in the step (1) by utilizing a screen printing technology;
(3) growing polypyrrole conductive polymer in a working area of a carbon electrode by using an in-situ chemical synthesis method, which comprises the following specific steps: firstly, measuring 20 mu L of pyrrole monomer, dripping the pyrrole monomer on a working area of an electrode (2), placing the electrode at room temperature for 10 min, then transferring 20 mu L of newly-configured mixed solution of ferric chloride and 0.3M hydrochloric acid with the molar concentration of 0.49M onto the surface of the electrode, placing the electrode at 4 ℃ for 3h, then sequentially and carefully cleaning the surface of the working electrode by using 50 mu L of 0.3M hydrochloric acid solution, 0.1M sodium chloride solution and deionized water, and finally drying the electrode at room temperature;
(4) pouring 10 mL of 0.1M cadmium nitrate methanol solution into 10 mL of chitosan solution (0.5% M/v), stirring for 30min at room temperature, then adding 10 mL of 0.1M sodium sulfate methanol/water solution (1:1, v/v), stirring strongly for 3h, centrifuging the synthesized product, washing with methanol and water, and re-dispersing in 5mL of water to prepare the chitosan modified cadmium sulfide quantum dot; fixing cadmium sulfide quantum dots in the working area of the electrode in the step (3), after carefully cleaning the electrode, placing 20 mu L of glutaraldehyde solution on the surface of the electrode to activate the chitosan modified cadmium sulfide quantum dots, and then cleaning the cadmium sulfide quantum dots for three times by PBS (phosphate buffer solution) with the pH value of 7.4;
(5) mu.L of 1. mu.M capture DNA strand was immobilized on the working region of the electrode obtained in step (4), followed by blocking the non-immobilized active sites with BSA, washing with PBS pH7.4, and then 20. mu.L of the electrode containing miRNA at different concentrations and 0.03U. mu.L-1The mixed solution of DSN and 5 mM magnesium chloride was placed on a paper chip, incubated at room temperature for 2 hours, and then applied with PB at pH7.4S, cleaning for three times;
(6) mu.L of 1. mu.M hemin-labeled DNA was added to a paper chip, which was carefully washed with PBS pH7.4, followed by 9.79M H2O2(i.e., 30% by weight) was diluted to 0.03M, 4-chloro-1-naphthol was dissolved in ethanol, and then diluted to 1mL in 0.01M PBS pH7.4, where the volume fraction of ethanol was 2% and the molar concentration of 4-chloro-1-naphthol was 1 mM, and 5. mu.L of diluted H was taken2O2Adding into the above solution, shaking for 1min to obtain BCP solution, adding 20 μ L of the above BCP solution into the working region of the electrode obtained in step (5), standing at room temperature for 10 min, washing with PBS of pH7.4, and drying at room temperature;
(7) and (4) obtaining a working area of the electrode in the step (6), dripping 20 mu L of PBS (phosphate buffer solution) containing 0.01M ascorbic acid and having pH of 7.4, and carrying out photoelectrochemical signal detection through a time-current curve under the assistance of a xenon lamp by using a three-electrode system.
Sequence listing
<110> university of Jinan
<120> efficient miRNA detection based on hemin-induced biocatalysis photoelectric sensitive interface
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Claims (3)
1. A miRNA high-efficiency detection method based on hemin-induced biocatalysis photoelectric sensitive interface is characterized by comprising the following steps:
(1) firstly, designing a pattern of a microfluidic paper chip by using Adobe illustrator CS4 software, printing the designed pattern on A4 chromatographic paper by using a wax printer, then putting the printed chromatographic paper in an oven, and heating for 60 s at 100 ℃ to form a hydrophobic region and a hydrophilic working region;
(2) printing a three-electrode system (a carbon working electrode, a carbon counter electrode and an Ag/AgCl reference electrode) on the paper chip obtained in the step (1) by utilizing a screen printing technology;
(3) growing polypyrrole conductive polymer in a working area of a carbon electrode by using an in-situ chemical synthesis method, which comprises the following specific steps: firstly, measuring 20 mu L-30 mu L of pyrrole monomer, dripping the pyrrole monomer on a working area of an electrode (2), standing for 10 min at room temperature, then taking 20 mu L of newly-configured mixed solution of ferric chloride with the molar concentration of 0.49M and hydrochloric acid with the molar concentration of 0.3M, transferring the mixed solution to the surface of the electrode, standing for 2-3 h at 4 ℃, then sequentially and carefully cleaning the surface of the working electrode with 50 mu L of 0.3M hydrochloric acid solution, 0.1M sodium chloride solution and deionized water, and finally drying at room temperature;
(4) fixing 20 mu L of chitosan modified cadmium sulfide quantum dots in the working area of the electrode in the step (3), after carefully cleaning the electrode, placing 20 mu L of glutaraldehyde solution on the surface of the electrode to activate the chitosan modified cadmium sulfide quantum dots, and then cleaning the electrode three times by PBS (phosphate buffer solution) with the pH value of 7.4;
(5) mu.L of 1. mu.M capture DNA was immobilized on the working region of the electrode obtained in step (4), followed by blocking of the non-immobilized active sites with BSAWashing with PBS (pH 7.4), collecting 20 μ L of the mixture containing miRNA at different concentrations and 0.03U μ L-1Placing the mixed solution of DSN and 5 mM magnesium chloride on a paper chip, incubating for 2 h at room temperature, and washing with PBS (pH 7.4) for three times;
(6) adding 20 μ L of 1 μ M hemin-labeled DNA onto a paper chip, carefully washing with PBS (pH 7.4), adding 50 μ L of BCP solution into the working area of the electrode obtained in step (5), standing at room temperature for 10 min, washing with PBS (pH 7.4), and drying at room temperature;
(7) and (4) obtaining a working area of the electrode in the step (6), dripping 20 mu L of PBS (phosphate buffer solution) containing 0.01M ascorbic acid and having pH of 7.4, and carrying out photoelectrochemical signal detection through a time-current curve under the assistance of a xenon lamp by using a three-electrode system.
2. The efficient miRNA detection method based on hemin-induced biocatalysis photoelectric sensitive interface as claimed in claim 1, wherein the preparation process of the chitosan-modified cadmium sulfide quantum dot is as follows: pouring 10 mL of 0.1M cadmium nitrate methanol solution into 10 mL of chitosan solution (0.5% M/v), stirring for 30min at room temperature, then adding 10 mL of 0.1M sodium sulfate methanol/water solution (1:1, v/v), stirring vigorously for 3h, centrifuging the synthesized product, washing with methanol and water, and re-dispersing in 5mL of water to prepare the chitosan modified cadmium sulfide quantum dot.
3. The efficient miRNA detection method based on hemin-induced biocatalysis photoelectric sensitive interface as claimed in claim 1, wherein the BCP solution preparation process is as follows: 9.79M of H2O2(namely the mass fraction is 30%) to 0.03M; dissolving 4-chloro-1-naphthol in ethanol, diluting with 0.01M PBS (pH 7.4) to volume of 1mL, wherein the volume fraction of ethanol is 2% and the molar concentration of 4-chloro-1-naphthol is 1 mM, and collecting 5 μ L of diluted H2O2Adding into the above solution, and shaking for 1 min.
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