CN111024788B - Preparation method of paper-based ratio photoelectrochemical biosensor for detecting microRNA - Google Patents
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Abstract
The invention discloses a preparation method of a paper-based ratio photoelectrochemical biosensor for detecting microRNA. Growing gold nanoparticles on the surface of a paper-based double electrode consisting of a working electrode and an internal reference electrode, and then electrodepositing cuprous oxide and sensitizing graphene quantum dots and silver iodide nanoparticles to enhance a photocurrent signal; when the target microRNA exists, DNA probes output by double-strand specific nuclease reaction induced by microRNA with different concentrations and constant concentrations are respectively incubated on the surfaces of a working electrode and an inner reference electrode, DNA hairpins H1 and H2 on the surfaces of a DNA bridging chain and the electrodes are combined to induce and form a DNA bridge nano structure, so that silver iodide nanoparticles marked at the ends of H1 and H2 are far away from the surfaces of the electrodes, a photocurrent signal is reduced, and the sensitive detection of the microRNA is realized based on the ratio of a working current signal to an inner reference current signal.
Description
Technical Field
The invention relates to the technical fields of photoelectrochemical signal detection technology, paper-based sensing device preparation technology, nano composite material synthesis technology and microRNA analysis and detection, in particular to a preparation method of a paper-based ratio photoelectrochemical biosensor for detecting microRNA.
Background
In recent years, various paper-based photoelectrochemical analysis devices have been prepared based on the combined advantages of microfluidic paper analysis devices and photoelectrochemical analysis methods, such as low cost, portability, simple operation, high sensitivity, and the like. In the paper-based photoelectrochemical analysis process, a single signal reading mode is generally adopted, and the single signal analysis strategy is easily interfered by external environment, analysis equipment, target analytes and the like, so that false positive/negative signals are generated, and the analysis accuracy is reduced. Compared with a single-signal analysis detection method, the double-signal ratio analysis detection method can provide self-correction, effectively reduce interference between an inner boundary and an outer boundary, and show higher analysis accuracy and sensitivity.
microRNA is an endogenous non-coding ribonucleic acid consisting of about 18-25 nucleotides, which is closely related to certain pathological processes. A large number of studies indicate that the occurrence of malignant tumors is related to the abnormal expression of microRNA. However, the content of microRNA is low, and in order to realize quantitative detection of microRNA, it is necessary to improve the accuracy and sensitivity of the analysis method. Therefore, the advantages of high sensitivity and accuracy of the double-signal ratio analysis detection method are fully utilized, and the development of the paper-based ratio photoelectrochemical biosensor for sensitively detecting microRNA has important significance for clinical diagnosis application.
Disclosure of Invention
The invention aims to construct a paper-based ratio photoelectrochemical sensing platform for sensitively detecting microRNA by combining a spatially-resolved double-cathode array and an electron transfer tunnel distance regulation and control strategy coordinated by a DNA bridge nano structure. The double-cathode array consists of a working electrode and an internal reference electrode, gold nanoparticles are functionalized on the surface of the electrode array, and then a cascade sensitization structure formed by cuprous oxide, graphene quantum dots and silver iodide nanoparticles is assembled under the assistance of DNA hairpins H1 and H2, so that a large photocurrent signal can be generated. In the presence of the target microRNA, a target object circulating reaction catalyzed by the double-strand specific nuclease is initiated, and a large amount of DNA probes can be output. The output DNA probe combined with the DNA bridging chain can induce the formation of a DNA bridging nano structure, so that silver iodide nano particles marked at the tail ends of H1 and H2 are far away from the surface of an electrode, and a photocurrent signal is reduced. The DNA probes output by the reaction of the microRNA induced double-strand specific nuclease with different concentrations are incubated on the working electrode to finally obtain a variable working current signal, and the DNA probes output by the reaction of the microRNA induced double-strand specific nuclease with constant concentration are incubated on the internal reference electrode to finally obtain a constant internal reference current signal. And the microRNA sensitivity detection is realized by calculating the ratio value of the working current signal and the internal reference current signal.
In order to solve the technical problem, the invention is realized by the following measures:
(1) firstly, designing a batik pattern of a paper auxiliary plate and a paper detection plate by using Adobe illustrator CS4 software on a computer, then printing the designed batik pattern on chromatographic paper by using a wax printer with the model of Color Qube 8580, and finally heating the chromatographic paper printed with wax in an oven at 130 ℃ for 50 s to obtain a hydrophilic auxiliary area of the paper auxiliary plate and a hydrophilic working electrode area and an inner reference electrode area of the paper detection plate;
(2) the functionalized gold nanoparticle conducting layer of the hydrophilic working electrode area and the internal reference electrode area of the paper detection board obtained in the step (1) comprises the following specific steps: firstly, preparing gold seeds, adding 100-200 mu L of mercaptoethylamine with the concentration of 0.2-0.25M into 10-15 mL of chloroauric acid solution with the concentration of 1.0-1.5 mM, magnetically stirring at room temperature for 10-30 min, adding 2-5 mu L of sodium borohydride solution with the concentration of 10-20 mM into the obtained mixed solution, and continuously magnetically stirring under the dark condition for 5-10 min to obtain wine red solution; then respectively dripping 80-100 mu L of prepared gold seed solution in a working electrode area and an internal reference electrode area, drying in a 60 ℃ drying oven, repeating the dripping-drying process for 3-5 times, respectively dripping 80-100 mu L of growth solution in the working electrode area and the internal reference electrode area, wherein the growth solution consists of 20-25 mM chloroauric acid and 200-300 mM hydroxylamine hydrochloride, reacting at room temperature for 20-40 min, washing the surfaces of the working electrode area and the internal reference electrode area with secondary water, and drying in the 60 ℃ drying oven to obtain a working electrode and an internal reference electrode coated by gold nanoparticles;
(3) firstly, utilizing Adobe illustrator CS4 software to design printing patterns of an Ag/AgCl reference electrode and a carbon counter electrode on a computer, and then printing the designed printing patterns on the hydrophilic auxiliary area of the paper auxiliary plate obtained in the step (1) by a screen printing technology to obtain the Ag/AgCl reference electrode and the carbon counter electrode;
(4) firstly, electrodepositing rhombus cuprous oxide in a working electrode area coated by the gold nanoparticles obtained in the step (2) by using a three-electrode system consisting of the working electrode coated by the gold nanoparticles obtained in the step (2), the Ag/AgCl reference electrode obtained in the step (3) and a carbon counter electrode, wherein the deposition voltage is-0.2V, the deposition time is 40 min, the deposition temperature is 70 ℃, the deposition electrolyte is a mixed solution consisting of copper acetate with the concentration of 0.02M and lactic acid with the concentration of 0.4M, the pH of the deposition electrolyte is adjusted to 9 by sodium hydroxide with the concentration of 1M, washing the surface of the working electrode area by using secondary water after the electrodeposition is finished, and drying in a 60 ℃ oven; then, electrodepositing cuprous oxide in a rhombus shape in the area of the internal reference electrode coated with the gold nanoparticles obtained in the step (2) by using a three-electrode system consisting of the internal reference electrode coated with the gold nanoparticles obtained in the step (2), the Ag/AgCl reference electrode obtained in the step (3) and a carbon counter electrode under the same deposition conditions, and finally obtaining a working electrode modified by the cuprous oxide and the internal reference electrode;
(5) preparing graphene quantum dots: dissolving 0.25 g of carbon powder into 50 mL of 6M nitric acid solution, heating and refluxing for 24 h at 130 ℃ after magnetic stirring for 1 h, cooling to room temperature, centrifuging the obtained mixed solution for 20 min and collecting supernatant, heating the collected supernatant at 100 ℃ to remove water and nitric acid to obtain a reddish brown solid, finally dissolving the obtained reddish brown solid in secondary water, and dialyzing in a dialysis bag for 72 h to obtain a graphene quantum dot solution;
(6) preparing DNA hairpins H1 and H2 marked by silver iodide nanoparticles, wherein the process comprises two steps: the first step is to synthesize silver iodide nanoparticles, 1-2 mmol KI and 0.2-0.8 g polyvinylpyrrolidone are dissolved in 20-40 mL secondary water, after magnetic stirring is carried out for 40-60 min, 5-10 mL silver nitrate solution with the concentration of 0.1-0.3M is added drop by drop, and the obtained yellow mixed solution is dialyzed in a dialysis bag for 24-36 h to obtain silver iodide nanoparticles; labeling the obtained silver iodide nanoparticles on DNA hairpins H1 and H2, labeling an amino group at the H15 'end, a sulfhydryl group at the 3' end, a sulfhydryl group at the H25 'end and an amino group at the 3' end, firstly, utilizing trihydroxymethyl aminomethane (2-carboxymethyl) phosphine solution with the concentration of 10 mM to break disulfide bonds in the sulfhydryl groups at the H1 end and the H2 end, then mixing the obtained H1 and H2 with 2 mL prepared silver iodide nanoparticles and incubating for 1H at room temperature, and finally centrifugally washing the obtained mixed solution to remove redundant H1 and H2 to obtain the silver iodide nanoparticle labeled H1 and H2;
(7) target cycling reaction catalyzed by double-strand specific nuclease: mixing 10 mu L of DNA hairpin HP with the concentration of 0.3 mu M with 10 mu L of target microRNA with different concentrations, 4 mu L of double-strand specific nuclease with the concentration of 0.5U and 6 mu L of Tris-HCl buffer solution with the pH of 8.0, wherein the used Tris-HCl buffer solution with the pH of 8.0 consists of Tris-HCl with the concentration of 50 mM, dimercaptothreitol with the concentration of 1 mM and magnesium chloride with the concentration of 5 mM, heating and reacting the obtained mixed solution at 50 ℃ for 60 min, then adding 5 mu L of ethylenediamine tetraacetic acid with the concentration of 15 mM, and continuously heating and reacting at 50 ℃ for 20 min to obtain double-strand specific nuclease reaction products induced by the target microRNA with different concentrations; meanwhile, carrying out double-strand specific nuclease reaction induced by the target microRNA with constant concentration under the same double-strand specific nuclease reaction condition to obtain a double-strand specific nuclease reaction product induced by the target microRNA with constant concentration;
(8) construction of paper-based ratio photoelectrochemical biosensor: respectively dripping 20 mu L of 3-aminopropyltriethoxysilane with the mass fraction of 2% on the surfaces of the cuprous oxide modified working electrode and the internal reference electrode obtained in the step (4), reacting at room temperature for 50 min, washing with a phosphate buffer solution with the pH of 7.4, wherein the phosphate buffer solution is abbreviated as PBS, then dripping 20 mu L of a mixed solution consisting of the graphene quantum dot solution obtained in the step (5), 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride with the concentration of 20 mM and hydroxythiosuccinimide with the concentration of 20 mM, reacting at room temperature for 1H, washing with PBS with the pH of 7.4, dripping 20 mu L of H1 and H2 marked by the silver iodide nanoparticles obtained in the step (6), incubating for 2H, and washing with PBS with the pH of 7.4, mu.L of mercaptohexanol of 1 mM concentration was added dropwise thereto and reacted at room temperature for 1 hour, and after washing with PBS of pH 7.4 to remove the excess mercaptohexanol, dripping 20 mu L of target microRNA-induced double-strand specific nuclease reaction products with different concentrations obtained in the step (7) onto the surface of a working electrode, dripping 20 mu L of target microRNA-induced double-strand specific nuclease reaction products with constant concentration obtained in the step (7) onto the surface of an internal reference electrode, reacting at 37 deg.C for 1 h, washing with PBS (pH 7.4), dripping 20 μ L DNA bridging chain with concentration of 5.0 μ M on the surfaces of working electrode and internal reference electrode, after incubation reaction for 1 h at room temperature, washing with PBS (phosphate buffer solution) with pH 7.4 to remove redundant DNA bridging chains, and finally forming DNA bridging nano structures on the surfaces of the working electrode and the internal reference electrode;
(9) double signal ratio detection: firstly, working current signal detection is carried out by a time-current method by using a three-electrode system consisting of the working electrode obtained in the step (8), the Ag/AgCl reference electrode obtained in the step (3) and a carbon counter electrode, the excitation wavelength range used in the detection process is 200-2500 nm, the detection voltage used is 0.0V, the detection electrolyte used is PBS with the concentration of 0.1M and the pH value of 7.4, after the working current signal is recorded for 10 s, the working electrode is disconnected, then the internal reference current signal is recorded by using the internal reference electrode obtained in the step (8) and the three-electrode system consisting of the Ag/AgCl reference electrode obtained in the step (3) and the carbon counter electrode through the condition of detecting the working current signal, the recording time is 10 s, and finally the ratio of the working current signal and the internal reference current signal is calculated, and sensitive detection of microRNA is realized.
The invention has the beneficial effects that:
(1) based on the comprehensive advantages of an integration ratio analysis strategy and a photoelectrochemical analysis technology, the constructed paper-based ratio photoelectrochemical biosensor can effectively reduce interference between internal and external boundaries, reduce false positive/negative signals, show higher analysis sensitivity and accuracy, and has important significance for diagnosis of clinical microRNA.
(2) The cascade sensitization structure composed of cuprous oxide, graphene quantum dots and silver iodide nanoparticles not only has strong photoresponse capability, but also can effectively accelerate photo-generated charge separation and transfer, greatly enhance photocurrent signals and improve signal amplification capability.
(3) A target object is subjected to cyclic reaction catalyzed by double-strand specific nuclease to output a large number of DNA probes, the DNA probes are combined with a DNA bridging chain to induce the formation of a DNA bridge nano structure, so that silver iodide nano particles marked at the ends of DNA hairpins H1 and H2 are far away from the surface of an electrode, the electron transfer process is blocked, a photocurrent signal is greatly reduced, and further signal amplification is realized.
The specific implementation mode is as follows:
in order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
Example 1: application of paper-based ratio photoelectrochemical biosensor in detection of microRNA-141
(1) Firstly, designing a batik pattern of a paper auxiliary plate and a paper detection plate by using Adobe illustrator CS4 software on a computer, then printing the designed batik pattern on chromatographic paper by using a wax printer with the model of Color Qube 8580, and finally heating the chromatographic paper printed with wax in an oven at 130 ℃ for 50 s to obtain a hydrophilic auxiliary area of the paper auxiliary plate and a hydrophilic working electrode area and an inner reference electrode area of the paper detection plate;
(2) the functionalized gold nanoparticle conducting layer of the hydrophilic working electrode area and the internal reference electrode area of the paper detection board obtained in the step (1) comprises the following specific steps: firstly, preparing gold seeds, adding 100 mu L of mercaptoethylamine with the concentration of 0.24M into 10 mL of chloroauric acid solution with the concentration of 1.5 mM, magnetically stirring at room temperature for 20 min, adding 5 mu L of sodium borohydride solution with the concentration of 15 mM into the obtained mixed solution, and continuously magnetically stirring under the dark condition for 5 min to obtain wine red solution; then respectively dripping 80 mu L of prepared gold seed solution into a working electrode area and an internal reference electrode area, drying in a 60 ℃ drying oven, repeating the dripping-drying process for 4 times, respectively dripping 80 mu L of growth solution into the working electrode area and the internal reference electrode area, wherein the growth solution consists of 20 mM chloroauric acid and 200 mM hydroxylamine hydrochloride, reacting at room temperature for 35 min, washing the surfaces of the working electrode area and the internal reference electrode area with secondary water, and drying in the 60 ℃ drying oven to obtain a working electrode and an internal reference electrode coated by gold nanoparticles;
(3) firstly, utilizing Adobe illustrator CS4 software to design printing patterns of an Ag/AgCl reference electrode and a carbon counter electrode on a computer, and then printing the designed printing patterns on the hydrophilic auxiliary area of the paper auxiliary plate obtained in the step (1) by a screen printing technology to obtain the Ag/AgCl reference electrode and the carbon counter electrode;
(4) firstly, electrodepositing rhombus cuprous oxide in a working electrode area coated by the gold nanoparticles obtained in the step (2) by using a three-electrode system consisting of the working electrode coated by the gold nanoparticles obtained in the step (2), the Ag/AgCl reference electrode obtained in the step (3) and a carbon counter electrode, wherein the deposition voltage is-0.2V, the deposition time is 40 min, the deposition temperature is 70 ℃, the deposition electrolyte is a mixed solution consisting of copper acetate with the concentration of 0.02M and lactic acid with the concentration of 0.4M, the pH of the deposition electrolyte is adjusted to 9 by sodium hydroxide with the concentration of 1M, washing the surface of the working electrode area by using secondary water after the electrodeposition is finished, and drying in a 60 ℃ oven; then, electrodepositing cuprous oxide in a rhombus shape in the area of the internal reference electrode coated with the gold nanoparticles obtained in the step (2) by using a three-electrode system consisting of the internal reference electrode coated with the gold nanoparticles obtained in the step (2), the Ag/AgCl reference electrode obtained in the step (3) and a carbon counter electrode under the same deposition conditions, and finally obtaining a working electrode modified by the cuprous oxide and the internal reference electrode;
(5) preparing graphene quantum dots: dissolving 0.25 g of carbon powder into 50 mL of 6M nitric acid solution, heating and refluxing for 24 h at 130 ℃ after magnetic stirring for 1 h, cooling to room temperature, centrifuging the obtained mixed solution for 20 min and collecting supernatant, heating the collected supernatant at 100 ℃ to remove water and nitric acid to obtain a reddish brown solid, finally dissolving the obtained reddish brown solid in secondary water, and dialyzing in a dialysis bag for 72 h to obtain a graphene quantum dot solution;
(6) preparing DNA hairpins H1 and H2 marked by silver iodide nanoparticles, wherein the process comprises two steps: the first step is to synthesize silver iodide nanoparticles, 1.5 mmol KI and 0.52 g polyvinylpyrrolidone are dissolved in 20 mL secondary water, after magnetic stirring is carried out for 60 min, 10 mL silver nitrate solution with the concentration of 0.1M is added dropwise, and the obtained yellow mixed solution is dialyzed in a dialysis bag for 24 h to obtain silver iodide nanoparticles; labeling the obtained silver iodide nanoparticles on DNA hairpins H1 and H2, labeling an amino group at the H15 'end, a sulfhydryl group at the 3' end, a sulfhydryl group at the H25 'end and an amino group at the 3' end, firstly, utilizing trihydroxymethyl aminomethane (2-carboxymethyl) phosphine solution with the concentration of 10 mM to break disulfide bonds in the sulfhydryl groups at the H1 end and the H2 end, then mixing the obtained H1 and H2 with 2 mL prepared silver iodide nanoparticles and incubating for 1H at room temperature, and finally centrifugally washing the obtained mixed solution to remove redundant H1 and H2 to obtain the silver iodide nanoparticle labeled H1 and H2;
(7) target cycling reaction catalyzed by double-strand specific nuclease: mixing 10 mu L of DNA hairpin HP with the concentration of 0.3 mu M with 10 mu L of target microRNA-141 with different concentrations, 4 mu L of double-strand specific nuclease with the concentration of 0.5U and 6 mu L of Tris-HCl buffer solution with the pH of 8.0, wherein the used Tris-HCl buffer solution with the pH of 8.0 consists of Tris-HCl with the concentration of 50 mM, dimercaptothreitol with the concentration of 1 mM and magnesium chloride with the concentration of 5 mM, heating and reacting the obtained mixed solution at 50 ℃ for 60 min, then adding 5 mu L of ethylenediamine tetraacetic acid with the concentration of 15 mM, and continuously heating and reacting at 50 ℃ for 20 min to obtain double-strand specific nuclease reaction products induced by the target microRNA-141 with different concentrations; meanwhile, carrying out double-strand specific nuclease reaction induced by the target microRNA-141 with constant concentration under the same double-strand specific nuclease reaction condition to obtain a double-strand specific nuclease reaction product induced by the target microRNA-141 with constant concentration;
(8) construction of paper-based ratio photoelectrochemical biosensor: respectively dripping 20 mu L of 3-aminopropyltriethoxysilane with the mass fraction of 2% on the surfaces of the cuprous oxide modified working electrode and the internal reference electrode obtained in the step (4), reacting at room temperature for 50 min, washing with a phosphate buffer solution with the pH of 7.4, wherein the phosphate buffer solution is abbreviated as PBS, then dripping 20 mu L of a mixed solution consisting of the graphene quantum dot solution obtained in the step (5), 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride with the concentration of 20 mM and hydroxythiosuccinimide with the concentration of 20 mM, reacting at room temperature for 1H, washing with PBS with the pH of 7.4, dripping 20 mu L of H1 and H2 marked by the silver iodide nanoparticles obtained in the step (6), incubating for 2H, and washing with PBS with the pH of 7.4, mu.L of mercaptohexanol of 1 mM concentration was added dropwise thereto and reacted at room temperature for 1 hour, and after washing with PBS of pH 7.4 to remove the excess mercaptohexanol, dripping 20 mu L of target microRNA-141 induced double-strand specific nuclease reaction products with different concentrations obtained in the step (7) onto the surface of a working electrode, dripping 20 mu L of target microRNA-141 induced double-strand specific nuclease reaction products with constant concentration obtained in the step (7) onto the surface of an internal reference electrode, reacting at 37 deg.C for 1 h, washing with PBS (pH 7.4), dripping 20 μ L DNA bridging chain with concentration of 5.0 μ M on the surfaces of working electrode and internal reference electrode, after incubation reaction for 1 h at room temperature, washing with PBS (phosphate buffer solution) with pH 7.4 to remove redundant DNA bridging chains, and finally forming DNA bridging nano structures on the surfaces of the working electrode and the internal reference electrode;
(9) double signal ratio detection: firstly, working current signal detection is carried out by a time-current method by using a three-electrode system consisting of the working electrode obtained in the step (8), the Ag/AgCl reference electrode obtained in the step (3) and a carbon counter electrode, the excitation wavelength range used in the detection process is 200-2500 nm, the detection voltage used is 0.0V, the detection electrolyte used is PBS with the concentration of 0.1M and the pH value of 7.4, after the working current signal is recorded for 10 s, the working electrode is disconnected, then the internal reference current signal is recorded by using the internal reference electrode obtained in the step (8) and the three-electrode system consisting of the Ag/AgCl reference electrode obtained in the step (3) and the carbon counter electrode through the condition of detecting the working current signal, the recording time is 10 s, and finally the ratio of the working current signal and the internal reference current signal is calculated, and sensitive detection of the microRNA-141 is realized.
Sequence listing
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Claims (1)
1. A preparation method of a paper-based ratio photoelectrochemical biosensor for detecting microRNA is characterized by comprising the following steps:
(1) firstly, designing a batik pattern of a paper auxiliary plate and a paper detection plate by using Adobe illustrator CS4 software on a computer, then printing the designed batik pattern on chromatographic paper by using a wax printer with the model of Color Qube 8580, and finally heating the chromatographic paper printed with wax in an oven at 130 ℃ for 50 s to obtain a hydrophilic auxiliary area of the paper auxiliary plate and a hydrophilic working electrode area and an inner reference electrode area of the paper detection plate;
(2) the functionalized gold nanoparticle conducting layer of the hydrophilic working electrode area and the internal reference electrode area of the paper detection board obtained in the step (1) comprises the following specific steps: firstly, preparing gold seeds, adding 100-200 mu L of mercaptoethylamine with the concentration of 0.2-0.25M into 10-15 mL of chloroauric acid solution with the concentration of 1.0-1.5 mM, magnetically stirring at room temperature for 10-30 min, adding 2-5 mu L of sodium borohydride solution with the concentration of 10-20 mM into the obtained mixed solution, and continuously magnetically stirring under the dark condition for 5-10 min to obtain wine red solution; then respectively dripping 80-100 mu L of prepared gold seed solution in a working electrode area and an internal reference electrode area, drying in a 60 ℃ drying oven, repeating the process of dripping-drying for 3-5 times, respectively dripping 80-100 mu L of growth solution in the working electrode area and the internal reference electrode area, wherein the growth solution consists of 20-25 mM chloroauric acid and 200-300 mM hydroxylamine hydrochloride, reacting at room temperature for 20-40 min, washing the surfaces of the working electrode area and the internal reference electrode area with secondary water, and drying in the 60 ℃ drying oven to obtain a working electrode and an internal reference electrode coated by gold nanoparticles;
(3) firstly, utilizing Adobe illustrator CS4 software to design printing patterns of an Ag/AgCl reference electrode and a carbon counter electrode on a computer, and then printing the designed printing patterns on the hydrophilic auxiliary area of the paper auxiliary plate obtained in the step (1) by a screen printing technology to obtain the Ag/AgCl reference electrode and the carbon counter electrode;
(4) firstly, electrodepositing rhombus cuprous oxide in a working electrode area coated by the gold nanoparticles obtained in the step (2) by using a three-electrode system consisting of the working electrode coated by the gold nanoparticles obtained in the step (2), the Ag/AgCl reference electrode obtained in the step (3) and a carbon counter electrode, wherein the deposition voltage is-0.2V, the deposition time is 40 min, the deposition temperature is 70 ℃, the deposition electrolyte is a mixed solution consisting of copper acetate with the concentration of 0.02M and lactic acid with the concentration of 0.4M, the pH of the deposition electrolyte is adjusted to 9 by sodium hydroxide with the concentration of 1M, washing the surface of the working electrode area by using secondary water after the electrodeposition is finished, and drying in a 60 ℃ oven; then, electrodepositing cuprous oxide in a rhombus shape in the area of the internal reference electrode coated with the gold nanoparticles obtained in the step (2) by using a three-electrode system consisting of the internal reference electrode coated with the gold nanoparticles obtained in the step (2), the Ag/AgCl reference electrode obtained in the step (3) and a carbon counter electrode under the same deposition conditions, and finally obtaining a working electrode modified by the cuprous oxide and the internal reference electrode;
(5) preparing graphene quantum dots: dissolving 0.25 g of carbon powder into 50 mL of 6M nitric acid solution, heating and refluxing for 24 h at 130 ℃ after magnetic stirring for 1 h, cooling to room temperature, centrifuging the obtained mixed solution for 20 min and collecting supernatant, heating the collected supernatant at 100 ℃ to remove water and nitric acid to obtain a reddish brown solid, finally dissolving the obtained reddish brown solid in secondary water, and dialyzing in a dialysis bag for 72 h to obtain a graphene quantum dot solution;
(6) preparing DNA hairpins H1 and H2 marked by silver iodide nanoparticles, wherein the process comprises two steps: the first step is to synthesize silver iodide nanoparticles, 1-2 mmol KI and 0.2-0.8 g polyvinylpyrrolidone are dissolved in 20-40 mL secondary water, after magnetic stirring is carried out for 40-60 min, 5-10 mL silver nitrate solution with the concentration of 0.1-0.3M is added drop by drop, and the obtained yellow mixed solution is dialyzed in a dialysis bag for 24-36 h to obtain silver iodide nanoparticles; the second step is to label the obtained silver iodide nanoparticles on DNA hairpins H1 and H2, the used H15 'end is labeled with amino, the 3' end is labeled with mercapto, the H25 'end is labeled with mercapto, the 3' end is labeled with amino, firstly, the disulfide bonds in the mercapto at the ends of H1 and H2 are broken by using trihydroxymethyl aminomethane (2-carboxymethyl) phosphine solution with the concentration of 10 mM, then the obtained H1 and H2 are mixed with 2 mL of prepared silver iodide nanoparticles and incubated for 1H at room temperature, finally the obtained mixed solution is centrifugally washed to remove the redundant H1 and H2, and the DNA hairpins H1 and H2 labeled with the silver iodide nanoparticles are obtained;
(7) target cycling reaction catalyzed by double-strand specific nuclease: mixing 10 mu L of DNA hairpin HP with the concentration of 0.3 mu M with 10 mu L of target microRNA with different concentrations, 4 mu L of double-strand specific nuclease with the concentration of 0.5U and 6 mu L of Tris-HCl buffer solution with the pH of 8.0, wherein the used Tris-HCl buffer solution with the pH of 8.0 consists of Tris-HCl with the concentration of 50 mM, dimercaptothreitol with the concentration of 1 mM and magnesium chloride with the concentration of 5 mM, heating and reacting the obtained mixed solution at 50 ℃ for 60 min, then adding 5 mu L of ethylenediamine tetraacetic acid with the concentration of 15 mM, and continuously heating and reacting at 50 ℃ for 20 min to obtain double-strand specific nuclease reaction products induced by the target microRNA with different concentrations; meanwhile, carrying out double-strand specific nuclease reaction induced by the target microRNA with constant concentration under the same double-strand specific nuclease reaction condition to obtain a double-strand specific nuclease reaction product induced by the target microRNA with constant concentration;
(8) construction of paper-based ratio photoelectrochemical biosensor: respectively dripping 20 mu L of 3-aminopropyltriethoxysilane with the mass fraction of 2% on the surfaces of the cuprous oxide modified working electrode and the internal reference electrode obtained in the step (4), reacting at room temperature for 50 min, washing with a phosphate buffer solution with the pH of 7.4, wherein the phosphate buffer solution is abbreviated as PBS, then dripping 20 mu L of a mixed solution consisting of the graphene quantum dot solution obtained in the step (5), 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide hydrochloride with the concentration of 20 mM and hydroxythiosuccinimide with the concentration of 20 mM, reacting at room temperature for 1H, washing with PBS with the pH of 7.4, dripping 20 mu L of DNA hairpins H1 and H2 marked by the silver iodide nanoparticles obtained in the step (6), incubating for 2H, washing with PBS with the pH of 7.4, mu.L of mercaptohexanol of 1 mM concentration was added dropwise thereto and reacted at room temperature for 1 hour, and after washing with PBS of pH 7.4 to remove the excess mercaptohexanol, dripping 20 mu L of target microRNA-induced double-strand specific nuclease reaction products with different concentrations obtained in the step (7) onto the surface of a working electrode, dripping 20 mu L of target microRNA-induced double-strand specific nuclease reaction products with constant concentration obtained in the step (7) onto the surface of an internal reference electrode, reacting at 37 deg.C for 1 h, washing with PBS (pH 7.4), dripping 20 μ L DNA bridging chain with concentration of 5.0 μ M on the surfaces of working electrode and internal reference electrode, after incubation reaction for 1 h at room temperature, washing with PBS (phosphate buffer solution) with pH 7.4 to remove redundant DNA bridging chains, and finally forming DNA bridging nano structures on the surfaces of the working electrode and the internal reference electrode;
(9) double signal ratio detection: firstly, working current signal detection is carried out by a time-current method by using a three-electrode system consisting of the working electrode obtained in the step (8), the Ag/AgCl reference electrode obtained in the step (3) and a carbon counter electrode, the excitation wavelength range used in the detection process is 200-2500 nm, the detection voltage used is 0.0V, the detection electrolyte used is PBS with the concentration of 0.1M and the pH value of 7.4, after the working current signal is recorded for 10 s, the working electrode is disconnected, then the internal reference current signal is recorded by using the internal reference electrode obtained in the step (8) and the three-electrode system consisting of the Ag/AgCl reference electrode obtained in the step (3) and the carbon counter electrode through the condition of detecting the working current signal, the recording time is 10 s, and finally the ratio of the working current signal and the internal reference current signal is calculated, and sensitive detection of microRNA is realized.
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