CN110412097B - Preparation method of a photoelectrochemical sensor for ultrasensitive detection of microRNA - Google Patents
Preparation method of a photoelectrochemical sensor for ultrasensitive detection of microRNA Download PDFInfo
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- 108700011259 MicroRNAs Proteins 0.000 title claims abstract description 39
- 239000002679 microRNA Substances 0.000 title claims abstract description 39
- 238000001514 detection method Methods 0.000 title claims abstract description 29
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- 108020004414 DNA Proteins 0.000 claims abstract description 51
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- JCXGWMGPZLAOME-UHFFFAOYSA-N bismuth atom Chemical compound [Bi] JCXGWMGPZLAOME-UHFFFAOYSA-N 0.000 claims abstract description 30
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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Abstract
本发明公开了一种高灵敏的检测microRNA的纸基光电化学传感器。利用蜡打印技术制备纸芯片，并在其亲水工作区域原位生长金纳米颗粒实现纸芯片的功能化，随后修饰氧化亚铜/硫化铋/钒酸铋三级敏化物，通过固定的发夹DNA链对microRNA进行捕获，通过双链特异性核酸酶的特异性的识别和酶切作用，实现对microRNA的信号放大，随后通过多支杂交链反应，在链的主干部分嵌入铂纳米粒子，枝干部分形成氯化血红素/G‑四联体结构，形成具有类过氧化氢酶生物特性的DNA多联体，进一步实现信号的放大，从而完成光电化学传感器的制备，实现对microRNA的超灵敏、准确检测。The invention discloses a paper-based photoelectrochemical sensor for detecting microRNA with high sensitivity. The paper chips were prepared by wax printing technology, and gold nanoparticles were grown in situ in their hydrophilic working areas to realize the functionalization of the paper chips, and then the cuprous oxide/bismuth sulfide/bismuth vanadate tertiary sensitizers were modified through the fixed hairpins The DNA chain captures the microRNA, and through the specific recognition and enzymatic cleavage of the double-strand-specific nuclease, the signal amplification of the microRNA is realized, and then through the multi-branch hybridization chain reaction, platinum nanoparticles are embedded in the backbone of the chain, and the branch The dry part forms a hemin/G-quadruplex structure, forming a DNA concatemer with catalase-like biological properties, further realizing signal amplification, thereby completing the preparation of photoelectrochemical sensors and realizing ultra-sensitivity to microRNA , Accurate detection.
Description
Technical Field
The invention relates to the technical field of microRNA analysis and detection technology, multi-hybrid-strand signal amplification technology, DNA mimic enzyme catalysis technology, paper chip technology and composite nano materials, in particular to preparation of a photoelectrochemical sensor for ultrasensitive detection of microRNA.
Background
In recent years, the incidence of cancer has increased dramatically worldwide, which greatly threatens human health, and early detection of cancer is attracting more and more attention. The change of microRNA can regulate the expression of genes, and is a potential tumor marker, so that the detection of microRNA draws wide attention of people. The high-sensitivity and high-selectivity determination of microRNA has very important significance for gene screening, early diagnosis and treatment of genetic diseases. To date, several detection methods like real-time fluorescent quantitative polymerase chain reaction, nether hybridization, DNA microarray have been developed and used for the detection of cancer cells. However, these methods are time-consuming, require complicated sample pretreatment and expensive instruments, and thus, development of an ultra-sensitive, portable, and inexpensive assay for detecting microRNA is still urgently required.
Cuprous oxide, a commonly used photoelectric material, is widely used in photoelectrochemical sensors due to its non-toxicity, good catalytic activity, good biocompatibility, and unique optical and electrical properties. The key to improving the sensitivity of the photoelectrochemical sensor is to improve the photoelectrochemical response of the photoelectrochemical material. The ternary complex formed by compounding the bismuth vanadate/bismuth sulfide and cuprous oxide can reduce the recombination rate of electrons and holes and effectively enhance photoelectric signals.
At present, the DNA mimic enzyme has wide application in the field of biological catalysis, and compared with other mimic enzymes, the DNA mimic enzyme has the characteristics of good stability, easiness in synthesis and replication, difficulty in hydrolysis and the like, so that the DNA mimic enzyme is favored. hemin/G-quadruplex peroxidase mimic enzyme is an emerging DNA mimic enzyme rapidly developed in recent years and capable of catalyzing H2O2Since many reactions are involved, signal amplification techniques based on these reactions are widely used in bioassays. In addition, a signal amplification technique of multiple hybrid strands has attracted much attention in recent years, and a longer DNA double helix structure with multiple branches can be obtained by a reaction of multiple hybrid strands. In order to further amplify and analyze detection signals, the two signal amplification technologies are combined, more platinum nanoparticles are adsorbed on a DNA double-helix framework by utilizing the electrostatic adsorption effect between the platinum nanoparticles and a DNA chain, and branched chains on a plurality of hybrid chains can form a hemin/G-tetrad structure in the presence of hemin, so that the photoelectrochemical response of a photoelectric material is enhanced more, and the sensitivity of analysis and detection is improved.
Disclosure of Invention
The invention aims to prepare a bismuth vanadate/bismuth sulfide/cuprous oxide paper electrode with large specific surface area, good biocompatibility and photoelectric performance by an electrodeposition method, fix hairpin DNA chains on the surface of the electrode, capture target microRNA by using the hairpin DNA chains, realize the cyclic amplification of signals by the specific recognition and the enzyme digestion of double-chain specific nuclease, then embed platinum nanoparticles into the trunk part of the chains by a cascade multi-branch hybrid chain reaction, form a hemin/G-tetrad structure on the branch part, further realize the amplification of the signals, complete the preparation of a photoelectrochemical sensor and realize the ultrasensitive detection of the microRNA.
In order to solve the technical problem, the invention is realized by the following measures:
(1) designing a hydrophobic wax printing pattern of the microfluidic paper chip on a computer by using Adobe illustrator CS4 software, printing the designed printing pattern on chromatographic paper by using a wax printer, then putting the printed chromatographic paper in an oven, heating for 50 seconds at 130 ℃ to melt the wax and penetrate the thickness of the whole chromatographic paper to form a hydrophobic region and a hydrophilic working region;
(2) designing printing patterns of a working electrode, a counter electrode and a reference electrode which are matched with the wax printing patterns obtained in the step (1) on a computer, and printing a carbon working electrode, a carbon counter electrode and an Ag/AgCl reference electrode on the wax printing chromatographic paper obtained in the step (1) by utilizing a screen printing technology;
(3) growing gold nanoparticles in the working area of the carbon working electrode obtained in the step (2) by using an in-situ growth method, which comprises the following specific steps: firstly, gold nanoparticles are synthesized: measuring 60-90 mL of secondary water, placing the secondary water in a three-neck flask, heating the secondary water to 90 ℃, adding 0.5-0.9 mL of chloroauric acid with the mass fraction of 1%, continuing to heat the secondary water in a water bath to 96 ℃, immediately adding 2.5-2.9 mL of sodium citrate with the mass fraction of 1% after the reaction is carried out for 1 min, and reacting for 15-20 min under magnetic stirring to obtain a gold nanoparticle solution; then, firstly, 10-20 mu L of gold nanoparticle solution is dripped on a working area of a carbon working electrode by adopting a dripping-drying method, natural drying is carried out at room temperature, the dripping-drying operation is repeated for 3-5 times, 10-20 mu L of freshly prepared mixed solution of chloroauric acid with the mass fraction of 1% and 0.2M hydroxylamine hydrochloride is dripped on the working area, wherein the volume ratio of the chloroauric acid to the hydroxylamine hydrochloride is 1:1, after the gold nanoparticle solution grows for 20-40 min at room temperature, the working area is cleaned by secondary water and natural drying is carried out for 30 min at room temperature;
(4) modifying small-surface cuprous oxide on the working area of the carbon working electrode obtained in the step (3) by using an electrodeposition method, and modifying a bismuth vanadate/bismuth sulfide compound on the cuprous oxide;
(5) fixing the hairpin DNA chain S1 in the working area of the carbon working electrode obtained in the step (4), then adopting 6-mercapto-1-hexanol to block the active site, and then utilizing the S1 chain to capture microRNA;
(6) sequentially incubating the microRNA of the target detection object and the double-stranded specific nuclease in the working area of the carbon working electrode obtained in the step (5) and washing the carbon working electrode by using a phosphate buffer solution with the pH of 7.4;
(7) formation of DNA concatemers: dissolving the hairpin DNA molecule H1 and the hairpin DNA molecule H2 in a phosphate buffer solution with the pH value of 7.4, dripping the mixture into the working area of the carbon working electrode obtained in the step (6), carrying out in-situ proliferation to form a multi-branched hybrid chain, continuously dripping chlorhematin to form a chlorhematin/G-tetrad structure, embedding platinum nano particles into the main chain of the multi-branched hybrid chain by utilizing electrostatic adsorption to form a DNA (deoxyribonucleic acid) concatemer with biological catalysis, and catalyzing H2O2Decomposition to O2；
(8) In the working region of the carbon working electrode obtained in step (7), 10. mu.L of a solution containing 0.01M H was added dropwise2O2Under the irradiation of a xenon lamp, taking a printed Ag/AgCl electrode as a reference electrode, a printed carbon electrode as a counter electrode, and the carbon working electrode obtained in the step (7) as a working electrode, performing photoelectrochemical signal detection under the voltage of 0.0V by using a three-electrode system through a time-current curve method, and drawing a standard curve of photocurrent intensity and microRNA concentration to realize the detection of the microRNA.
The invention relates to an electrodeposition method for modifying small-area cuprous oxide in a working area of a carbon working electrode and modifying a bismuth vanadate/bismuth sulfide compound on the cuprous oxide, which comprises the following specific steps:
a. depositing cuprous oxide on the working area of the carbon working electrode obtained in the step (3): using an Ag/AgCl electrode as a reference electrode, a platinum electrode as a counter electrode, using the carbon working electrode obtained in the step (3) as a working electrode, using a three-electrode system, depositing small-surface cuprous oxide in the working area of the carbon working electrode obtained in the step (3) by a potential dissolution analysis method, wherein the deposited electrolyte consists of copper acetate with the concentration of 0.02M and lactic acid with the concentration of 0.3-0.5M, the pH of the electrolyte is adjusted to 9-11 by sodium hydroxide with the concentration of 2M, the deposition voltage is-0.3V and-0.5V, the deposition temperature is 60 ℃, the deposition time is 25-45 min, and after the deposition is finished, washing the surface of the working area by secondary water and naturally drying at room temperature;
b. synthesis of bismuth vanadate/bismuth sulfide complexes: firstly, synthesizing bismuth vanadate by a hydrothermal method, dissolving 1 mmol of bismuth nitrate pentahydrate in 40-60 mL of mixed solution of secondary water and ethylene glycol, wherein the volume ratio of the secondary water to the ethylene glycol is 1:1, sequentially adding 1 mmol of sodium metavanadate and 2.5-3.5 mmol of diisooctyl succinate sodium sulfonate, violently stirring for 5-10 min, then transferring into a 50mL reaction kettle, reacting for 16-18 h at 160 ℃, cooling to room temperature, respectively centrifugally cleaning for 3-5 times by using the secondary water and absolute ethyl alcohol, and then drying for 4 h at 60 ℃ in vacuum to obtain bismuth vanadate; dissolving 0.25 mmol of prepared bismuth vanadate in 40 mL of secondary water, adding 0.05-0.2 mmol of thioacetamide, stirring for 5-10 min, transferring to a 50mL reaction kettle, reacting at 120 ℃ for 8-10 h, cooling to room temperature, respectively centrifugally cleaning for 3-5 times by using secondary water and absolute ethyl alcohol, and then drying at 60 ℃ for 4 h in vacuum to obtain a bismuth vanadate/bismuth sulfide compound;
c. compounding a bismuth vanadate/bismuth sulfide compound with cuprous oxide: dripping 10 mu L of 3-aminopropyl triethoxysilane with mass fraction of 2 percent into the mixture to be oxidized
And (3) washing the working area on the surface of the cuprous by using a phosphate buffer solution with the pH value of 7.4, drying, then dropwise adding 10 mu L of mixed solution containing bismuth vanadate/bismuth sulfide compound and 2.5% glutaraldehyde by mass, drying, and then washing by using a phosphate buffer solution with the pH value of 7.4.
The specific steps of fixing the hairpin DNA chain S1 in the working area of the carbon working electrode obtained in the step (4), then adopting 6-mercapto-1-hexanol to block the active site, and then utilizing S1 to capture microRNA are as follows: mu.L of a mixture containing 2.5% by mass of glutaraldehyde and the DNA chain S1 was added dropwise to the working area of the carbon working electrode obtained in step (4), after incubation for 2 hours, washed with a phosphate buffer solution of pH 7.4 containing 0.05% Tween 20, 10. mu.L of 6-mercapto-1-hexanol at a concentration of 2 mM was added dropwise for blocking non-specific binding sites, and after incubation for 30 minutes, unbound 6-mercapto-1-hexanol was removed by washing with a phosphate buffer solution of pH 7.4 and dried naturally.
The specific steps of sequentially incubating the microRNA of the target detection object and the double-stranded specific nuclease in the working area of the carbon working electrode obtained in the step (5) and washing the incubated object with the phosphate buffer solution with the pH value of 7.4 are as follows: and (3) dropwise adding 10 mu L of microRNA-141 with different concentrations to the working area of the carbon working electrode obtained in the step (5), incubating at 37 ℃ for 40 min, washing with a phosphate buffer solution with pH 7.4 to remove unbound microRNA-141, continuously dropwise adding 10 mu L of 0.05U of double-stranded specific nuclease solution, incubating at 37 ℃ for 40 min, and washing with a phosphate buffer solution with pH 7.4.
The specific steps of the invention for forming the DNA concatemer are as follows:
a. synthesizing platinum nano particles: adding 500-1000 mu L of chloroplatinic acid with the concentration of 20 mM into 20-50mL of mixed solution of water and ethanol, wherein the volume ratio of the water to the ethanol is 1:1, adding 0.4-0.9 mol of polyvinylpyrrolidone, and refluxing for 4 hours at 80 ℃ to obtain a platinum nanoparticle solution;
preparation of DNA concatemers: adding 10 mu L of mixed liquor containing the hairpin DNA molecule H1 and the hairpin DNA molecule H2 into the working area of the carbon working electrode obtained in the step (6), wherein the concentrations of the hairpin DNA molecule H1 and the hairpin DNA molecule H2 are both 5 mu M, incubating at room temperature for 80 min, and washing with phosphate buffer solution with pH 7.4 to remove the hairpin DNA molecule H1 and the hairpin DNA molecule H2 which are not combined to react; then 10. mu.L of a 0.2 mM hemin solution was added dropwise, incubated at room temperature for 50 min, washed with a pH 7.4 phosphate buffer, dried, and then 10. mu.L of the prepared platinum nanoparticles were further added dropwise, and finally, excess platinum nanoparticles were removed by washing with a pH 7.4 phosphate buffer.
The invention has the beneficial effects that:
(1) cuprous oxide prepared by a simple electrodeposition method has a large surface area, can load more bismuth vanadate/bismuth sulfide compounds, and the formed three-stage sensitized structure can effectively enhance photoelectric signals, thereby providing a good platform for fixing a large number of hairpin DNA chains, facilitating amplification of detection signals and enhancing analysis sensitivity.
(2) The double-stranded specific nuclease can be used for identifying and enzyme-cutting DNA in a DNA/RNA hybrid chain with high selectivity, so that the cyclic utilization of the target microRNA is realized, and the amplification of signals is further realized.
(3) The long multi-branch hybrid chain with multiple branches is obtained by utilizing the multi-branch hybrid chain reaction, the long branch DNA provides a large amount of active sites for loading the platinum nanoparticles, and the multi-branch hybrid chain branches can form a large amount of hemin/G-tetrad structures and can catalyze and decompose more H2O2Production of O2As an electron acceptor, the cathode photocurrent is enhanced, and the analysis detection signal is greatly improved.
(4) By utilizing a multiple signal amplification technology, an ultra-sensitive photoelectrochemical sensor for detecting microRNA is constructed, the microRNA can be simply, quickly and accurately detected, and the method has great significance in early detection, metastasis and treatment of clinical cancers.
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 ultra-sensitive photoelectrochemical sensor in detection of microRNA-141
(1) Designing a hydrophobic wax printing pattern of the microfluidic paper chip on a computer by using Adobe illustrator CS4 software, printing the designed printing pattern on chromatographic paper by using a wax printer, then putting the printed chromatographic paper in an oven, heating for 50 seconds at 130 ℃ to melt the wax and penetrate the thickness of the whole chromatographic paper to form a hydrophobic region and a hydrophilic working region;
(2) designing printing patterns of a working electrode, a counter electrode and a reference electrode which are matched with the wax printing patterns obtained in the step (1) on a computer, and printing a carbon working electrode, a carbon counter electrode and an Ag/AgCl reference electrode on the wax printing chromatographic paper obtained in the step (1) by utilizing a screen printing technology;
(3) growing gold nanoparticles in the working area of the carbon working electrode obtained in the step (2) by using an in-situ growth method, which comprises the following specific steps: firstly, gold nanoparticles are synthesized: weighing 80 mL of secondary water, placing the secondary water in a three-neck flask, heating the secondary water to 90 ℃, adding 0.8 mL of 1% chloroauric acid, continuing heating the secondary water in a water bath to 96 ℃, immediately adding 2.8 mL of 1% sodium citrate after the reaction is carried out for 1 min, and reacting for 15 min under magnetic stirring to obtain a gold nanoparticle solution; then, a dripping-drying method is adopted, 10 mu L of gold nanoparticle solution is firstly dripped on a working area of a carbon working electrode, natural drying is carried out at room temperature, the dripping-drying operation is repeated for 3 times, 5 mu L of freshly prepared 1% chloroauric acid and 5 mu L of 0.2M hydroxylamine hydrochloride solution are respectively taken, mixed and dripped on the working area, after the mixture grows at room temperature for 30 min, the working area is cleaned by secondary water and natural drying is carried out at room temperature for 30 min;
(4) modifying small-surface cuprous oxide on the working area of the carbon working electrode obtained in the step (3) by using an electrodeposition method, modifying a bismuth vanadate/bismuth sulfide compound on the cuprous oxide, and firstly preparing a cuprous oxide material: using an Ag/AgCl electrode as a reference electrode, a platinum electrode as a counter electrode, using the carbon working electrode obtained in the step (3) as a working electrode, using a three-electrode system, depositing small-surface cuprous oxide in the working area of the carbon working electrode obtained in the step (3) by a potential dissolution analysis method, wherein the deposited electrolyte consists of copper acetate with the concentration of 0.02M and lactic acid with the concentration of 0.4M, the pH of the electrolyte is adjusted to 11 by sodium hydroxide with the concentration of 2M, the deposition voltage is-0.4V, the deposition temperature is 60 ℃, the deposition time is 27 min, and after the deposition is finished, washing the surface of the working area by secondary water and naturally drying at room temperature; subsequently, a bismuth vanadate/sulfide complex was prepared: firstly, synthesizing bismuth vanadate by a hydrothermal method, dissolving 1 mmol of bismuth nitrate pentahydrate in 40 mL of mixed solution of secondary water and ethylene glycol, wherein the volume ratio of the secondary water to the ethylene glycol is 1:1, sequentially adding 1 mmol of sodium metavanadate and 3 mmol of diisooctyl succinate sodium sulfonate, violently stirring for 5 min, transferring into a 50mL reaction kettle, reacting for 18 hours at 160 ℃, cooling to room temperature, respectively centrifugally cleaning for 3 times by using the secondary water and absolute ethyl alcohol, and then drying for 4 hours at 60 ℃ in vacuum to obtain bismuth vanadate; dissolving 0.25 mmol of prepared bismuth vanadate in 40 mL of secondary water, adding 0.1 mmol of thioacetamide, stirring for 5 min, transferring to a 50mL reaction kettle, reacting at 120 ℃ for 8 hours, cooling to room temperature, respectively centrifugally cleaning with secondary water and absolute ethyl alcohol for 3 times, and then drying at 60 ℃ for 4 hours in vacuum to obtain a bismuth vanadate/bismuth sulfide compound; and finally compounding the bismuth vanadate/bismuth sulfide compound with cuprous oxide: dripping 10 mu L of 3-aminopropyltriethoxysilane with the mass fraction of 2% on the surface of cuprous oxide, washing a working area by using a phosphate buffer solution with the pH of 7.4, drying, dripping 10 mu L of mixed solution containing a bismuth vanadate/bismuth sulfide compound and glutaraldehyde with the mass fraction of 2.5%, drying, and washing by using a phosphate buffer solution with the pH of 7.4;
(5) dripping 10 mu L of mixed solution containing 2.5 mass percent of glutaraldehyde and a DNA chain S1 into the working area of the carbon working electrode obtained in the step (4), after incubating for 2 hours, washing with phosphate buffer solution with the pH of 7.4 and containing 0.05 percent of Tween 20, dripping 10 mu L of 6-mercapto-1-hexanol with the concentration of 2 mM for blocking non-specific binding sites, after incubating for 30 minutes, washing with phosphate buffer solution with the pH of 7.4 to remove unbound 6-mercapto-1-hexanol, and naturally drying;
(6) then dripping 10 mu L of microRNA-141 with different concentrations into the working area of the carbon working electrode obtained in the step (5), incubating for 40 min at 37 ℃, washing with phosphate buffer solution with pH 7.4 to remove unbound microRNA-141, continuously dripping 10 mu L of 0.05U of double-stranded specific nuclease solution, incubating for 40 min at 37 ℃, and washing with phosphate buffer solution with pH 7.4;
(7) formation of DNA concatemers: firstly, preparing platinum nanoparticles: adding 750 mu L of chloroplatinic acid with the concentration of 20 mM into 30 mL of mixed solution of water and ethanol, wherein the volume ratio of the water to the ethanol is 1:1, adding 0.6 mol of polyvinylpyrrolidone, and refluxing for 4 hours at the temperature of 80 ℃ to obtain a platinum nanoparticle solution; then preparing DNA concatemers: adding 10 mu L of mixed liquor containing the hairpin DNA molecule H1 and the hairpin DNA molecule H2 into the working area of the carbon working electrode obtained in the step (6), wherein the concentrations of the hairpin DNA molecule H1 and the hairpin DNA molecule H2 are both 5 mu M, incubating at room temperature for 80 min, and washing with phosphate buffer solution with pH 7.4 to remove the hairpin DNA molecule H1 and the hairpin DNA molecule H2 which are not combined to react; then dropwise adding 10 mu L of chlorhematin solution with the concentration of 0.2 mM, incubating for 50 min at room temperature, washing by using a phosphate buffer solution with the pH of 7.4, continuously dropwise adding 10 mu L of prepared platinum nanoparticles after drying, and finally washing by using a phosphate buffer solution with the pH of 7.4 to remove redundant platinum nanoparticles;
(8) in the working region of the carbon working electrode obtained in the step (7), 10. mu.L of a solution containing 0.01M H was added dropwise2O2And (3) under the irradiation of a xenon lamp, taking a printed Ag/AgCl electrode as a reference electrode, a printed carbon electrode as a counter electrode, and the carbon working electrode obtained in the step (7) as a working electrode, performing photoelectrochemical signal detection under the voltage of 0.0V by using a three-electrode system through a time-current curve method, and drawing a standard curve of photocurrent intensity and microRNA concentration to realize the detection of the microRNA.
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Claims (5)
1. A preparation method of a photoelectric chemical sensor for detecting microRNA in an ultrasensitive manner is characterized by comprising the following steps:
(1) designing a hydrophobic wax printing pattern of the microfluidic paper chip on a computer by using Adobe illustrator CS4 software, printing the designed printing pattern on chromatographic paper by using a wax printer, then putting the printed chromatographic paper in an oven, heating at 130 ℃ for 60 seconds to melt the wax and permeate the whole chromatographic paper to form a hydrophobic region and a hydrophilic working region;
(2) designing printing patterns of a working electrode, a counter electrode and a reference electrode which are matched with the wax printing patterns obtained in the step (1) on a computer, and printing a carbon working electrode, a carbon counter electrode and an Ag/AgCl reference electrode on the wax printing chromatographic paper obtained in the step (1) by utilizing a screen printing technology;
(3) growing gold nanoparticles in the working area of the carbon working electrode obtained in the step (2) by using an in-situ growth method, which comprises the following specific steps: firstly, gold nanoparticles are synthesized: measuring 60-90 mL of secondary water, placing the secondary water in a three-neck flask, heating the secondary water to 90 ℃, adding 0.5-0.9 mL of chloroauric acid with the mass fraction of 1%, continuing to heat the secondary water in a water bath to 96 ℃, immediately adding 2.5-2.9 mL of sodium citrate with the mass fraction of 1% after the reaction is carried out for 1 min, and reacting for 15-20 min under magnetic stirring to obtain a gold nanoparticle solution; then, firstly, 10-20 mu L of gold nanoparticle solution is dripped on a working area of a carbon working electrode by adopting a dripping-drying method, natural drying is carried out at room temperature, the dripping-drying operation is repeated for 3-5 times, 10-20 mu L of freshly prepared mixed solution of chloroauric acid with the mass fraction of 1% and 0.2M hydroxylamine hydrochloride is dripped on the working area, wherein the volume ratio of the chloroauric acid to the hydroxylamine hydrochloride is 1:1, after the gold nanoparticle solution grows for 20-40 min at room temperature, the working area is cleaned by secondary water and natural drying is carried out for 30 min at room temperature;
(4) modifying small-surface cuprous oxide on the working area of the carbon working electrode obtained in the step (3) by using an electrodeposition method, and modifying a bismuth vanadate/bismuth sulfide compound on the cuprous oxide;
(5) fixing the hairpin DNA chain S1 in the working area of the carbon working electrode obtained in the step (4), then adopting 6-mercapto-1-hexanol to block the active site, and then utilizing the S1 chain to capture microRNA;
(6) sequentially incubating the microRNA of the target detection object and the double-stranded specific nuclease in the working area of the carbon working electrode obtained in the step (5) and washing the carbon working electrode by using a phosphate buffer solution with the pH of 7.4;
(7) formation of DNA concatemers: DNA of hairpinDissolving the molecule H1 and the hairpin DNA molecule H2 in a phosphate buffer solution with the pH value of 7.4, dripping the solution into the working area of the carbon working electrode obtained in the step (6), carrying out in-situ proliferation to form a plurality of hybrid chains, continuously dripping chlorhematin to form a chlorhematin/G-tetrad structure, embedding platinum nano particles into the main chain of the plurality of hybrid chains by utilizing the electrostatic adsorption effect to form a DNA (deoxyribonucleic acid) concatemer with the biological catalysis effect, and catalyzing H2O2Decomposition to O2；
(8) In the working region of the carbon working electrode obtained in the step (7), 10. mu.L of a solution containing 0.01M H was added dropwise2O2And (3) under the irradiation of a xenon lamp, taking a printed Ag/AgCl electrode as a reference electrode, a printed carbon electrode as a counter electrode, and the carbon working electrode obtained in the step (7) as a working electrode, performing photoelectrochemical signal detection under the voltage of 0.0V by using a three-electrode system through a time-current curve method, and drawing a standard curve of photocurrent intensity and microRNA concentration to realize the detection of the microRNA.
2. The preparation method of the photoelectric chemical sensor for the ultrasensitive detection of the microRNA according to claim 1, wherein the specific steps of modifying a small-area cuprous oxide on an electrode working area and modifying a bismuth vanadate/bismuth sulfide compound on the cuprous oxide by an electrodeposition method are as follows:
a. depositing cuprous oxide on the working area of the carbon working electrode obtained in the step (3): using an Ag/AgCl electrode as a reference electrode, a platinum electrode as a counter electrode, using the carbon working electrode obtained in the step (3) as a working electrode, using a three-electrode system, depositing small-surface cuprous oxide in the working area of the carbon working electrode obtained in the step (3) by a potential dissolution analysis method, wherein the deposited electrolyte consists of copper acetate with the concentration of 0.02M and lactic acid with the concentration of 0.3-0.5M, the pH of the electrolyte is adjusted to 9-11 by sodium hydroxide with the concentration of 2M, the deposition voltage is-0.3V and-0.5V, the deposition temperature is 60 ℃, the deposition time is 25-45 min, and after the deposition is finished, washing the surface of the working area by secondary water and naturally drying at room temperature;
b. synthesis of bismuth vanadate/bismuth sulfide complexes: firstly, synthesizing bismuth vanadate by a hydrothermal method, dissolving 1 mmol of bismuth nitrate pentahydrate in 40-60 mL of mixed solution of secondary water and ethylene glycol, wherein the volume ratio of the secondary water to the ethylene glycol is 1:1, sequentially adding 1 mmol of sodium metavanadate and 2.5-3.5 mmol of diisooctyl succinate sodium sulfonate, violently stirring for 5-10 min, then transferring into a 50mL reaction kettle, reacting for 16-18 h at 160 ℃, cooling to room temperature, respectively centrifugally cleaning for 3-5 times by using the secondary water and absolute ethyl alcohol, and then drying for 4 h at 60 ℃ in vacuum to obtain bismuth vanadate; dissolving 0.25 mmol of prepared bismuth vanadate in 40 mL of secondary water, adding 0.05-0.2 mmol of thioacetamide, stirring for 5-10 min, transferring to a 50mL reaction kettle, reacting at 120 ℃ for 8-10 h, cooling to room temperature, respectively centrifugally cleaning for 3-5 times by using secondary water and absolute ethyl alcohol, and then drying at 60 ℃ for 4 h in vacuum to obtain a bismuth vanadate/bismuth sulfide compound;
c. compounding a bismuth vanadate/bismuth sulfide compound with cuprous oxide: and dripping 10 mu L of 3-aminopropyltriethoxysilane with the mass fraction of 2% on the surface of cuprous oxide, washing a working area by using a phosphate buffer solution with the pH of 7.4, drying, dripping 10 mu L of a mixed solution containing a bismuth vanadate/bismuth sulfide compound and glutaraldehyde with the mass fraction of 2.5%, drying, and washing by using a phosphate buffer solution with the pH of 7.4.
3. The method for preparing the photoelectrochemical sensor for the ultrasensitive detection of the microRNA according to claim 1, wherein the specific steps of fixing the hairpin DNA strand S1 in the working region of the carbon working electrode obtained in the step (4), then blocking the active site with 6-mercapto-1-hexanol, and then capturing the microRNA with the S1 strand are as follows: mu.L of a mixture containing 2.5% by mass of glutaraldehyde and the DNA chain S1 was added dropwise to the working area of the carbon working electrode obtained in step (4), after incubation for 2 hours, washed with a phosphate buffer solution of pH 7.4 containing 0.05% Tween 20, 10. mu.L of 6-mercapto-1-hexanol at a concentration of 2 mM was added dropwise for blocking non-specific binding sites, and after incubation for 30 minutes, unbound 6-mercapto-1-hexanol was removed by washing with a phosphate buffer solution of pH 7.4 and dried naturally.
4. The method for preparing the photoelectrochemical sensor for detecting the microRNA with the ultra sensitivity as claimed in claim 1, wherein the specific steps of sequentially incubating the microRNA as the target detection object and the double-stranded specific nuclease in the working area of the carbon working electrode obtained in the step (5) and washing the incubated object with the phosphate buffer solution with the pH of 7.4 are as follows: and (3) dripping 10 mu L of microRNA with different concentrations to the working area of the carbon working electrode in the step (5), incubating for 40 min at 37 ℃, washing out the unbound microRNA by using a phosphate buffer solution with pH 7.4, dripping 10 mu L of 0.05U of double-stranded specific nuclease for incubating for 40 min, washing by using a phosphate buffer solution with pH 7.4, and drying at room temperature.
5. The method for preparing the photoelectrochemical sensor for the ultrasensitive detection of the microRNA according to claim 1, wherein the specific steps for forming the DNA concatemer are as follows:
a. synthesizing platinum nano particles: adding 500-1000 mu L of chloroplatinic acid with the concentration of 20 mM into 20-50mL of mixed solution of water and ethanol, wherein the volume ratio of the water to the ethanol is 1:1, adding 0.4-0.9 mol of polyvinylpyrrolidone, and refluxing for 4 hours at 80 ℃ to obtain a platinum nanoparticle solution;
preparation of DNA concatemers: adding 10 mu L of mixed liquor containing the hairpin DNA molecule H1 and the hairpin DNA molecule H2 into the working area of the carbon working electrode obtained in the step (6), wherein the concentrations of the hairpin DNA molecule H1 and the hairpin DNA molecule H2 are both 5 mu M, incubating at room temperature for 80 min, and washing with phosphate buffer solution with pH 7.4 to remove the hairpin DNA molecule H1 and the hairpin DNA molecule H2 which are not combined to react; then 10. mu.L of a 0.2 mM hemin solution was added dropwise, incubated at room temperature for 50 min, washed with a pH 7.4 phosphate buffer, dried, and then 10. mu.L of the prepared platinum nanoparticles were further added dropwise, and finally, excess platinum nanoparticles were removed by washing with a pH 7.4 phosphate buffer.
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