CN111830107A - Method for detecting prostate specific antigen based on enzyme biofuel cell - Google Patents
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- 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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- 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
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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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
- G01N27/327—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
Abstract
The invention discloses a method for detecting prostate specific antigen based on an enzyme biofuel cell. Hydrophobic and hydrophilic areas were prepared on paper using wax printing and laser cutting techniques, and carbon electrodes were printed by means of screen printing techniques. Different areas of the paper chip are functionalized by different methods. The zinc oxide nanorod is used for increasing the adsorption of glucose oxidase, so that the specific catalysis of glucose is accelerated, the bilirubin oxidase/PtNi nanocube compound has a synergistic effect on the reduction of oxygen, and the open-circuit voltage is recorded by means of an electrochemical workstation, so that the ultra-sensitive detection of the prostate specific antigen is realized. Meanwhile, the consumption of the hydrogen peroxide can be proved by utilizing the color development of the 3,3',5,5' -tetramethyl benzidine.
Description
Technical Field
The invention relates to a method for detecting prostate specific antigen based on an enzyme biofuel cell, belonging to the technical field of detection of prostate specific antigen.
Background
Prostate cancer is the most common malignancy of the male genitourinary system, accounting for the second place in the mortality of men with malignancies, and the most common examination is prostate-specific antigen screening, i.e., determining the concentration level of prostate-specific antigen in serum. The prostate specific antigen is used as a marker of prostate cancer, has the characteristics of strong sensitivity and specificity, noninvasive detection and the like, and plays an important role in diagnosis of prostate cancer.
In recent years, many techniques for detecting prostate-specific antigens, such as electrochemiluminescence, surface enhanced raman spectroscopy, colorimetric analysis, photoelectrochemical immunoassay, etc., have been developed, but these methods require expensive equipment, professional operators, and complicated processes, and are time-consuming.
Self-powered sensors based on biofuel cells as a powerful strategy have been widely used in the fields of detection of biomolecules, toxic pollutants, immunoassays and the like, by virtue of simple instruments, easy miniaturization, independence and sustainability of working advantages.
Disclosure of Invention
Aiming at the problems existing at present, the technical problem to be solved by the invention is to provide a construction method of an enzyme biofuel sensor with low cost, simple and convenient operation, environmental protection and high sensitivity, which is characterized by comprising the following steps:
1. hydrophobic wax print patterns were designed on a computer using Adobe Illustrator CS6 software and printed in bulk using a wax jet printer onto cut a4 size filter paper, which was then heated on a hot plate until the wax melted and penetrated the entire thickness of the paper, forming hydrophobic areas, the paper device containing a Y-type solution transfer chip (layer i), a hollow channel chip (layer ii), a working chip (layer iii) containing a bioanode, a biocathode, a colorimetric area and a water absorbing area, and a slidable chip (layer iv).
2. And printing a carbon working electrode on the biological anode area and the biological cathode area by adopting a screen printing method, and cutting off a hydrophilic area surrounded by hydrophobic wax in the hollow area.
3. Functionalizing a biological anode and a biological cathode hydrophilic working area of a working chip, firstly growing gold nanoparticles by a seed solution growth method, wherein the specific growth steps are as follows: pouring 80 mL of secondary water into a three-neck flask, heating to 90 ℃, adding 800 mu L of chloroauric acid solution with the mass fraction of 1%, continuously heating to 96 ℃ for 1 min, finally adding 2.8mL of sodium citrate with the mass fraction of 1%, continuously heating for 15 min, naturally cooling to obtain gold seed solution, dropwise adding 20 mu L of the obtained gold seed solution into a working area, standing and airing, repeating for three times, dissolving 0.0139 g of hydroxylamine hydrochloride in 1 mL of water, mixing 667 mu L of chloroauric acid solution with the mass fraction of 1%, dropwise adding 20 mu L of the mixed solution into the working area modified with gold seeds, standing for 30 min, and washing with the secondary water for 3 times.
4. Growing a zinc oxide nano rod in a hydrophilic area of a biological anode of a working chip, wherein the specific growth steps are as follows: 0.439 g of zinc acetate is firstly dissolved in 50 mL of ethanol, dissolved at 90 ℃ and spin-coated on the bioanode hydrophilic area, then 50 mL of zinc nitrate containing 25 mM of hexamethylenetetramine and the same proportion is stirred for 30 min, transferred into an autoclave, the prepared bioanode hydrophilic area is placed inside, kept for 6 h at 90 ℃, finally washed with water for three times, and 1 mu mol/L of glucose oxidase is dripped and kept for a whole night at 4 ℃.
5. Modifying a bilirubin oxidase/PtNi nanocube biological conjugate aptamer in a biological cathode hydrophilic region of a working chip, and the specific steps are as follows: 20.0. mu. L c-DNA was added dropwise, incubated overnight at 4 ℃ and the surface was washed with phosphate buffer in order to eliminate the liberated c-DNA, and then 20. mu.L of 10 mM monoethanolamine was added dropwise at room temperature, held for 50 minutes to block and then washed with phosphate buffer. And then, 20.0 mu L of bilirubin oxidase/PtNi nanocube-labeled aptamer is dripped to react for 110 min at 37 ℃ to realize hybridization reaction, and then the hybridization reaction is washed by phosphate buffer solution, wherein the base sequences of the c-DNA and the aptamer are shown in a nucleotide sequence table, wherein the 5 'end of the c-DNA is modified with 1 methylene, and the 5' end of the aptamer is modified with 1 sulfydryl.
6. Modifying in a colorimetric region of a working chip, and specifically comprising the following steps: 20 mu L of 20 mM 3,3',5,5' -tetramethyl benzidine and 20 mu L of gold nanocluster liquid are dripped, and the mixture is kept stand and dried.
7. Performing bioassay in a hydrophilic area of a biological cathode of a working chip, dripping prostate specific antigens with different concentrations at 37 ℃, washing the released bilirubin oxidase/PtNi nanocubes with phosphate buffer, naturally airing, stacking the layers I to III, clamping the assembled device with a folding back clamp, injecting 100 mu L of phosphate buffer containing 50 mM glucose into a round inlet of the layer I to activate the device, enabling the solution to simultaneously reach two semicircular areas from a Y-shaped channel and finally vertically reach the working chip, thereby initiating redox reactions on the biological anode and the biological cathode, recording open-circuit voltage related to the concentration of the prostate specific antigens through an electrochemical workstation, and simultaneously, adjusting the slidable chip, wherein the hydrophilic area in the slidable chip serves as a bridge between the biological anode and a colorimetric area, hydrogen peroxide generated by the oxidation reaction of glucose in the bioanode flows into the colorimetric region, so that the gold nanoclusters pre-loaded in the colorimetric region can catalyze the color development reaction of 3,3',5,5' -tetramethylbenzidine in the presence of hydrogen peroxide, the color of the gold nanoclusters changes from colorless to blue, and then the solution flows along the fluid path until the solution reaches the absorption region. Thus, the consumption of hydrogen peroxide was demonstrated using a colorimetric method.
8. And drawing a standard curve of the open-circuit voltage and the concentration of the prostate specific antigen to finish the measurement of the prostate specific antigen.
The invention has the beneficial effects that:
1. the sensor based on the enzyme biofuel cell can detect the prostate specific antigen without external energy, thereby reducing the experiment cost
2. The paper background is white, the interference signal is low, and the color development is obvious.
3. The enzyme is used as a catalyst to convert chemical energy in the fuel into electric energy, and cannot pollute the environment.
4. Compared with the traditional glassy carbon electrode and glass electrode, the paper substrate has the advantages of rich raw materials, light weight, low price, easy folding and degradability.
Description of the drawings:
the invention is described in further detail below with reference to the figures and specific embodiments:
FIG. 1 is a schematic view of a paper-based device fold.
Fig. 2 is a hydrophobic wax printing pattern, 1 being a Y-type solution transfer chip, 2 being a hollow channel chip, 3 being a biocathode, 4 being a bioanode, 5 being a slidable chip.
Detailed Description
Example 1
A method for detecting prostate specific antigen based on an enzyme biofuel cell, comprising the steps of:
1. hydrophobic wax print patterns were designed on a computer using Adobe Illustrator CS6 software and printed in bulk using a wax jet printer onto cut a4 size filter paper, which was then heated on a hot plate until the wax melted and penetrated the entire thickness of the paper, forming hydrophobic areas, the paper device containing a Y-type solution transfer chip (layer i), a hollow channel chip (layer ii), a working chip (layer iii) containing a bioanode, a biocathode, a colorimetric area and a water absorbing area, and a slidable chip (layer iv).
2. And printing a carbon working electrode on the biological anode area and the biological cathode area by adopting a screen printing method, and cutting off a hydrophilic area surrounded by hydrophobic wax in the hollow area.
3. Functionalizing a biological anode and a biological cathode hydrophilic working area of a working chip, firstly growing gold nanoparticles by a seed solution growth method, wherein the specific growth steps are as follows: pouring 80 mL of secondary water into a three-neck flask, heating to 90 ℃, adding 800 mu L of chloroauric acid solution with the mass fraction of 1%, continuously heating to 96 ℃ for 1 min, finally adding 2.8mL of sodium citrate with the mass fraction of 1%, continuously heating for 15 min, naturally cooling to obtain gold seed solution, dropwise adding 20 mu L of the obtained gold seed solution into a working area, standing and airing, repeating for three times, dissolving 0.0139 g of hydroxylamine hydrochloride in 1 mL of water, mixing 667 mu L of chloroauric acid solution with the mass fraction of 1%, dropwise adding 20 mu L of the mixed solution into the working area modified with gold seeds, standing for 30 min, and washing with the secondary water for 3 times.
4. Growing a zinc oxide nano rod in a hydrophilic area of a biological anode of a working chip, wherein the specific growth steps are as follows: 0.439 g of zinc acetate is firstly dissolved in 50 mL of ethanol, dissolved at 90 ℃ and spin-coated on the bioanode hydrophilic area, then 50 mL of zinc nitrate containing 25 mM of hexamethylenetetramine and the same proportion is stirred for 30 min, transferred into an autoclave, the prepared bioanode hydrophilic area is placed inside, kept for 6 h at 90 ℃, finally washed with water for three times, and 1 mu mol/L of glucose oxidase is dripped and kept for a whole night at 4 ℃.
5. Modifying a bilirubin oxidase/PtNi nanocube biological conjugate aptamer in a biological cathode hydrophilic region of a working chip, and the specific steps are as follows: 20.0. mu. L c-DNA was added dropwise, incubated overnight at 4 ℃ and the surface was washed with phosphate buffer in order to eliminate the liberated c-DNA, and then 20. mu.L of 10 mM monoethanolamine was added dropwise at room temperature, held for 50 minutes to block and then washed with phosphate buffer. And then, 20.0 mu L of bilirubin oxidase/PtNi nanocube-labeled aptamer is dripped to react for 110 min at 37 ℃ to realize hybridization reaction, and then the hybridization reaction is washed by phosphate buffer solution, wherein the base sequences of the c-DNA and the aptamer are shown in a nucleotide sequence table, wherein the 5 'end of the c-DNA is modified with 1 methylene, and the 5' end of the aptamer is modified with 1 sulfydryl.
6. Modifying in a colorimetric region of a working chip, and specifically comprising the following steps: 20 mu L of 20 mM 3,3',5,5' -tetramethyl benzidine and 20 mu L of gold nanocluster liquid are dripped, and the mixture is kept stand and dried.
7. Performing biological measurement in a biological cathode hydrophilic region of a working chip, dripping prostate specific antigens with different concentrations at 37 ℃, then flushing the released bilirubin oxidase/PtNi nanocubes with phosphate buffer solution, and naturally airing.
8. Stacking layers I to III and clamping the assembled device with fold-back clamps, injecting 100. mu.L of phosphate buffer containing 50 mM glucose into the circular inlet of layer I to activate the device, the solution can simultaneously reach two semicircular areas from the Y-channel and finally vertically reach the working chip, thereby initiating redox reactions on the bioanode and the biocathode electrode, recording open circuit voltage related to prostate specific antigen concentration through the electrochemical workstation, and simultaneously, by adjusting the slidable chip, the hydrophilic area in the slidable chip acts as a bridge between the bioanode and the colorimetric area, hydrogen peroxide generated by glucose oxidation reaction in the bioanode flows into the colorimetric area, so that pre-loaded gold nanoclusters in the colorimetric area can catalyze 3,3',5,5' -tetramethylbenzidine color development reaction in the presence of hydrogen peroxide, the color changes from colorless to blue, and the solution then flows along the fluid path until it reaches the absorption region. Thus, the consumption of hydrogen peroxide was demonstrated using a colorimetric method.
9. And drawing a standard curve of the open-circuit voltage and the concentration of the prostate specific antigen to finish the measurement of the prostate specific antigen.
Sequence listing
<110> university of Jinan
<120> method for detecting prostate specific antigen based on enzyme biofuel cell
<130>2020
<160>2
<170>SIPOSequenceListing 1.0
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<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
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tttttttttg ctatttgatg gcg 23
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
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ttaattaaag ctcgccatca aatagc 26
Claims (2)
1. The method for detecting the prostate specific antigen based on the enzyme biofuel cell is characterized by comprising the following steps:
(1) designing a hydrophobic wax printing pattern on a computer by using Adobe Illustrator CS6 software and printing the pattern on a cut A4 size filter paper in batch by using a wax-jet printer, and then heating the paper on a heating plate until the wax is melted and permeates the thickness of the whole paper to form a hydrophobic area, wherein the paper device comprises a Y-shaped solution transfer chip (layer I), a hollow channel chip (layer II), a working chip (layer III) comprising a bioanode, a biocathode, a colorimetric area and a water absorption area and a slidable chip (layer IV);
(2) printing a carbon working electrode on a biological anode area and a biological cathode area by adopting a screen printing method, and cutting a hydrophilic area surrounded by hydrophobic wax in a hollow area;
(3) functionalizing a biological anode and a biological cathode hydrophilic working area of a working chip, firstly growing gold nanoparticles by a seed solution growth method, wherein the specific growth steps are as follows: pouring 80 mL of secondary water into a three-neck flask, heating to 90 ℃, adding 800 mu L of chloroauric acid solution with the mass fraction of 1%, continuously heating to 96 ℃ for 1 min, finally adding 2.8mL of sodium citrate with the mass fraction of 1%, continuously heating for 15 min, naturally cooling to obtain gold seed solution, dropwise adding 20 mu L of the obtained gold seed solution into a working area, standing and airing for three times, dissolving 0.0139 g of hydroxylamine hydrochloride in 1 mL of water, mixing 667 mu L of chloroauric acid solution with the mass fraction of 1%, dropwise adding 20 mu L of the mixed solution into the working area decorated with gold seeds, standing for 30 min, and washing with the secondary water for 3 times;
(4) growing a zinc oxide nano rod in a hydrophilic area of a biological anode of a working chip, wherein the specific growth steps are as follows: dissolving 0.439 g of zinc acetate in 50 mL of ethanol, dissolving at 90 ℃ and spin-coating to a biological anode hydrophilic region, then stirring 50 mL of zinc nitrate containing 25 mM of hexamethylenetetramine and the same proportion for 30 min, transferring to a high-pressure kettle, placing the prepared biological anode hydrophilic region in the high-pressure kettle, keeping for 6 h at 90 ℃, finally washing with secondary water for three times, dropwise adding 1 mu mol/L glucose oxidase, and keeping for a whole night at 4 ℃;
(5) modifying a bilirubin oxidase/PtNi nanocube biological conjugate aptamer in a biological cathode hydrophilic region of a working chip, and the specific steps are as follows: 20.0. mu. L c-DNA was added dropwise, incubated overnight at 4 ℃ and the surface was washed with phosphate buffer in order to eliminate the liberated c-DNA, and then 20. mu.L of 10 mM monoethanolamine was added dropwise at room temperature, held for 50 minutes to block and then washed with phosphate buffer. Then, 20.0 mu L of bilirubin oxidase/PtNi nanocube-labeled aptamer is dripped to react for 110 min at 37 ℃ to realize hybridization reaction, and then the hybridization reaction is washed by phosphate buffer solution, the base sequences of the c-DNA and the aptamer are shown in a nucleotide sequence table, wherein the 5 'end of the c-DNA is modified with 1 methylene, and the 5' end of the aptamer is modified with 1 sulfydryl;
(6) modifying in a colorimetric region of a working chip, and specifically comprising the following steps: 20 mu L of 20 mM 3,3',5,5' -tetramethyl benzidine and 20 mu L of gold nanocluster liquid are dripped, and the mixture is kept stand and dried;
(7) performing biological measurement in a biological cathode hydrophilic region of a working chip, dripping prostate specific antigens with different concentrations at 37 ℃, washing the released bilirubin oxidase/PtNi nanocubes by using a phosphate buffer solution, and naturally airing;
(8) stacking layers I to III and clamping the assembled device with fold-back clamps, injecting 100. mu.L of phosphate buffer containing 50 mM glucose into the circular inlet of layer I to activate the device, the solution can simultaneously reach two semicircular areas from the Y-channel and finally vertically reach the working chip, thereby initiating redox reactions on the bioanode and the biocathode electrode, recording open circuit voltage related to prostate specific antigen concentration through the electrochemical workstation, and simultaneously, by adjusting the slidable chip, the hydrophilic area in the slidable chip acts as a bridge between the bioanode and the colorimetric area, hydrogen peroxide generated by glucose oxidation reaction in the bioanode flows into the colorimetric area, so that pre-loaded gold nanoclusters in the colorimetric area can catalyze 3,3',5,5' -tetramethylbenzidine color development reaction in the presence of hydrogen peroxide, the color changes from colorless to blue, and the solution then flows along the fluid path until it reaches the absorption region. Thus, the consumption of hydrogen peroxide was demonstrated using a colorimetric method;
(9) and drawing a standard curve of the open-circuit voltage and the concentration of the prostate specific antigen to finish the measurement of the prostate specific antigen.
2. The paper-based dual-mode electrochemical sensor for detecting adenosine triphosphate according to claim 1, characterized in that the total size of the paper device is 60 mm-70 mm x 130 mm-140 mm.
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