CN113092452A - Preparation method and application of biochemical sensor - Google Patents
Preparation method and application of biochemical sensor Download PDFInfo
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- CN113092452A CN113092452A CN202110440786.9A CN202110440786A CN113092452A CN 113092452 A CN113092452 A CN 113092452A CN 202110440786 A CN202110440786 A CN 202110440786A CN 113092452 A CN113092452 A CN 113092452A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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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
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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
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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
Abstract
The invention relates to a preparation method and application of a biochemical sensor. The cerium and silver doped antimony tungstate is used as a substrate material, the large specific surface area of the cerium and silver doped antimony tungstate can increase light capture and biomolecule load, the cerium and silver doped antimony tungstate can provide an electronic path, a plasma effect is caused, and the separation efficiency of photo-generated charges is improved. A dipping method is adopted to consume partial silver ions in situ on cerium and silver doped antimony tungstate to grow silver sulfide, and meanwhile, an energy band matching structure formed by modifying indium sulfide on the cerium and silver doped antimony tungstate/silver sulfide has good photoelectric response. On the other hand, cadmium sulfide/polydopamine with excellent conductivity is used as a signal marker, the polydopamine can be directly combined with biomolecules through Michael addition or Schiff alkali, the cadmium sulfide can be better matched with energy bands of cerium and silver doped antimony tungstate/silver sulfide/indium sulfide, and the photoelectric conversion efficiency of the sensor is effectively improved. The biochemical sensor constructed based on the method has wider linear range and higher sensitivity, and has important significance for detecting the small cell lung cancer marker neuron specific enolase.
Description
Technical Field
The invention relates to the technical field of novel nanometer functional materials, immunoassay and biochemical sensor detection, and provides a preparation method of a photoelectrochemical sensor based on a cerium and silver co-doped antimony tungstate/silver sulfide/indium sulfide composite material.
Background
Neuron Specific Enolase (NSE), also called neuroenolase, is an isomer of enolase and is present only in neurons and neuroendocrine cells. The positive of serum NSE of a Small Cell Lung Cancer (SCLC) patient can reach 65-100%, and for healthy people, the serum NSE level is 5-12 ng/mL, and the cerebrospinal fluid is less than 20 ng/mL. Patients with lung cancer have altered levels of NSE in the blood. It is currently recognized that NSE can be used as a tumor marker with high specificity and high sensitivity for small cell lung cancer. Therefore, monitoring NSE is of great importance for routine examination, diagnosis and treatment of SCLC. To date, a number of assays have been developed for measuring NSE, such as fluorescence immunoassay, radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescent immunoassay, and electrochemical immunoassay. However, the above method has the disadvantages of expensive apparatus and complicated operation. Therefore, the establishment of a detection method with low cost, quick detection, high sensitivity and strong specificity has important scientific significance and practical value.
In recent years, the photoelectric chemical sensor has been paid more and more attention by researchers due to the characteristics of low background signal, high sensitivity, simple equipment, low detection cost, easy miniaturization and the like. The photoelectrochemical sensor is based on that an external light source excites a photoelectric sensitive material to cause electron-hole pairs to be separated, and under the condition of proper bias potential, the rapid transfer of electrons on an electrode, a semiconductor, a modifier and an analyte is realized, and photocurrent is formed. Under the optimal condition, the change of the concentration of the analyte can directly influence the magnitude of the photocurrent, and then qualitative and quantitative analysis on the analyte can be realized according to the change of the photocurrent by utilizing the biological immune combination or the specific cross-linking of the aptamer. The research of the photoelectrochemical sensor mainly focuses on improving the sensitivity, selectivity, stability and the like of detection, and the most key technology is to improve the size and stability of photocurrent and the adsorption, fixation and other performances of biomolecules capable of realizing specific binding to antibodies and the like.
Photoactive materials are key components of photoelectrochemical sensors. Antimony tungstate, which is a photoelectric conversion semiconductor material with excellent performance, is widely used in the fields of photocatalysis, fuel cells and the like. However, its application is limited by the higher recombination rate of photogenerated electron-hole pairs and the lower charge transfer capability. Therefore, the antimony tungstate prepared by the hydrothermal reaction and the cerium and silver co-doped antimony tungstate can provide a direct electronic path, can also cause a plasma effect, effectively enhances the separation efficiency of photo-generated charges, and meanwhile, the three components of the substrate material have good energy band matching and are beneficial to charge separation. In addition, cadmium sulfide/polydopamine with excellent conductivity is used as a signal marker, so that the photoelectric conversion efficiency of the sensor is effectively improved, and the sensitive detection of the nerve enolase is realized.
Disclosure of Invention
One of the purposes of the invention is to synthesize cerium and silver co-doped antimony tungstate, provide a direct electronic pathway, cause a surface plasma effect, promote the transfer of photo-generated electrons and improve the photoelectric conversion efficiency;
the invention aims to sacrifice part of silver ions of cerium and silver co-doped antimony tungstate to grow silver sulfide in situ, and simultaneously modify indium sulfide on the cerium and silver co-doped antimony tungstate/silver sulfide to form an energy band matching structure, so that the material has stronger and more stable photoelectric response under the irradiation of visible light;
the invention also aims to construct a sandwich-type photoelectrochemical sensor for rapid and sensitive detection of the nerve enolase by using cadmium sulfide/polydopamine with excellent conductivity as a signal marker.
The technical scheme adopted by the invention is as follows:
1. a preparation method of a biochemical sensor is characterized in that the biochemical sensor is a photoelectrochemical sensor based on a cerium and silver co-doped antimony tungstate/silver sulfide/indium sulfide composite material, and the preparation method comprises the following steps:
(1) ultrasonically cleaning ITO conductive glass of 2.4 cm multiplied by 0.8 cm by using liquid detergent, acetone, ethanol and ultrapure water for 30 min in sequence, and drying by using nitrogen;
(2) dropwise adding 10 mu L of cerium and silver co-doped antimony tungstate microspheres with the concentration of 4-6 mg/mL to the conductive surface of the ITO conductive glass, airing at room temperature to prepare a cerium and silver co-doped antimony tungstate electrode, and immersing the electrode into Na with the concentration of 0.1-0.5 mol/L2S·9H2Performing O reaction for 30-50 s to obtain a cerium and silver co-doped antimony tungstate/silver sulfide electrode, continuously dropwise adding 4 mu L of indium sulfide solution with the concentration of 3-5 mg/mL to the surface of the cerium and silver co-doped antimony tungstate/silver sulfide electrode, drying at room temperature, and washing with ultrapure water to obtain the cerium and silver co-doped antimony tungstate/silver sulfide/indium sulfide electrode;
(3) dropwise adding 4 mu L of thioglycolic acid with the concentration of 3 mmol/L onto the surface of the electrode to fix the nerve enolase capture antibody, drying in a refrigerator at 4 ℃, washing with ultrapure water, continuously dropwise adding 3 mu L of a mixed solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (10 mmol/L/2 mmol/L) to activate carboxyl, drying in the refrigerator at 4 ℃, and washing with ultrapure water;
(4) dripping 6 mu L of nerve enolase capture antibody solution with the concentration of 6-10 mu g/mL to the surface of the modified electrode, incubating for 1 h in a refrigerator at 4 ℃, and washing with phosphate buffer solution with the pH of 7.4;
(5) dripping 3 mu L of bovine serum albumin solution with the mass fraction of 1.0% on the surface of the modified electrode to seal the nonspecific active site on the surface of the electrode, airing in a refrigerator at 4 ℃, and cleaning by phosphate buffer solution with the pH of 7.4;
(6) dripping 6 mu L of a series of nerve enolase antigen solutions with different concentrations and the concentration of 0.0001 ng/mL-50 ng/mL, incubating for 1 h in a refrigerator at 4 ℃, and washing with a phosphate buffer solution with the pH of 7.4;
(7) and dripping 6 mu L of a cadmium sulfide/polydopamine composite substance labeled nerve enolase antibody solution with the concentration of 3.0-5.0 mg/mL onto the surface of the electrode, incubating for 1 h in a refrigerator at 4 ℃, and cleaning by using a phosphate buffer solution with the pH of 7.4 to prepare the biochemical sensor.
2. The preparation method according to claim 1, wherein the cerium and silver co-doped antimony tungstate microsphere is prepared by the following steps:
dissolving 0.3-0.5 g of sodium tungstate hydrate in 8 mL of ultrapure water, and referring to the solution A; dissolving 0.4-0.6 g of antimony trichloride in 8 mL of absolute ethyl alcohol to obtain a solution B, slowly dripping the solution B into the solution A, stirring for 10 min to obtain a light yellow solution, adding 0.03-0.05 mg of silver nitrate and 0.08-0.12 mg of cerous nitrate hydrate into the solution, stirring for 30 min, adjusting the pH to 2 by using 1 mol/L of sodium hydroxide solution or nitric acid solution, continuously stirring for 10 min, transferring the obtained solution to a high-pressure reaction kettle, reacting at 180 ℃ for 18-24 h, taking out a product after the high-pressure reaction kettle is gradually cooled to room temperature, washing with ethanol and ultrapure water for 3-4 times, and vacuum drying for 10-16 h.
3. The preparation method according to claim 1, wherein the indium sulfide is prepared by the following steps:
dissolving 0.2-0.5 g of indium nitrate hydrate in 80 mL of ultrapure water, adding 0.10-0.12 g of thioacetamide into the solution, stirring for 30 min, transferring the obtained solution to a high-pressure reaction kettle, reacting at 120 ℃ for 10-12 h, taking out a product after the high-pressure reaction kettle is gradually cooled to room temperature, washing with ethanol for 3-4 times, and vacuum drying for 6-8 h.
4. The preparation method according to claim 1, wherein the preparation steps of the cadmium sulfide/polydopamine complex labeled neuroenolase antibody solution are as follows:
(1) preparation of polydopamine:
dissolving 0.1-0.3 g of dopamine in 100 mL of Tris-buffer (pH is 8.8), adding 50 mL of isopropanol into the solution, stirring for 24 hours, centrifuging the obtained solution at 9000/min for 20 min, washing with ultrapure water for 4-6 times to remove unreacted substances, distributing the product in 10 mL of ultrapure water, and placing in a refrigerator at 4 ℃;
(2) preparation of cadmium sulfide/polydopamine composite:
dissolving 0.008-0.01 g of cadmium acetate hydrate and 6-8 mL of polydopamine in 10 mL of absolute ethyl alcohol, stirring for 1 h, transferring the obtained mixed solution into an oil bath, heating at 80-100 ℃ for 10-15 min, adding 10 mL of thioacetamide (2-5 mg) aqueous solution into the solution in batches under stirring, continuing heating for 1 h, cooling to room temperature, washing with ultrapure water and absolute ethyl alcohol, and vacuum drying overnight;
(3) preparing a cadmium sulfide/polydopamine complex labeled nerve enolase antibody solution:
dispersing 2 mg of cadmium sulfide/polydopamine in 1 mL of neural enolase antibody with the concentration of 5 mu g/mL, adding 100 mu L of bovine serum albumin solution with the mass fraction of 1.0% into the solution, shaking at 4 ℃ for 12 h, centrifugally washing for 3 times by using phosphate buffer solution with the pH value of 7.4, and dispersing in 1 mL of phosphate buffer solution with the pH value of 7.4.
5. The biochemical sensor prepared by the preparation method in the technical scheme 1 is used for detecting nerve enolase, and the detection steps are as follows:
(1) an electrochemical workstation is used for testing by a three-electrode system, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, a completely modified ITO electrode is used as a working electrode, and the test is carried out in 10 mL of phosphate buffer solution with the pH value of 7.4 and containing 0.1 mol/L ascorbic acid;
(2) detecting the nerve enolase by a time-current method, setting the voltage to be 0V, running the time to be 200 s, and irradiating by an LED lamp;
(3) when the background current tends to be stable, turning on the lamp every 10-20 s for continuously irradiating for 10-20 s, recording the photocurrent, and drawing a working curve;
(4) and (3) replacing the nerve enolase standard solution with the nerve enolase sample solution to be detected for detection, and checking a detection result through a working curve.
Advantageous results of the invention
(1) The large specific surface area of the synthesized cerium and silver co-doped antimony tungstate can increase the load of biomolecules, and the doping of the cerium and silver co-elements can provide a direct electronic path, so that the surface plasma effect is caused, and the separation efficiency of photo-generated charges is improved. Partial silver ions are consumed in situ on cerium and silver co-doped antimony tungstate to grow silver sulfide, and indium sulfide is modified on the cerium and silver co-doped antimony tungstate/silver sulfide to form an energy band matching structure, so that the absorption of visible light can be increased, and good photoelectric response is shown;
(2) the cadmium sulfide/polydopamine composite synthesized by the invention is used as a detection antibody marker to construct a signal enhancement type photoelectrochemical sensor, on one hand, the excellent conductivity of cadmium sulfide and the abundant functional groups on the surface of polydopamine can be directly combined with biomolecules, so that the photoelectric conversion efficiency of the sensor is effectively improved; on the other hand, better energy band matching is realized between the cadmium sulfide/polydopamine and cerium and silver co-doped antimony tungstate, so that the strength and stability of photocurrent are greatly improved; thereby greatly reducing background signals and realizing the construction of a high-sensitivity biosensor;
(3) the photoelectric chemical sensor prepared by the invention is used for detecting the nerve enolase, has short response time and good stability, and can realize simple, quick, high-sensitivity and specific detection of the nerve enolase.
Detailed Description
Example 1 preparation of cerium and silver co-doped antimony tungstate microspheres:
dissolving 0.3 g of sodium tungstate hydrate in 8 mL of ultrapure water, and obtaining solution A; dissolving 0.4 g of antimony trichloride in 8 mL of absolute ethyl alcohol, namely solution B, slowly dripping the solution B into the solution A, stirring for 10 min to obtain a light yellow solution, adding 0.03 mg of silver nitrate and 0.08 mg of cerium nitrate hydrate into the solution, stirring for 30 min, adjusting the pH to 2 by using 1 mol/L sodium hydroxide solution or nitric acid solution, continuously stirring for 10 min, transferring the obtained solution to a high-pressure reaction kettle, reacting for 18 h at 180 ℃, taking out a product after the high-pressure reaction kettle is gradually cooled to room temperature, washing for 3 times by using ethanol and ultrapure water, and vacuum drying for 10 h.
Example 2 preparation of cerium and silver co-doped antimony tungstate microspheres:
dissolving 0.5 g of sodium tungstate hydrate in 8 mL of ultrapure water, and obtaining solution A; dissolving 0.6 g of antimony trichloride in 8 mL of absolute ethyl alcohol, namely solution B, slowly dripping the solution B into the solution A, stirring for 10 min to obtain a light yellow solution, adding 0.05 mg of silver nitrate and 0.12 mg of cerium nitrate hydrate into the solution, stirring for 30 min, adjusting the pH value to 2 by using 1 mol/L sodium hydroxide solution or nitric acid solution, continuously stirring for 10 min, transferring the obtained solution to a high-pressure reaction kettle, reacting for 24 h at 180 ℃, taking out a product after the high-pressure reaction kettle is gradually cooled to room temperature, washing for 4 times by using ethanol and ultrapure water, and vacuum drying for 16 h.
Example 3 preparation of indium sulfide:
dissolving 0.2 g of indium nitrate hydrate in 80 mL of ultrapure water, adding 0.10 g of thioacetamide into the solution, stirring for 30 min, transferring the obtained solution to a high-pressure reaction kettle, reacting at 120 ℃ for 10 h, gradually cooling the high-pressure reaction kettle to room temperature, taking out the product, washing with ethanol for 3 times, and drying in vacuum for 6 h.
Example 4 preparation of indium sulfide:
dissolving 0.4 g of indium nitrate hydrate in 80 mL of ultrapure water, adding 0.12 g of thioacetamide into the solution, stirring for 30 min, transferring the obtained solution to a high-pressure reaction kettle, reacting for 12 h at 120 ℃, taking out a product after the high-pressure reaction kettle is gradually cooled to room temperature, washing for 4 times by using ethanol, and drying for 8 h in vacuum.
Example 5 preparation of a cadmium sulfide/polydopamine complex labeled neuroenolase antibody solution:
(1) preparation of polydopamine:
dissolving 0.1 g dopamine in 100 mL Tris-buffer (pH 8.8), adding 50 mL isopropanol to the solution, stirring for 24 h, centrifuging the resulting solution at 9000/min for 20 min, washing with ultrapure water 4 times to remove unreacted substances, distributing the product in 10 mL ultrapure water, and placing in a 4 ℃ refrigerator;
(2) preparation of cadmium sulfide/polydopamine composite:
dissolving 0.008 g of cadmium acetate hydrate and 6 mL of polydopamine in 10 mL of absolute ethyl alcohol, stirring for 1 h, transferring the obtained mixed solution into an oil bath, heating at 80 ℃ for 10 min, adding 10 mL of thioacetamide (2 mg) aqueous solution into the solution in batches under stirring, continuously heating for 1 h, cooling to room temperature, washing with ultrapure water and absolute ethyl alcohol, and vacuum-drying overnight;
(3) preparing a cadmium sulfide/polydopamine complex labeled nerve enolase antibody solution:
dispersing 2 mg of cadmium sulfide/polydopamine in 1 mL of neural enolase antibody with the concentration of 5 mu g/mL, adding 100 mu L of bovine serum albumin solution with the mass fraction of 1.0% into the solution, shaking at 4 ℃ for 12 h, centrifugally washing for 3 times by using phosphate buffer solution with the pH value of 7.4, and dispersing in 1 mL of phosphate buffer solution with the pH value of 7.4.
Example 6 preparation of a cadmium sulfide/polydopamine complex labeled neuroenolase antibody solution:
(1) preparation of polydopamine:
dissolving 0.2 g dopamine in 100 mL Tris-buffer (pH 8.8), adding 50 mL isopropanol to the solution, stirring for 24 h, centrifuging the resulting solution at 9000/min for 20 min, washing with ultrapure water for 5 times to remove unreacted substances, distributing the product in 10 mL ultrapure water, and placing in a 4 ℃ refrigerator;
(2) preparation of cadmium sulfide/polydopamine composite:
dissolving 0.009 g of cadmium acetate hydrate and 7 mL of polydopamine in 10 mL of absolute ethyl alcohol, stirring for 1 h, transferring the obtained mixed solution into an oil bath, heating at 90 ℃ for 12 min, adding 10 mL of thioacetamide (3 mg) aqueous solution into the solution in batches under stirring, continuing to heat for 1 h, cooling to room temperature, washing with ultrapure water and absolute ethyl alcohol, and vacuum-drying overnight;
(3) preparing a cadmium sulfide/polydopamine complex labeled nerve enolase antibody solution:
dispersing 2 mg of cadmium sulfide/polydopamine in 1 mL of neural enolase antibody with the concentration of 5 mu g/mL, adding 100 mu L of bovine serum albumin solution with the mass fraction of 1.0% into the solution, shaking at 4 ℃ for 12 h, centrifugally washing for 3 times by using phosphate buffer solution with the pH value of 7.4, and dispersing in 1 mL of phosphate buffer solution with the pH value of 7.4.
Example 7 preparation of a photoelectrochemical sensor based on a cerium and silver co-doped antimony tungstate/silver sulfide/indium sulfide composite material:
(1) ultrasonically cleaning ITO conductive glass of 2.4 cm multiplied by 0.8 cm by using liquid detergent, acetone, ethanol and ultrapure water for 30 min in sequence, and drying by using nitrogen;
(2) 10 mu L of the cerium and silver codoped antimony tungstate microsphere prepared in the example 1 with the concentration of 4 mg/mL is dripped on the conductive surface of ITO conductive glass, air drying is carried out at room temperature to prepare a cerium and silver codoped antimony tungstate electrode, and the electrode is immersed in 0.2 mol/L of Na2S·9H2Carrying out O reaction for 30 s to obtain a cerium and silver co-doped antimony tungstate/silver sulfide electrode, dripping 4 mu L of the indium sulfide solution prepared in the example 3 with the concentration of 3 mg/mL onto the surface of the electrode, airing at room temperature, and washing with ultrapure water to obtain the cerium and silver co-doped antimony tungstate/silver sulfide/indium sulfide electrode;
(3) dropwise adding 4 mu L of thioglycolic acid with the concentration of 3 mmol/L onto the surface of the electrode to fix the nerve enolase capture antibody, drying in a refrigerator at 4 ℃, washing with ultrapure water, continuously dropwise adding 3 mu L of a mixed solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (10 mmol/L/2 mmol/L) to activate carboxyl, drying in the refrigerator at 4 ℃, and washing with ultrapure water;
(4) dripping 6 mu L of nerve enolase capture antibody solution with the concentration of 6 mu g/mL to the surface of the modified electrode, incubating for 1 h in a refrigerator at 4 ℃, and washing with phosphate buffer solution with the pH of 7.4;
(5) dripping 3 mu L of bovine serum albumin solution with the mass fraction of 1.0% on the surface of the modified electrode to seal the nonspecific active site on the surface of the electrode, airing in a refrigerator at 4 ℃, and cleaning by phosphate buffer solution with the pH of 7.4;
(6) dripping 6 mu L of a series of nerve enolase antigen solutions with different concentrations and the concentration of 0.0001 ng/mL-50 ng/mL, incubating for 1 h in a refrigerator at 4 ℃, and washing with a phosphate buffer solution with the pH of 7.4;
(7) 6 mul of the mixed solution of cadmium sulfide/polydopamine complex labeled neuroenolase antibody prepared in example 5 with the concentration of 3.0 mg/mL was dropped on the surface of the electrode, incubated in a refrigerator at 4 ℃ for 1 h, and washed with phosphate buffer solution of pH 7.4, to prepare a biochemical sensor.
Example 8 preparation of a photoelectrochemical sensor based on a cerium and silver co-doped antimony tungstate/silver sulfide/indium sulfide composite material:
(1) ultrasonically cleaning ITO conductive glass of 2.4 cm multiplied by 0.8 cm by using liquid detergent, acetone, ethanol and ultrapure water for 30 min in sequence, and drying by using nitrogen;
(2) 10 mu L of the cerium and silver codoped antimony tungstate prepared in the embodiment 2 with the concentration of 5 mg/mL is dripped on an ITO conductive glass conductive surface and dried at room temperature to prepare a cerium and silver codoped antimony tungstate electrode, and the electrode is immersed in 0.2 mol/L of Na2S·9H2Performing O reaction for 40 s to obtain a cerium and silver co-doped antimony tungstate/silver sulfide electrode, dripping 4 mu L of the indium sulfide solution prepared in the example 4 with the concentration of 4 mg/mL onto the surface of the electrode, airing at room temperature, and washing with ultrapure water to obtain the cerium and silver co-doped antimony tungstate/silver sulfide/indium sulfide electrode;
(3) dropwise adding 4 mu L of thioglycolic acid with the concentration of 3 mmol/L onto the surface of the electrode to fix the nerve enolase capture antibody, drying in a refrigerator at 4 ℃, washing with ultrapure water, continuously dropwise adding 3 mu L of a mixed solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (10 mmol/L/2 mmol/L) to activate carboxyl, drying in the refrigerator at 4 ℃, and washing with ultrapure water;
(4) dripping 6 mu L of neural enolase capture antibody solution with the concentration of 8 mu g/mL to the surface of the modified electrode, incubating for 1 h in a refrigerator at 4 ℃, and washing with phosphate buffer solution with the pH of 7.4;
(5) dripping 3 mu L of bovine serum albumin solution with the mass fraction of 1.0% on the surface of the modified electrode to seal the nonspecific active site on the surface of the electrode, airing in a refrigerator at 4 ℃, and cleaning by phosphate buffer solution with the pH of 7.4;
(6) dripping 6 mu L of a series of nerve enolase antigen solutions with different concentrations and the concentration of 0.0001 ng/mL-50 ng/mL, incubating for 1 h in a refrigerator at 4 ℃, and washing with a phosphate buffer solution with the pH of 7.4;
(7) 6 mu L of the mixed solution of the cadmium sulfide/polydopamine composite labeled neuroenolase antibody prepared in the embodiment 6 with the concentration of 4.0 mg/mL is dripped on the surface of an electrode, incubated for 1 h in a refrigerator at 4 ℃, and cleaned by phosphate buffer solution with the pH of 7.4, so that the photoelectrochemical sensor for detecting the neuroenolase is prepared.
The photoelectrochemical sensor constructed in example 9 was used for the detection of nerve enolase by the following steps:
(1) an electrochemical workstation is used for testing by using a three-electrode system, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the biochemical sensor prepared in example 7 is used as a working electrode, and the test is carried out in 10 mL of phosphate buffer solution with pH 7.4 and containing 0.1 mol/L ascorbic acid;
(2) detecting the nerve enolase by a time-current method, setting the voltage to be 0V, running the time to be 200 s, and irradiating by an LED lamp;
(3) when the background current tends to be stable, turning on the lamp every 10 s for continuously irradiating for 10 s, recording the photocurrent, and drawing a working curve, wherein the linear detection range is 0.0001 ng/mL-50 ng/mL;
(4) and (3) replacing the nerve enolase standard solution with the nerve enolase sample solution to be detected for detection, and checking a detection result through a working curve.
The photoelectrochemical sensor constructed in example 10 was used for the detection of nerve enolase by the following steps:
(1) the electrochemical workstation is used for testing by a three-electrode system, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the biochemical sensor prepared in the example 8 is used as a working electrode, and the test is carried out in 10 mL of phosphate buffer solution with pH 7.4 and containing 0.1 mol/L ascorbic acid;
(2) detecting the nerve enolase by a time-current method, setting the voltage to be 0V, running the time to be 200 s, and irradiating by an LED lamp;
(3) when the background current tends to be stable, turning on the lamp every 20 s for continuously irradiating for 20 s, recording the photocurrent, and drawing a working curve, wherein the linear detection range is 0.0001 ng/mL-50 ng/mL;
(4) and (3) replacing the nerve enolase standard solution with the nerve enolase sample solution to be detected for detection, and checking a detection result through a working curve.
Claims (2)
1. A preparation method of a biochemical sensor is characterized in that the biochemical sensor is a photoelectrochemical sensor based on a cerium and silver co-doped antimony tungstate/silver sulfide/indium sulfide composite material, and the preparation method comprises the following steps:
(1) ultrasonically cleaning ITO conductive glass of 2.4 cm multiplied by 0.8 cm by using liquid detergent, acetone, ethanol and ultrapure water for 30 min in sequence, and drying by using nitrogen;
(2) dropwise adding 10 mu L of cerium and silver co-doped antimony tungstate microspheres with the concentration of 4-6 mg/mL to the conductive surface of the ITO conductive glass obtained in the step (1), airing at room temperature to obtain a cerium and silver co-doped antimony tungstate electrode, and then immersing the electrode into Na with the concentration of 0.1-0.5 mol/L2S·9H2Performing O reaction for 30-50 s to obtain a cerium and silver co-doped antimony tungstate/silver sulfide electrode;
(3) dropwise adding 4 mu L of indium sulfide solution with the concentration of 3-5 mg/mL to the electrode surface of the cerium and silver co-doped antimony tungstate/silver sulfide electrode obtained in the step (2), airing at room temperature, and washing with ultrapure water to obtain the cerium and silver co-doped antimony tungstate/silver sulfide/indium sulfide electrode;
(4) dropwise adding 4 mu L of thioglycolic acid with the concentration of 3 mmol/L onto the electrode surface of the cerium and silver co-doped antimony tungstate/silver sulfide/indium sulfide electrode obtained in the step (3) for fixing a nerve enolase capture antibody, drying in a refrigerator at 4 ℃, continuously dropwise adding 3 mu L of a mixed solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (10 mmol/L/2 mmol/L) for activating carboxyl after washing with ultrapure water, drying in the refrigerator at 4 ℃, and washing with ultrapure water;
(5) dripping 6 mu L of nerve enolase capture antibody solution with the concentration of 6-10 mu g/mL to the surface of the modified electrode obtained in the step (4), incubating for 1 h in a refrigerator at 4 ℃, and washing with a phosphate buffer solution with the pH value of 7.4;
(6) dripping 3 mu L of bovine serum albumin solution with the mass fraction of 1.0% to the surface of the modified electrode obtained in the step (5) to seal the non-specific active sites on the surface of the electrode, airing in a refrigerator at 4 ℃, and washing with a phosphate buffer solution with the pH value of 7.4;
(7) dripping 6 mu L of a series of nerve enolase antigen solutions with different concentrations and the concentration of 0.0001 ng/mL-50 ng/mL to the surface of the modified electrode obtained in the step (6), incubating for 1 h in a refrigerator at 4 ℃, and washing with a phosphate buffer solution with the pH of 7.4;
(8) dripping 6 mu L of cadmium sulfide/polydopamine composite labeled nerve enolase antibody solution with the concentration of 3.0-5.0 mg/mL to the surface of the modified electrode obtained in the step (7), incubating for 1 h in a refrigerator at 4 ℃, and cleaning with a phosphate buffer solution with the pH of 7.4 to prepare a biochemical sensor which can be used for photoelectric detection of nerve enolase;
the cerium and silver co-doped antimony tungstate microsphere prepared in the step (2) is prepared by the following steps: dissolving 0.3-0.5 g of sodium tungstate hydrate in 8 mL of ultrapure water, and referring to the solution A; dissolving 0.4-0.6 g of antimony trichloride in 8 mL of absolute ethyl alcohol to obtain a solution B, slowly dripping the solution B into the solution A, stirring for 10 min to obtain a light yellow solution, adding 0.03-0.05 mg of silver nitrate and 0.08-0.12 mg of cerous nitrate hydrate into the solution A, stirring for 30 min, adjusting the pH to 2 by using 1 mol/L of sodium hydroxide solution or nitric acid solution, continuously stirring for 10 min, transferring the obtained solution to a high-pressure reaction kettle, reacting at 180 ℃ for 18-24 h, after the high-pressure reaction kettle is gradually cooled to room temperature, taking out a product, washing with ethanol and ultrapure water for 3-4 times, and vacuum drying for 10-16 h;
the cerium and silver co-doped antimony tungstate/silver sulfide electrode in the step (2) is prepared by consuming partial silver ions on the surface of the cerium and silver co-doped antimony tungstate electrode and growing in situ;
the indium sulfide solution in the step (3) is a sol water solution of indium sulfide, and the indium sulfide preparation step is as follows: dissolving 0.2-0.5 g of indium nitrate hydrate in 80 mL of ultrapure water, adding 0.10-0.12 g of thioacetamide into the solution, stirring for 30 min, transferring the obtained solution to a high-pressure reaction kettle, reacting at 120 ℃ for 10-12 h, taking out a product after the high-pressure reaction kettle is gradually cooled to room temperature, washing with ethanol for 3-4 times, and vacuum drying for 6-8 h to obtain indium sulfide;
the preparation method of the solution of the cadmium sulfide/polydopamine complex labeled neuroenolase antibody in the step (8) comprises the following steps:
preparing polydopamine, comprising the following steps: dissolving 0.1-0.3 g of dopamine in 100 mL of Tris-buffer with pH 8.8, adding 50 mL of isopropanol into the solution, stirring for 24 hours, centrifuging the obtained solution at 9000/min for 20 min, washing with ultrapure water for 4-6 times to remove unreacted substances, distributing the product in 10 mL of ultrapure water, and placing in a refrigerator at 4 ℃;
preparing a cadmium sulfide/polydopamine compound, comprising the following steps: dissolving 0.008-0.01 g of cadmium acetate hydrate and 6-8 mL of polydopamine in 10 mL of absolute ethyl alcohol, stirring for 1 h, transferring the obtained mixed solution into an oil bath, heating at 80-100 ℃ for 10-15 min, adding 10 mL of thioacetamide (2-5 mg) aqueous solution into the solution in batches under stirring, continuing heating for 1 h, cooling to room temperature, washing with ultrapure water and absolute ethyl alcohol, and vacuum drying overnight;
preparing a cadmium sulfide/polydopamine composite substance-labeled nerve enolase antibody solution, comprising the following steps: dispersing 2 mg of cadmium sulfide/polydopamine composite in 1 mL of neural enolase antibody with the concentration of 5 mu g/mL, adding 100 mu L of bovine serum albumin solution with the mass fraction of 1.0% into the solution, shaking at 4 ℃ for 12 h, centrifugally washing for 3 times by using a phosphate buffer solution with the pH of 7.4, and dispersing in 1 mL of phosphate buffer solution with the pH of 7.4.
2. The biochemical sensor prepared by the preparation method according to claim 1, wherein the biochemical sensor is used for detecting nerve enolase, and the detection steps are as follows:
(1) an electrochemical workstation is used for testing by a three-electrode system, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, a biochemical sensor is used as a working electrode, and the test is carried out in 10 mL of phosphate buffer solution with the pH value of 7.4 and containing 0.1 mol/L ascorbic acid;
(2) detecting the nerve enolase by a time-current method, setting the voltage to be 0V, running the time to be 200 s, and irradiating by an LED lamp;
(3) when the background current tends to be stable, turning on the lamp every 10-20 s for continuously irradiating for 10-20 s, recording the photocurrent, and drawing a working curve;
(4) and (3) replacing the nerve enolase standard solution with the nerve enolase sample solution to be detected for detection, and checking a detection result through a working curve.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114047235A (en) * | 2021-10-22 | 2022-02-15 | 济南大学 | Based on Cs2AgBiBr6Preparation method of constructed neuron-specific enolase photoelectrochemical sensor |
| CN114324520A (en) * | 2021-12-20 | 2022-04-12 | 济南大学 | Preparation method of photoelectrochemical sensor based on silver sulfide enhanced PTCA glutathione detection |
| CN114674896A (en) * | 2022-03-28 | 2022-06-28 | 济南大学 | Preparation method of photoelectrochemical NSE sensor based on controlled release ZIF-8 shielding shell |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114047235A (en) * | 2021-10-22 | 2022-02-15 | 济南大学 | Based on Cs2AgBiBr6Preparation method of constructed neuron-specific enolase photoelectrochemical sensor |
| CN114047235B (en) * | 2021-10-22 | 2023-06-20 | 济南大学 | Preparation method of neuron-specific enolase photoelectrochemical sensor |
| CN114324520A (en) * | 2021-12-20 | 2022-04-12 | 济南大学 | Preparation method of photoelectrochemical sensor based on silver sulfide enhanced PTCA glutathione detection |
| CN114324520B (en) * | 2021-12-20 | 2023-07-04 | 济南大学 | Preparation method of photoelectrochemical sensor based on silver sulfide enhanced PTCA glutathione detection |
| CN114674896A (en) * | 2022-03-28 | 2022-06-28 | 济南大学 | Preparation method of photoelectrochemical NSE sensor based on controlled release ZIF-8 shielding shell |
| CN114674896B (en) * | 2022-03-28 | 2023-07-04 | 济南大学 | Preparation method of photoelectrochemical NSE sensor based on controlled release ZIF-8 shielding shell |
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