CN110231336B - Preparation method of graphene/polyaniline nanowire array immunosensor - Google Patents
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Abstract
The invention relates to a preparation method of a graphene/polyaniline nanowire array immunosensor, belonging to the technical field of biosensing; the invention adopts an electrochemiluminescence technology, takes procalcitonin as an analyte model, takes a palladium hybridized graphene/polyaniline nano-array structure as a sensing platform, luminol is taken as an electrochemical luminescence signal source, carboxylated cerium doped tin disulfide is taken as a second antibody marker, the preparation method of the electrochemiluminescence sensor is provided, the method has the advantages of simple preparation and low cost, overcomes the defects of complex operation, high cost and long time consumption of the traditional detection technology, and the sensor developed based on the method is applied to the detection of the actual sample of procalcitonin, has the advantages of high sensitivity, strong specificity, quick response and portability, the detection limit is as low as 1.2 fg/mL, the linear range is as wide as 5 fg/mL-50 ng/mL, and the method has great potential application value in the early clinical detection of procalcitonin.
Description
Technical Field
A preparation method of a graphene/polyaniline nanowire array immunosensor belongs to the technical field of biosensing.
Background
The expert consensus on procalcitonin emergency clinical application published in 9 months of 2012 states that: the level of procalcitonin of a sepsis patient is obviously higher than that of a non-sepsis patient, and the rising of procalcitonin has high specificity to sepsis caused by bacterial infection, so that the procalcitonin can be used as a biomarker for diagnosing sepsis and identifying serious bacterial infection; the existing method for detecting procalcitonin is mainly an electrochemical biosensor, and the electrochemiluminescence technology combines the advantages of two technologies, namely chemiluminescence and electrochemistry, so that the method has the characteristics of easiness in operation, strong controllability, high response speed and the like, and has higher sensitivity and lower detection limit than electrochemistry; the biosensor prepared based on the electrochemiluminescence technology has the advantages of being stronger than that of an electrochemical technology, and provides a stronger technical support for the new immunization method.
Disclosure of Invention
One of the technical tasks of the invention is to make up the defects of the existing detection technology, and provide a preparation method of a graphene/polyaniline array biosensor, wherein the preparation method has the advantages of simple preparation process, low cost and rapid signal response, greatly shortens the detection time, and saves time and labor;
the second technical task of the invention is to provide the application of the immunosensor, the immunosensor can rapidly detect procalcitonin, and the immunosensor has the advantages of high sensitivity, strong specificity and good reproducibility, the detection limit is 1.2 fg/mL, the linear range is 5 fg/mL-50 ng/mL, and the immunosensor has great potential application value in clinical detection.
The technical scheme of the invention is as follows
A preparation method of a graphene/polyaniline nanowire array immunosensor comprises the following steps:
(1) polishing glassy carbon electrodes with the diameter of 4 mm by using alumina with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;
(2) dropping 6 mu L of capture antibody Ab with the concentration of 2-4 mg/mL on the surface of the glassy carbon electrode1Placing the marked palladium hybridized graphene/polyaniline solution at 4 ℃ for airing;
(3) dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 1-3% to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode by phosphate buffer solution with the pH of 7.4, and airing at 4 ℃;
(4) Dripping 6 mu L of procalcitonin standard solution or solution with unknown concentration, incubating for 0.5-2 h at 37 ℃, washing the surface of the electrode by using phosphate buffer solution with pH of 7.4, and airing at 4 ℃;
(5) dropwise adding 6 mu L of detection antibody Ab with the concentration of 2-4 mg/mL2And (3) washing the surface of the electrode by using a marked cerium-doped tin disulfide-luminol solution and a phosphate buffer solution with the pH value of 7.4, placing the electrode at 4 ℃, airing, and finishing the construction of the sensor.
Preparation method of graphene/polyaniline nanowire array immunosensor, and capture antibody Ab1A labeled palladium-hybridized graphene/polyaniline solution is prepared by the following steps:
(1) adding 30-50 mL of deionized water into 0.13-0.15 g of sodium tetrachloropalladate and 0.13-0.15 g of sodium citrate solid, uniformly dissolving the mixed solid, then dropwise adding 0.5-0.7 mL of sodium borohydride solution into the mixed solution, finally, continuously stirring to obtain black palladium nanoparticles, and storing at 4 ℃ in a dark place;
(2) adding 0.4-0.6 g of graphene oxide into a beaker containing 90-110 mL of anhydrous dimethylformamide, then adding 2.78-2.98 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into the upper suspension under the condition of ice-water bath, stirring for 1-3 h under the nitrogen atmosphere, then adding 1.8-2.0 mL of 1, 3-diaminopropane, stirring overnight at room temperature, finally washing with water and ethanol respectively, and drying to obtain aminated graphene oxide;
(3) Dispersing 5-15 mg of aminated graphene oxide in 5-15 mL of hydrochloric acid solution, performing ultrasonic treatment for 15-45 min, uniformly dispersing the aminated graphene oxide, cooling the suspension in an ice bath to 0 ℃, then adding 0.4-0.6 mL of aniline into the aminated graphene oxide, continuously stirring, slowly adding 5-15 mL of solution dissolved with 1.25-1.45 g of potassium persulfate, after the reaction lasts for 4-6 h in the ice bath, adding 0.1 mol/L hydrochloric acid solution to remove all unreacted reactants, washing 3 times with deionized water and hexane respectively, and drying for 12 h to obtain graphene/polyaniline solution;
(4) mixing 1-3 mL of graphene/polyaniline solution with the concentration of 2 mg/mL and 1-3 mL of palladium nanoparticle solution, oscillating for 24-48 h in a dark place, centrifugally separating, removing supernatant, dispersing into 1 mL of ultrapure water to prepare palladium hybrid graphene/polyaniline solution, and then adding 50-150 uL of capture antibody Ab with the concentration of 1 ug/mL1After the solution is shaken for 6-18 h, the solution is dispersed into 2 mL phosphate buffer solution with pH of 7.4 after centrifugal separation to obtain a capture antibody Ab1A labeled palladium-hybridized graphene/polyaniline solution.
Preparation method of graphene/polyaniline nanowire array immunosensor, and detection antibody Ab 2A labeled cerium doped tin disulfide-luminol solution prepared by the steps of:
(1) completely dissolving 0.01-0.03 mol/L tin pentahydrate tetrachloride, 0.001-0.003 mol/L cerium nitrate hexahydrate, 0.05-0.07 mol/L thioacetamide and 0.4-0.6 g hexadecyltrimethylammonium bromide in a mixture of ethanol and distilled water, transferring the mixture into a 200 mL high-temperature reaction kettle with a polytetrafluoroethylene lining, keeping the temperature at 180 ℃, reacting for 24-48 h, and then cooling to room temperature; centrifugally washing the obtained solid for multiple times, drying the solid in a vacuum drying oven at 60 ℃ for 12-36 hours, and then sintering the solid for 2-4 hours at 500 ℃ under the protection of argon to obtain cerium-doped tin disulfide solid;
(2) weighing 1-3 mg of cerium-doped tin disulfide, dispersing the cerium-doped tin disulfide into 0.5-1.5 mL of deionized water to prepare a solution, then adding 5-15 mu L of luminol with the concentration of 5 mmol/L, oscillating for 6-18 h, obtaining a precipitate through centrifugal separation, and finally dispersing the precipitate into 1 mL of deionized water again to obtain a target solution cerium-doped tin disulfide-luminol solution;
(3) adding 100-300 mu L of detection antibody Ab with the concentration of 10 mg/mL into the solution 2The solution is incubated for 6-18 h under the oscillation at 4 ℃, centrifuged and dispersed into 1 mL of phosphate buffer solution with the pH value of 7.4 to obtainTo the detection antibody Ab2The marked cerium doped tin disulfide-luminol solution is stored at 4 ℃ for later use.
An application of a sensor obtained by a preparation method of a graphene/polyaniline nanowire array immunosensor in procalcitonin detection comprises the following operation steps:
(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to be 800V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-0.7V, and the scanning rate is set to be 0.1V/s;
(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared in claim 1 as a working electrode, immersing the sensor for incubating a series of procalcitonins with standard concentrations into 10 mL of phosphate buffer solution containing 35-65 mmol/L of hydrogen peroxide for performing electrochemiluminescence test to obtain corresponding electrochemiluminescence signal intensity when the procalcitonins with different concentrations are incubated, and drawing a working curve, wherein the detection limit is 1.2 fg/mL, and the linear range is 5 fg/mL-50 ng/mL;
(3) and testing the electrochemiluminescence sensor for incubating the procalcitonin actual sample with unknown concentration to obtain corresponding signal intensity, and calculating according to the working curve to obtain the procalcitonin concentration in the actual sample.
Advantageous results of the invention
(1) The palladium functionalized graphene/polyaniline array is used as a sensor substrate for the first time, the array structure has a highly ordered three-dimensional structure and a large specific surface area, a large number of binding sites can be provided for immobilized capture antibodies, and meanwhile, the array can form an electron transport network with a three-dimensional structure by combining the excellent conductivity of graphene and polyaniline, so that the electron transfer of the sensor substrate is accelerated, and the conductivity of the sensor is improved;
(2) the carboxylated cerium-doped tin disulfide nanosheet is used as a beacon immobilized luminescent molecule luminol for the first time, the generation of superoxide radical is the main reason of luminol luminescence, the tin disulfide has good catalytic effect on the decomposition of hydrogen peroxide into superoxide radical, and the doping of cerium element can further accelerate the rapid decomposition of hydrogen peroxide, so that more superoxide radical can be obtained, and the electrochemiluminescence excitation of luminol is obviously enhanced;
(3) the invention provides a preparation method of an immunosensor based on a graphene/polyaniline array for the first time based on an electrochemiluminescence technology, the method has the advantages of simple preparation, low cost and high response speed, the defects of the existing detection technology are overcome, the immunosensor prepared by the method is used for detecting procalcitonin, the immunosensor has the advantages of high sensitivity, good reproducibility and portability, the detection limit can reach 1.2 fg/mL, the linear range is as wide as 5 fg/mL-50 ng/mL, and a brand new analysis method is provided for early clinical detection of procalcitonin.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Embodiment 1 a method for preparing a graphene/polyaniline nanowire array immunosensor, comprising the following steps:
(1) polishing glassy carbon electrodes with the diameter of 4 mm by using alumina with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;
(2) 6 mu L of capture antibody Ab with the concentration of 2 mg/mL is dripped on the surface of the glassy carbon electrode1Placing the marked palladium hybridized graphene/polyaniline solution at 4 ℃ for airing;
(3) dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 1% to seal the nonspecific active sites on the surface of the electrode, washing the surface of the electrode with phosphate buffer solution with the pH of 7.4, and airing at 4 ℃;
(4) dripping 6 μ L of procalcitonin standard solution or unknown concentration solution, incubating at 37 deg.C for 0.5 h, washing electrode surface with phosphate buffer solution with pH of 7.4, and air drying at 4 deg.C;
(5) 6 mu L of detection antibody Ab with the concentration of 2 mg/mL is dripped2The labeled cerium doped tin disulfide-luminol solution was washed with a pH 7.4 phosphate buffer solution And (5) placing the electrode surface at 4 ℃ for airing, and finishing the construction of the sensor.
Embodiment 2 a method for preparing a graphene/polyaniline nanowire array immunosensor, comprising the following steps:
(1) polishing glassy carbon electrodes with the diameter of 4 mm by using alumina with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;
(2) 6 mu L of capture antibody Ab with the concentration of 3 mg/mL is dripped on the surface of the glassy carbon electrode1Placing the marked palladium hybridized graphene/polyaniline solution at 4 ℃ for airing;
(3) dropwise adding 3 mu L of 2% bovine serum albumin solution to seal the nonspecific active sites on the surface of the electrode, washing the surface of the electrode with a phosphate buffer solution with pH of 7.4, and airing at 4 ℃;
(4) dripping 6 μ L of procalcitonin standard solution or unknown concentration solution, incubating at 37 deg.C for 1.5 h, washing electrode surface with phosphate buffer solution with pH of 7.4, and air drying at 4 deg.C;
(5) 6 mu L of detection antibody Ab with the concentration of 3 mg/mL is dripped2And (3) washing the surface of the electrode by using a marked cerium-doped tin disulfide-luminol solution and a phosphate buffer solution with the pH value of 7.4, placing the electrode at 4 ℃, airing, and finishing the construction of the sensor.
Embodiment 3 a method for preparing a graphene/polyaniline nanowire array immunosensor, comprising the following steps:
(1) Polishing glassy carbon electrodes with the diameter of 4 mm by using alumina with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;
(2) 6 mu L of capture antibody Ab with concentration of 4 mg/mL is dripped on the surface of the glassy carbon electrode1Placing the marked palladium hybridized graphene/polyaniline solution at 4 ℃ for airing;
(3) dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 3% to seal the nonspecific active sites on the surface of the electrode, washing the surface of the electrode with phosphate buffer solution with the pH of 7.4, and airing at 4 ℃;
(4) dripping 6 μ L of procalcitonin standard solution or unknown concentration solution, incubating at 37 deg.C for 2 h, washing electrode surface with phosphate buffer solution with pH of 7.4, and air drying at 4 deg.C;
(5) 6 mu L of detection antibody Ab with the concentration of 4 mg/mL is dripped2And (3) washing the surface of the electrode by using a marked cerium-doped tin disulfide-luminol solution and a phosphate buffer solution with the pH value of 7.4, placing the electrode at 4 ℃, airing, and finishing the construction of the sensor.
Example 4 Capture antibody Ab1A labeled palladium-hybridized graphene/polyaniline solution is prepared by the following steps:
adding 30 mL of deionized water into 0.13 g of sodium tetrachloropalladate and 0.13 g of sodium citrate solid, uniformly dissolving the mixed solid, then dropwise adding 0.5 mL of sodium borohydride solution into the mixed solution, finally, continuously stirring to obtain black palladium nanoparticles, and storing at 4 ℃ in a dark place;
Adding 0.4 g of graphene oxide into a beaker containing 90 mL of anhydrous dimethylformamide, then adding 2.78 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into the upper suspension under the condition of ice-water bath, stirring for 1 h under the nitrogen atmosphere, then adding 1.8 mL of 1, 3-diaminopropane, stirring overnight at room temperature, finally washing with water and ethanol respectively, and drying to obtain aminated graphene oxide;
dispersing 5 mg of aminated graphene oxide in 5 mL of hydrochloric acid solution, performing ultrasonic treatment for 15 min, uniformly dispersing the aminated graphene oxide, cooling the suspension in an ice bath to 0 ℃, adding 0.4 mL of aniline into the aminated graphene oxide, continuously stirring, slowly adding 5 mL of solution dissolved with 1.25 g of potassium persulfate, adding 0.1 mol/L hydrochloric acid solution to remove all unreacted reactants after the reaction lasts for 4 hours in the ice bath, washing with deionized water and hexane for 3 times respectively, and drying for 12 hours to obtain graphene/polyaniline solution;
mixing 1 mL of graphene/polyaniline solution with the concentration of 2 mg/mL and 1 mL of palladium nanoparticle solution, shaking for 24 h in a dark place, centrifugally separating, discarding supernatant, dispersing into 1 mL of ultrapure water to prepare palladium hybrid graphene/polyaniline solution, and adding 50 uL of capture with the concentration of 1 ug/mL Obtaining antibody Ab1After shaking the solution for 6 h, the solution was centrifuged and dispersed in 2 mL of pH 7.4 phosphate buffer to obtain the capture antibody Ab1A labeled palladium-hybridized graphene/polyaniline solution.
Example 5 Capture antibody Ab1A labeled palladium-hybridized graphene/polyaniline solution is characterized by being prepared by the following steps:
adding 40 mL of deionized water into 0.14 g of sodium tetrachloropalladate and 0.14 g of sodium citrate solid, uniformly dissolving the mixed solid, then dropwise adding 0.6 mL of sodium borohydride solution into the mixed solution, finally, continuously stirring to obtain black palladium nanoparticles, and storing at 4 ℃ in a dark place;
adding 0.5 g of graphene oxide into a beaker containing 100 mL of anhydrous dimethylformamide, then adding 2.88 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into the upper suspension under the condition of ice-water bath, stirring for 2 hours under the nitrogen atmosphere, then adding 1.9 mL of 1, 3-diaminopropane, stirring overnight at room temperature, finally washing with water and ethanol respectively, and drying to obtain aminated graphene oxide;
dispersing 10 mg of aminated graphene oxide in 10 mL of hydrochloric acid solution, performing ultrasonic treatment for 35 min, uniformly dispersing the aminated graphene oxide, cooling the suspension in an ice bath to 0 ℃, adding 0.5 mL of aniline into the aminated graphene oxide, continuously stirring, slowly adding 10 mL of solution dissolved with 1.35 g of potassium persulfate, after the reaction lasts for 5 hours in the ice bath, adding 0.1 mol/L hydrochloric acid solution to remove all unreacted reactants, washing with deionized water and hexane for 3 times respectively, and drying for 12 hours to obtain graphene/polyaniline solution;
Mixing 2 mL of graphene/polyaniline solution with the concentration of 2 mg/mL with 2 mL of palladium nanoparticle solution, oscillating for 36 h in a dark place, centrifugally separating, discarding supernatant, dispersing into 1 mL of ultrapure water to prepare palladium hybrid graphene/polyaniline solution, and then adding 100 uL of capture antibody Ab with the concentration of 1 ug/mL1After the solution was shaken for 12 hours, the solution was centrifuged and dispersed in 2 mL of a phosphate buffer solution having a pH of 7.4 to obtain a capture antibody Ab1A labeled palladium-hybridized graphene/polyaniline solution.
Example 6 Capture antibody Ab1A labeled palladium-hybridized graphene/polyaniline solution is characterized by being prepared by the following steps:
adding 50 mL of deionized water into 0.15 g of sodium tetrachloropalladate and 0.15 g of sodium citrate solid, uniformly dissolving the mixed solid, then dropwise adding 0.7 mL of sodium borohydride solution into the mixed solution, finally, continuously stirring to obtain black palladium nanoparticles, and storing at 4 ℃ in a dark place;
adding 0.6 g of graphene oxide into a beaker containing 110 mL of anhydrous dimethylformamide, then adding 2.98 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into the upper suspension under the condition of ice-water bath, stirring for 3 hours under the nitrogen atmosphere, then adding 2.0 mL of 1, 3-diaminopropane, stirring overnight at room temperature, finally washing with water and ethanol respectively, and drying to obtain aminated graphene oxide;
Dispersing 15 mg of aminated graphene oxide in 15 mL of hydrochloric acid solution, performing ultrasonic treatment for 45 min, uniformly dispersing the aminated graphene oxide, cooling the suspension in an ice bath to 0 ℃, adding 0.6 mL of aniline into the aminated graphene oxide, continuously stirring, slowly adding 15 mL of solution dissolved with 1.45 g of potassium persulfate, after the reaction lasts for 6 hours in the ice bath, adding 0.1 mol/L hydrochloric acid solution to remove all unreacted reactants, washing with deionized water and hexane for 3 times respectively, and drying for 12 hours to obtain graphene/polyaniline solution;
mixing 3 mL of graphene/polyaniline solution with the concentration of 2 mg/mL and 3 mL of palladium nanoparticle solution, oscillating for 48 hours in a dark place, centrifugally separating, discarding supernatant, dispersing into 1 mL of ultrapure water to prepare palladium hybrid graphene/polyaniline solution, and then adding 150 uL of capture antibody Ab with the concentration of 1 ug/mL1After the solution was shaken for 18 hours, the solution was centrifuged and dispersed in 2 mL of a phosphate buffer solution having a pH of 7.4 to obtain a capture antibody Ab1A labeled palladium-hybridized graphene/polyaniline solution.
Example 7 detection antibody Ab2MarkedA cerium-doped tin disulfide-luminol solution is prepared by the following steps:
completely dissolving 0.01 mol/L tin tetrachloride pentahydrate, 0.001 mol/L cerium nitrate hexahydrate, 0.05 mol/L thioacetamide and 0.4 g hexadecyl trimethyl ammonium bromide in a mixture of ethanol and distilled water, transferring the mixture to a 200 mL high-temperature reaction kettle with a polytetrafluoroethylene lining, keeping the temperature at 180 ℃, reacting for 24 hours, and then cooling to room temperature; centrifugally washing the obtained solid for multiple times, drying the solid in a vacuum drying oven at 60 ℃ for 12 hours, and then sintering the solid for 2 hours at 500 ℃ under the protection of argon to obtain cerium-doped tin disulfide solid;
Weighing 1 mg of cerium-doped tin disulfide, dispersing the cerium-doped tin disulfide into 0.5 mL of deionized water to prepare a solution, then adding 5 mu L of luminol with the concentration of 5 mmol/L, oscillating for 6 h, obtaining a precipitate through centrifugal separation, and finally dispersing the precipitate into 1 mL of deionized water again to obtain a target solution cerium-doped tin disulfide-luminol solution;
to the above solution, 100. mu.L of detection antibody Ab at a concentration of 10 mg/mL was added2Incubating the solution at 4 ℃ for 6 h with shaking, centrifuging, and dispersing into 1 mL of phosphate buffer solution with pH of 7.4 to obtain detection antibody Ab2The marked cerium doped tin disulfide-luminol solution is stored at 4 ℃ for later use.
Example 8 detection of antibody Ab2A labeled cerium doped tin disulfide-luminol solution prepared by the steps of:
completely dissolving 0.02 mol/L tin tetrachloride pentahydrate, 0.002 mol/L cerium nitrate hexahydrate, 0.06 mol/L thioacetamide and 0.5 g hexadecyl trimethyl ammonium bromide in a mixture of ethanol and distilled water, transferring the mixture into a 200 mL high-temperature reaction kettle with a polytetrafluoroethylene lining, keeping the temperature at 180 ℃, reacting for 36 hours, and then cooling to room temperature; centrifugally washing the obtained solid for multiple times, drying the solid in a vacuum drying oven at 60 ℃ for 24 hours, and then sintering the solid for 3 hours at 500 ℃ under the protection of argon to obtain cerium-doped tin disulfide solid;
Weighing 2 mg of cerium-doped tin disulfide, dispersing the cerium-doped tin disulfide into 1 mL of deionized water to prepare a solution, then adding 10 mu L of luminol with the concentration of 5 mmol/L, oscillating for 12 h, obtaining a precipitate through centrifugal separation, and finally dispersing the precipitate into 1 mL of deionized water again to obtain a target solution cerium-doped tin disulfide-luminol solution;
to the above solution, 200. mu.L of detection antibody Ab at a concentration of 10 mg/mL was added2Incubating the solution at 4 ℃ for 12 h with shaking, centrifuging, and dispersing into 1 mL of phosphate buffer solution with pH 7.4 to obtain detection antibody Ab2The marked cerium doped tin disulfide-luminol solution is stored at 4 ℃ for later use.
Example 9 detection of antibody Ab2A labeled cerium doped tin disulfide-luminol solution prepared by the steps of:
completely dissolving 0.03 mol/L tin tetrachloride pentahydrate, 0.003 mol/L cerium nitrate hexahydrate, 0.07 mol/L thioacetamide and 0.6 g hexadecyl trimethyl ammonium bromide in a mixture of ethanol and distilled water, transferring the mixture to a 200 mL high-temperature reaction kettle with a polytetrafluoroethylene lining, keeping the temperature at 180 ℃, reacting for 48 hours, and then cooling to room temperature; centrifugally washing the obtained solid for multiple times, drying the solid in a vacuum drying oven at 60 ℃ for 36 hours, and then sintering the solid for 4 hours at 500 ℃ under the protection of argon to obtain cerium-doped tin disulfide solid;
Weighing 3 mg of cerium-doped tin disulfide, dispersing the cerium-doped tin disulfide into 1.5 mL of deionized water to prepare a solution, then adding 15 mu L of luminol with the concentration of 5 mmol/L, oscillating for 18 h, obtaining a precipitate through centrifugal separation, and finally dispersing the precipitate into 1 mL of deionized water again to obtain a target solution cerium-doped tin disulfide-luminol solution;
to the above solution, 300. mu.L of detection antibody Ab at a concentration of 10 mg/mL was added2Incubating the solution at 4 ℃ for 18 h with shaking, centrifuging, and dispersing into 1 mL of phosphate buffer solution with pH 7.4 to obtain detection antibody Ab2The marked cerium doped tin disulfide-luminol solution is stored at 4 ℃ for later use.
Example 10 a sensor was used for the detection of procalcitonin by the following steps:
(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to be 800V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-0.7V, and the scanning rate is set to be 0.1V/s;
(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared in claim 1 as a working electrode, immersing the sensor for incubating a series of procalcitonin with standard concentration into 10 mL of phosphate buffer solution containing 35 mmol/L of hydrogen peroxide for electrochemiluminescence test to obtain corresponding electrochemiluminescence signal intensity when incubating procalcitonin with different concentrations, and drawing a working curve, wherein the detection limit is 1.2 fg/mL, and the linear range is 5 fg/mL-50 ng/mL;
(3) And testing the electrochemiluminescence sensor for incubating the procalcitonin actual sample with unknown concentration to obtain corresponding signal intensity, and calculating according to the working curve to obtain the procalcitonin concentration in the actual sample. Example 11 a sensor was used for the detection of procalcitonin by the following steps:
(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to be 800V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-0.7V, and the scanning rate is set to be 0.1V/s;
(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared in claim 1 as a working electrode, immersing the sensor for incubating a series of procalcitonin with standard concentration into 10 mL of phosphate buffer solution containing 55 mmol/L of hydrogen peroxide for electrochemiluminescence test to obtain corresponding electrochemiluminescence signal intensity when incubating procalcitonin with different concentrations, and drawing a working curve, wherein the detection limit is 1.2 fg/mL, and the linear range is 5 fg/mL-50 ng/mL;
(3) and testing the electrochemiluminescence sensor for incubating the procalcitonin actual sample with unknown concentration to obtain corresponding signal intensity, and calculating according to the working curve to obtain the procalcitonin concentration in the actual sample.
Example 12 a sensor was used for the detection of procalcitonin by the following steps:
(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to be 800V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-0.7V, and the scanning rate is set to be 0.1V/s;
(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared in claim 1 as a working electrode, immersing the sensor for incubating a series of procalcitonin with standard concentration into 10 mL of phosphate buffer solution containing 65 mmol/L of hydrogen peroxide for electrochemiluminescence test to obtain corresponding electrochemiluminescence signal intensity when incubating procalcitonin with different concentrations, and drawing a working curve, wherein the detection limit is 1.2 fg/mL, and the linear range is 5 fg/mL-50 ng/mL;
(3) and testing the electrochemiluminescence sensor for incubating the procalcitonin actual sample with unknown concentration to obtain corresponding signal intensity, and calculating according to the working curve to obtain the procalcitonin concentration in the actual sample.
Claims (4)
1. A preparation method of a graphene/polyaniline nanowire array immunosensor is characterized by comprising the following steps:
(1) Polishing glassy carbon electrodes with the diameter of 4 mm by using alumina with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished glassy carbon electrodes with ultrapure water;
(2) dropping 6 mu L of capture antibody Ab with the concentration of 2-4 mg/mL on the surface of the glassy carbon electrode1Placing the marked palladium hybridized graphene/polyaniline solution at 4 ℃ for airing;
(3) dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 1-3% to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode by phosphate buffer solution with the pH of 7.4, and airing at 4 ℃;
(4) dripping 6 mu L of procalcitonin standard solution or solution with unknown concentration, incubating for 0.5-2 h at 37 ℃, washing the surface of the electrode by using phosphate buffer solution with pH of 7.4, and airing at 4 ℃;
(5) dripping 6 mu L of the mixture with the concentration of 2-4 mg/mLAb detection antibody2And (3) washing the surface of the electrode by using a marked cerium-doped tin disulfide-luminol solution and a phosphate buffer solution with the pH value of 7.4, placing the electrode at 4 ℃, airing, and finishing the construction of the sensor.
2. The method for preparing the graphene/polyaniline nanowire array immunosensor according to claim 1, wherein the capture antibody Ab1A labeled palladium-hybridized graphene/polyaniline solution is prepared by the following steps:
Adding 30-50 mL of deionized water into 0.13-0.15 g of sodium tetrachloropalladate and 0.13-0.15 g of sodium citrate solid, uniformly dissolving the mixed solid, then dropwise adding 0.5-0.7 mL of sodium borohydride solution into the mixed solution, finally, continuously stirring to obtain black palladium nanoparticles, and storing at 4 ℃ in a dark place;
adding 0.4-0.6 g of graphene oxide into a beaker containing 90-110 mL of anhydrous dimethylformamide, then adding 2.78-2.98 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into the upper suspension under the condition of ice-water bath, stirring for 1-3 h under the nitrogen atmosphere, then adding 1.8-2.0 mL of 1, 3-diaminopropane, stirring overnight at room temperature, finally washing with water and ethanol respectively, and drying to obtain aminated graphene oxide;
dispersing 5-15 mg of aminated graphene oxide in 5-15 mL of hydrochloric acid solution, performing ultrasonic treatment for 15-45 min, uniformly dispersing the aminated graphene oxide, cooling the suspension in an ice bath to 0 ℃, then adding 0.4-0.6 mL of aniline into the aminated graphene oxide, continuously stirring, slowly adding 5-15 mL of solution dissolved with 1.25-1.45 g of potassium persulfate, after the reaction lasts for 4-6 h in the ice bath, adding 0.1 mol/L hydrochloric acid solution to remove all unreacted reactants, washing 3 times with deionized water and hexane respectively, and drying for 12 h to obtain graphene/polyaniline solution;
Mixing 1-3 mL of graphene/polyaniline solution with the concentration of 2 mg/mL and 1-3 mL of palladium nanoparticle solution, oscillating for 24-48 h in a dark place, centrifugally separating, discarding supernatant, dispersing into 1 mL of ultrapure water, and preparing palladium impurityThe graphene/polyaniline solution is dissolved, and then 50-150 uL of capture antibody Ab with the concentration of 1 ug/mL is added1After the solution is shaken for 6-18 h, the solution is dispersed into 2 mL phosphate buffer solution with pH of 7.4 after centrifugal separation to obtain a capture antibody Ab1A labeled palladium-hybridized graphene/polyaniline solution.
3. The method for preparing the graphene/polyaniline nanowire array immunosensor according to claim 1, wherein the detection antibody Ab2A labeled cerium doped tin disulfide-luminol solution prepared by the steps of:
completely dissolving 0.01-0.03 mol/L tin pentahydrate tetrachloride, 0.001-0.003 mol/L cerium nitrate hexahydrate, 0.05-0.07 mol/L thioacetamide and 0.4-0.6 g hexadecyltrimethylammonium bromide in a mixture of ethanol and distilled water, transferring the mixture into a 200 mL high-temperature reaction kettle with a polytetrafluoroethylene lining, keeping the temperature at 180 ℃, reacting for 24-48 h, and then cooling to room temperature; centrifugally washing the obtained solid for multiple times, drying the solid in a vacuum drying oven at 60 ℃ for 12-36 hours, and then sintering the solid for 2-4 hours at 500 ℃ under the protection of argon to obtain cerium-doped tin disulfide solid;
Weighing 1-3 mg of cerium-doped tin disulfide, dispersing the cerium-doped tin disulfide into 0.5-1.5 mL of deionized water to prepare a solution, then adding 5-15 mu L of luminol with the concentration of 5 mmol/L, oscillating for 6-18 h, obtaining a precipitate through centrifugal separation, and finally dispersing the precipitate into 1 mL of deionized water again to obtain a target solution cerium-doped tin disulfide-luminol solution;
adding 100-300 mu L of detection antibody Ab with the concentration of 10 mg/mL into the solution2The solution is incubated for 6-18 h under the condition of oscillation at 4 ℃, centrifuged and dispersed into 1 mL of phosphate buffer solution with pH of 7.4 to obtain a detection antibody Ab2The marked cerium doped tin disulfide-luminol solution is stored at 4 ℃ for later use.
4. The application of the sensor obtained by the preparation method of the graphene/polyaniline nanowire array immunosensor according to claim 1 in procalcitonin detection is characterized in that the detection steps are as follows:
(1) setting parameters: the high voltage of a photomultiplier of the ultra-weak electrochemiluminescence instrument is set to be 800V, the cyclic volt-ampere scanning potential range of the electrochemical workstation is set to be 0-0.7V, and the scanning rate is set to be 0.1V/s;
(2) and (3) testing: taking a silver/silver chloride electrode as a reference electrode, a platinum wire electrode as a counter electrode, taking the sensor prepared in claim 1 as a working electrode, immersing the sensor for incubating a series of procalcitonins with standard concentrations into 10 mL of phosphate buffer solution containing 35-65 mmol/L of hydrogen peroxide for performing electrochemiluminescence test to obtain corresponding electrochemiluminescence signal intensity when the procalcitonins with different concentrations are incubated, and drawing a working curve, wherein the detection limit is 1.2 fg/mL, and the linear range is 5 fg/mL-50 ng/mL;
(3) And testing the electrochemiluminescence sensor for incubating the procalcitonin actual sample with unknown concentration to obtain corresponding signal intensity, and calculating according to the working curve to obtain the procalcitonin concentration in the actual sample.
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