CN111766289B - Oxygen-enriched vacancy CeO 2 Preparation method of electrochemiluminescence immunosensor - Google Patents
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Abstract
The invention relates to a CeO based on an oxygen-enriched vacancy 2 A preparation method of an electrochemiluminescence immunosensor belongs to the field of novel nano materials and the technical field of biosensing; firstly provides a high-concentration oxygen vacancy reinforced CeO 2 The method for preparing oxygen-rich vacant CeO by sodium borohydride normal temperature reduction method 2 The high concentration of oxygen vacancies can improve CeO 2 Electronic structure for remarkably enhancing electron mobility thereof, and CeO prepared by conventional method 2 Compared with the nanometer material, oxygen-rich vacancy CeO 2 The invention has higher luminous efficiency, and based on the excellent conductivity and biocompatibility of the nano-gold, the nano-gold is used for functionalizing the oxygen-rich vacancy CeO 2 The non-marker immunosensor is developed as a signal source and applied to the actual sample detection of the non-small cell lung cancer disease marker CYFRA21-1, the detection limit is 25 pg/mL, the linear range is 50 pg/mL-50 ng/mL, and the non-small cell lung cancer immunosensor has obvious potential application value in the early diagnosis of non-small cell lung cancer.
Description
Technical Field
The invention belongs to the field of novel nano materials and the technical field of biosensing.
Background
CYFRA21-1 is a soluble fragment of cytokeratin 19, is considered to be a tumor marker mainly used for detecting lung cancer, and has important value particularly for diagnosing non-small cell lung cancer. If unclear annular shadows exist in the lung, the concentration of CYFRA21-1 in serum is higher than 30 ng/mL, the possibility of primary bronchogenic carcinoma is very high, the positive detection rate of various non-small cell lung cancers is 70% -85%, the serum concentration level of CYFRA21-1 is positively correlated with the clinical stage of tumors, the serum concentration level can also be used as an effective index for tracking early relapse after lung cancer surgery and radiotherapy and chemotherapy, the serum high concentration level of CYFRA21-1 indicates that the disease is in the progressive stage and poor prognosis, the marker of successful quality is that the serum concentration of CYFRA21-1 is rapidly reduced, otherwise, the disease is not completely cleared, and the sensitivity of CYFRA21-1 to diagnosis of various lung cancers is sequentially as follows: squamous carcinoma > adenocarcinoma > large cell carcinoma > small cell carcinoma. So far, only several analysis methods such as fluorescence, electrochemical analysis, and chromatography have been developed. Therefore, a simple, rapid and accurate determination method is developed, and the method has important significance for the instant detection of CYFRA 21-1.
As a research hotspot which is raised by the interdigitation of various subjects such as biology, chemistry, medicine, electronic technology and the like, the electrochemiluminescence immunoassay is the organic combination of the electrochemiluminescence technology and an immunoassay method, and the prepared immunosensor has the advantages of low cost, good selectivity, high sensitivity, high analysis speed, easiness in automation, miniaturization, integration and the like, and is widely applied to the fields of disease marker analysis, food safety analysis, environmental pollution analysis and the like.
In recent years, more and more semiconductor nanomaterialsThe electrochemiluminescence properties are found, for example, in CeO 2 、TiO 2 However, their poor electrochemical activity and conductivity are disadvantageous, which makes their luminous efficiency unsatisfactory and limits their application in electrochemiluminescence sensors. Therefore, the invention provides a high-concentration oxygen vacancy reinforced CeO 2 The method for preparing oxygen-rich vacancy CeO by sodium borohydride normal-temperature reduction method 2 The high concentration of oxygen vacancies may improve CeO 2 Electronic structure for remarkably enhancing electron mobility thereof, and CeO prepared by conventional method 2 Compared with the nanometer material, oxygen-rich vacancy CeO 2 The high-efficiency and stable-luminous-efficiency electrochemical sensor has higher and stable luminous efficiency, can realize high-efficiency and stable output of signals by using the electrochemical sensor as a signal source to construct an electrochemical sensor, and is favorable for realizing rapid and sensitive detection of CYFRA21-1 in serum.
Disclosure of Invention
One of the tasks of the present invention is to broaden the CeO 2 The application of semiconductor oxide nano material in electrochemiluminescence sensing is to provide a high-concentration oxygen vacancy reinforced CeO 2 Method for electrochemiluminescence properties, which method allows significant enhancement of CeO 2 The luminous efficiency of the material greatly improves the application of the material in immunosensing;
the second technical task of the invention is to make up the defects of the existing CYFRA21-1 detection technology and provide CeO based on an oxygen-rich vacancy 2 The electrochemiluminescence immunosensor can be used for rapidly detecting CYFRA21-1, has the advantages of high sensitivity, strong specificity and good reproducibility, and has the detection limit of 25 pg/mL and the linear range of 50 pg/mL-50 ng/mL.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
(1) nano gold functionalized oxygen-enriched vacancy CeO 2 Preparation of the solution
Firstly, completely dissolving 5-8 mmol of asparagine in 40-60 mL of ultrapure water, stirring for 15 min at 45-60 ℃, then adding 5-8 mmol of cerium trichloride heptahydrate, continuously stirring for 10 min, and then transferring the obtained product to 50 mL of polytetrafluoroethyleneAnd (3) in a high-temperature reaction kettle with the lining, keeping the temperature at 160-180 ℃, 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 40-80 ℃ for 12-36 hours, and sintering the solid at 360 ℃ for 1-3 hours to obtain bright yellow CeO 2 Dissolving 80-100 mg of product in 10 mL of ultrapure water to prepare 2-4 mg/mL CeO nanoparticles 2 Adding 1 mol/L sodium borohydride solution into the solution, performing ultrasonic treatment for 3 hours, performing centrifugal separation, washing and drying to obtain oxygen-enriched vacancy CeO 2 A nanomaterial;
preparing oxygen-enriched vacancy CeO with concentration of 2-4 mg/mL 2 Adding 1-3 mL of nano gold sol into the solution, vibrating and combining for 12 h, and then carrying out centrifugal separation to obtain nano gold functionalized oxygen-enriched vacancy CeO 2 A solution;
(2) polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder with the diameter of 1.0 micron, 0.3 micron and 0.05 micron in sequence, and washing the polished electrodes with ultrapure water;
(3) dripping 6 mu L of nano-gold functionalized oxygen-enriched vacancy CeO with the concentration of 2-4 mg/mL on the surface of a glassy carbon electrode 2 The solution is used as a sensing substrate and is placed at 37 ℃ for airing;
(4) dripping 6 mu L of CYFRA21-1 antibody solution with the concentration of 100 mu g/mL, washing the surface of the electrode by phosphate buffer solution PBS with the pH value of 7.4, placing the electrode at 4 ℃, and airing;
(5) 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 PBS with the pH of 7.4, and placing the electrode at 4 ℃ for airing;
(6) dripping 6 mu L of CYFRA21-1 standard solution with a certain concentration, incubating for 0.5-2 h at 37 ℃, washing the surface of the electrode by phosphate buffer solution PBS with pH 7.4, placing the electrode at 4 ℃, airing, and finishing the construction of the sensor.
Advantageous results of the invention
(1) Since CeO 2 The application of the semiconductor oxide nano material in the field of electrochemiluminescence is limited due to low luminous efficiency of the semiconductor oxide nano material, and the invention firstly providesHigh-concentration oxygen vacancy reinforced CeO 2 The method for improving the electrogenerated chemiluminescence property can obviously improve CeO by introducing a high-concentration oxygen vacancy through a sodium borohydride reduction method 2 The luminous efficiency of the CeO is improved 2 Further application and research of similar semiconductor nano materials in the field of electrochemiluminescence;
(2) the invention promotes CeO based on oxygen-enriched vacancy 2 The principle of electrochemiluminescence efficiency provides a novel and reliable immunosensing technology. The method solves the problems of complex operation, low sensitivity and poor reproducibility of the existing electrochemical detection technology, has the detection limit of 25 pg/mL and the linear range of 50 pg/mL-50 ng/mL when being applied to the sample detection of CYFRA21-1, and has the advantages of high response speed, high sensitivity, good reproducibility, simplicity in preparation, low cost and environmental friendliness.
Detailed Description
The invention will now be further illustrated by reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
Example 1 CeO based on oxygen-rich vacancies 2 The preparation method of the electrochemiluminescence immunosensor is characterized by comprising the following steps:
(1) nano gold functionalized oxygen-enriched vacancy CeO 2 Preparation of the solution
First, asparagine with a molar mass of 5 mmol was completely dissolved in 40 mL of ultrapure water and stirred at 45 ℃ for 15 min, then cerium trichloride heptahydrate with a molar mass of 5 mmol was added and stirring was continued for 10 min, and then the resultant was transferred to a polytetrafluoroethylene-lined high-temperature reaction vessel with a volume of 50 mL, the temperature was maintained at 160 ℃, reacted for 24 h, and then cooled to room temperature. The obtained solid is centrifugally washed for multiple times, dried in a vacuum drying oven at 40 ℃ for 12 hours and then sintered at 360 ℃ for 1 hour to obtain bright yellow CeO 2 The nanometer particles are prepared by dissolving 80 mg of product in 10 mL of ultrapure water to prepare CeO with the concentration of 2 mg/mL 2 Adding 1 mol/L sodium borohydride solution into the solution, performing ultrasonic treatment for 3 hours, performing centrifugal separation, washing and drying to obtain oxygen-enriched vacancy CeO 2 A nanomaterial;
preparation of oxygen-rich vacancy CeO with concentration of 2 mg/mL 2 Adding 1 mL of nano gold sol into the solution, vibrating and combining for 12 h, and then carrying out centrifugal separation to obtain nano gold functionalized oxygen-enriched vacancy CeO 2 A solution;
(2) polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder 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;
(3) 6 mu L of nano-gold functionalized oxygen-enriched vacancy CeO with the concentration of 2 mg/mL is dripped on the surface of the glassy carbon electrode 2 The solution is used as a sensing substrate and is placed at 37 ℃ for airing;
(4) dripping 6 mu L of CYFRA21-1 antibody solution with the concentration of 100 mu g/mL, washing the surface of the electrode by phosphate buffer solution PBS with the pH value of 7.4, placing the electrode at 4 ℃, and airing;
(5) 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 by phosphate buffer solution PBS with pH of 7.4, and placing the electrode at 4 ℃ for airing;
(6) dripping 6 μ L of CYFRA21-1 standard solution with a certain concentration, incubating at 37 deg.C for 0.5 h, washing electrode surface with phosphate buffer solution PBS with pH of 7.4, air drying at 4 deg.C, and completing sensor construction.
Example 2 oxygen-rich vacancy-based CeO 2 The preparation method of the electrochemiluminescence immunosensor is characterized by comprising the following steps:
(1) nano gold functionalized oxygen-enriched vacancy CeO 2 Preparation of the solution
First, asparagine having a molar mass of 6 mmol was completely dissolved in 50 mL of ultrapure water and stirred at 50 ℃ for 15 min, then cerium trichloride heptahydrate having a molar mass of 6 mmol was added and stirring was continued for 10 min, and then the resultant was transferred to a polytetrafluoroethylene-lined high-temperature reaction vessel having a volume of 50 mL, the temperature was maintained at 170 ℃, reacted for 36 h, and then cooled to room temperature. Centrifugally washing the obtained solid for multiple times, drying the solid in a vacuum drying oven at 40-80 ℃ for 24 hours, sintering the solid at 360 ℃ for 2 hours,obtaining bright yellow CeO 2 The nanometer particles are prepared by dissolving 90 mg of product in 10 mL of ultrapure water to prepare CeO with the concentration of 3 mg/mL 2 Adding 1 mol/L sodium borohydride solution into the solution, performing ultrasonic treatment for 3 hours, performing centrifugal separation, washing and drying to obtain oxygen-enriched vacancy CeO 2 A nanomaterial;
preparation of oxygen-rich vacancy CeO with concentration of 3 mg/mL 2 Adding 2 mL of nano-gold sol into the solution, vibrating and combining for 12 h, and then carrying out centrifugal separation to obtain nano-gold functionalized oxygen-enriched vacancy CeO 2 A solution;
(2) polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder 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;
(3) 6 mu L of nano-gold functionalized oxygen-enriched vacancy CeO with the concentration of 3 mg/mL is dripped on the surface of the glassy carbon electrode 2 The solution is used as a sensing substrate and is placed at 37 ℃ for airing;
(4) dripping 6 mu L of CYFRA21-1 antibody solution with the concentration of 100 mu g/mL, washing the surface of the electrode by phosphate buffer solution PBS with the pH value of 7.4, and placing the electrode at 4 ℃ for airing;
(5) 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 phosphate buffer solution PBS (pH 7.4), and airing at 4 ℃;
(6) dripping 6 μ L of CYFRA21-1 standard solution with a certain concentration, incubating at 37 deg.C for 1 h, washing the electrode surface with phosphate buffer solution PBS with pH of 7.4, air drying at 4 deg.C, and finishing the construction of the sensor.
Example 3 oxygen-rich vacancy-based CeO 2 The preparation method of the electrochemiluminescence immunosensor is characterized by comprising the following steps of:
(1) nano gold functionalized oxygen-enriched vacancy CeO 2 Preparation of the solution
Firstly, completely dissolving asparagine with the molar mass of 8 mmol in 40-60 mL of ultrapure water, stirring for 15 min at 60 ℃, then adding cerous chloride heptahydrate with the molar mass of 8 mmol, continuously stirring for 10 min, and then adding cerous chloride heptahydrate with the molar mass of 8 mmolThe resultant was transferred to a polytetrafluoroethylene-lined high-temperature reaction kettle having a volume of 50 mL, the temperature was maintained at 180 ℃, reacted for 48 hours, and then cooled to room temperature. The obtained solid is centrifugally washed for multiple times, dried in a vacuum drying oven at 80 ℃ for 36 hours and then sintered at 360 ℃ for 3 hours to obtain bright yellow CeO 2 The nanometer particles are prepared by dissolving 100 mg of product in 10 mL of ultrapure water to prepare 4 mg/mL CeO 2 Adding 1 mol/L sodium borohydride solution into the solution, performing ultrasonic treatment for 3 hours, performing centrifugal separation, washing and drying to obtain oxygen-enriched vacancy CeO 2 A nanomaterial;
preparation of oxygen-rich vacancy CeO with concentration of 4 mg/mL 2 Adding nano gold sol with the volume of 3 mL into the solution, vibrating and combining for 12 h, and then carrying out centrifugal separation to obtain nano gold functionalized oxygen-enriched vacancy CeO 2 A solution;
(2) polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder 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;
(3) 6 mu L of nano-gold functionalized oxygen-enriched vacancy CeO with the concentration of 4 mg/mL is dripped on the surface of the glassy carbon electrode 2 The solution is used as a sensing substrate and is placed at 37 ℃ for airing;
(4) dripping 6 mu L of CYFRA21-1 antibody solution with the concentration of 100 mu g/mL, washing the surface of the electrode by phosphate buffer solution PBS with the pH value of 7.4, and placing the electrode at 4 ℃ for airing;
(5) 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 PBS with the pH of 7.4, and placing the electrode at 4 ℃ for airing;
(6) dripping 6 μ L of CYFRA21-1 standard solution with a certain concentration, incubating at 37 deg.C for 2 h, washing electrode surface with phosphate buffer solution PBS with pH of 7.4, air drying at 4 deg.C, and finishing sensor construction.
Claims (1)
1. Oxygen-enriched vacancy CeO 2 The preparation method of the electrochemiluminescence immunosensor is characterized by comprising the following steps:
(1) nano gold functionalized oxygen-enriched vacancy CeO 2 Preparation of the solution
Firstly, completely dissolving 5-8 mmol of asparagine in molar mass in 40-60 mL of ultrapure water, stirring for 15 min at 45-60 ℃, then adding 5-8 mmol of cerium trichloride heptahydrate, continuously stirring for 10 min, then transferring the obtained product into a high-temperature reaction kettle with a 50 mL polytetrafluoroethylene lining, keeping the temperature at 160-180 ℃, reacting for 24-48 h, cooling to room temperature, centrifugally washing the obtained solid for multiple times, drying for 12-36 h in a vacuum drying box at 40-80 ℃, and sintering for 1-3 h at 360 ℃ to obtain bright yellow CeO 2 Dissolving 80-100 mg of product in 10 mL of ultrapure water to prepare 2-4 mg/mL CeO nanoparticles 2 Adding 1 mol/L sodium borohydride solution into the solution, performing ultrasonic treatment for 3 hours, performing centrifugal separation, washing and drying to obtain oxygen-enriched vacancy CeO 2 A nanomaterial;
preparing oxygen-enriched vacancy CeO with concentration of 2-4 mg/mL 2 Adding 1-3 mL of nano gold sol into the solution, vibrating and combining for 12 h, and then carrying out centrifugal separation to obtain nano gold functionalized oxygen-enriched vacancy CeO 2 A solution;
(2) polishing glassy carbon electrodes with the diameter of 4 mm by using alumina polishing powder 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;
(3) dripping and coating 6 mu L of nano-gold functionalized oxygen-enriched vacancy CeO with concentration of 2-4 mg/mL on the surface of a glassy carbon electrode 2 The solution is used as a sensing substrate and is placed at 37 ℃ for airing;
(4) dripping 6 mu L of CYFRA21-1 antibody solution with the concentration of 100 mu g/mL, washing the surface of the electrode by phosphate buffer solution PBS with the pH value of 7.4, and placing the electrode at 4 ℃ for airing;
(5) 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 PBS with the pH of 7.4, and placing the electrode at 4 ℃ for airing;
(6) dripping 6 mu L of CYFRA21-1 standard solution with a certain concentration, incubating for 0.5-2 h at 37 ℃, washing the surface of the electrode by phosphate buffer solution PBS with pH 7.4, placing the electrode at 4 ℃, airing, and finishing the construction of the sensor.
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