CN113514512B - g-C based 3 N 4 Preparation and application of electrochemical luminescence sensor of CoPd - Google Patents

g-C based 3 N 4 Preparation and application of electrochemical luminescence sensor of CoPd Download PDF

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CN113514512B
CN113514512B CN202110471542.7A CN202110471542A CN113514512B CN 113514512 B CN113514512 B CN 113514512B CN 202110471542 A CN202110471542 A CN 202110471542A CN 113514512 B CN113514512 B CN 113514512B
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copdnps
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胡丽华
师腾飞
崔倩倩
宋翠
张勇
吴丹
马洪敏
范大伟
魏琴
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University of Jinan
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Abstract

The invention relates to a porous g-C-based catalyst 3 N 4 A preparation method of an electrochemical luminescence sensor of CoPdNPs belongs to the technical field of novel sensor construction. Based on good specificity between antigen and antibody, the sensor utilizes porous g-C modified by CoPd alloy nano particles 3 N 4 Composite g-C 3 N 4 The CoPdNPs are taken as a substrate luminescent material, the Fe MOFs is loaded with small-size CuO as a quencher, and the electrochemical luminescence sensor is constructed by layer-by-layer assembly. The electrochemical luminescence sensor constructed by the invention has wider detection range, higher sensitivity and lower detection limit, and has important significance for detecting the neuroenolase.

Description

g-C based 3 N 4 Preparation and application of electrochemical luminescence sensor of CoPd
Technical Field
The invention relates to a g-C based 3 N 4 -a method for preparing an electrochemical luminescence sensor of the CoPdNPs. The invention adopts CoPd alloy nano particle modified porous g-C 3 N 4 Composite g-C 3 N 4 The specific detection of the neuroenolase can be realized by using the CoPdNPs as a substrate luminescent material, using Fe MOFs loaded with small-size CuO as a quencher and using the electrochemical luminescence sensor constructed by layer-by-layer assembly, and the method belongs to the technical field of novel sensor construction.
Background
Lung cancer is one of the most deadly cancers known, causing a large number of deaths worldwide, and is largely classified into Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC). Small cell lung cancer is known for its early metastatic propensity and rapid doubling time, and is difficult to treat due to its initial high sensitivity to chemotherapy, frequent recurrence and resistance to chemotherapy. Studies report that neuron-specific enolase (NSE) is a reliable, specific, sensitive serum biomarker for early diagnosis of SCLC, and can assess rehabilitation progression in patients. Thus, the determination of NSE levels is of great importance for monitoring the progression and therapeutic effect of SCLC in clinical diagnosis. Electrochemical luminescence is an emerging product combining electrochemical and luminescent technologies, has the advantages of low background noise, wide dynamic range, simple and convenient instrument and equipment, high sensitivity and the like, and is widely focused in the fields of biological analysis, food safety analysis, environmental pollution monitoring and the like.
The invention constructs a novel electrochemical luminescence sensor based on nano functional materials, which is used for detecting the nervonic enolase. Porous g-C modified by CoPd alloy nano particles 3 N 4 Composite g-C 3 N 4 The detection of the neuroenolase is realized by taking CoPdNPs as a substrate luminescent material and Fe MOFs loaded with small-size CuO as a quencher. The test result shows that the electrochemical luminescence sensor has high sensitivity, low detection limit and good stability. Based on the above findings, the inventors have completed the present invention.
Disclosure of Invention
One of the objects of the present invention is a porous g-C based on CoPd alloy nanoparticle modification 3 N 4 Composite g-C 3 N 4 The CoPdNPs are taken as a substrate luminescent material, the Fe MOFs is loaded with small-size CuO as a quencher, and a novel electrochemical luminescence sensor is constructed.
The second purpose of the invention is to provide a novel preparation method of the electrochemical luminescence sensor based on resonance energy transfer, and the sensor prepared by the method has good stability, good selectivity, high sensitivity and good reproducibility.
The third object of the invention is to realize the construction of the electrochemical luminescence sensor and the effective detection of the nervonase, and to achieve the purpose of the electrochemical luminescence sensor in the measurement of the nervonase.
1. Porous g-C based on CoPd alloy nanoparticle modification 3 N 4 Porous g-C of composite material 3 N 4 Method for preparing electrochemical luminescence sensor of-CoPdNPs
(1) With Al 2 O 3 Polishing powder polishes glassy carbon electrode with diameter of 4mm, and cleaning with ultrapure water, and cleaning with 6 mu L and 0.5-3 mg mL -1 Porous g-C modified by CoPd alloy nano particles 3 N 4 Porous g-C of composite material 3 N 4 Porous g-C as primary antibody label binding to CoPdNPs and to a neuroenolase-recognizing antibody 3 N 4 -CoPdNPs-Ab 1 Dropwise adding the solution onto the surface of the electrode, and airing at room temperature to form a film;
(2) 3 mu L of BSA solution with the mass fraction of 0.1% is dripped on the surface of the electrode, the electrode is washed by ultrapure water, and the electrode is dried at room temperature;
(3) Dripping 6 mu L and 0.00005~100 ng mL -1 A series of different concentrations of the neuroenolase antigen are added on the surface of the electrode, incubated 2h, flushed by ultrapure water and dried at room temperature;
(4) Small-size CuO loaded Fe MOFs-sCuO-Ab combined with secondary antibody marker of neuroenolase recognition antibody and dropwise added with 6 mu L Fe MOFs 2 Washing the solution with ultrapure water, and airing at room temperature to prepare an electrochemical luminescence sensor;
the Fe MOFs loads small-size CuO and binds to a secondary antibody marker Fe MOFs-sCuO-Ab of a neuroenolase recognition antibody 2 The preparation steps of the solution are as follows:
(1) Preparation of Fe MOFs
0.126g of 2-amino terephthalic acid and 0.187g of FeCl 3 ·6H 2 O and 3.0. Mu.L of acetic acid were dissolved in 15mL of DMF, the mixed solution was transferred to a reaction vessel, and then heated at 120℃for 4. 4h to give a crude product at 8000r min -1 Centrifuging, washing 3 times with DMF, washing 1 time with ethanol, and drying overnight in a vacuum drying oven at 60 ℃ to obtain Fe MOFs;
(2) Preparation of small-sized CuO (sCuO)
150mL of 0.02M Cu (CH) 3 COO) 2 ·H 2 Mixing O aqueous solution with 0.5. 0.5mL glacial acetic acid, and placing in round bottom with reflux deviceIn a flask, the solution was heated to boiling with vigorous stirring and then 10mL of 0.04g mL was added rapidly -1 Immediately forming a large amount of black precipitate, centrifuging the precipitate, washing the precipitate with absolute ethyl alcohol three times, and drying the precipitate in a vacuum oven for 12h to obtain sCuO;
(3) Fe MOFs-sCuO-Ab loaded with small-sized CuO and combined with secondary antibody of neuroenolase recognition antibody 2 Preparation of the solution
Dissolving 40mg small-sized copper oxide and 20mg Fe MOFs in 150mL ethanol, uniformly dispersing by ultrasonic waves, stirring overnight, centrifuging, washing with absolute ethyl alcohol three times, and drying overnight in a vacuum oven at 60 ℃ to obtain Fe MOFs-sCuO;
100 mu L of the mixture was concentrated to 10 mu g mL -1 Is a neuroenolase-recognizing antibody Ab 2 Added to 1.5mg mL of 1mL -1 In Fe MOFs-sCuO water solution, and vibrating and incubating for 12h at 4 ℃, dispersing the obtained product in PBS with pH of 7.4 and 1mL after centrifugation, thus obtaining the secondary antibody marker Fe MOFs-sCuO-Ab with Fe MOFs loaded with small-size CuO and combined with a neuroenolase recognition antibody 2 A solution.
2. CoPd alloy nanoparticle modified porous g-C 3 N 4 Composite g-C 3 N 4 Porous g-C as primary antibody label binding to CoPdNPs and to a neuroenolase-recognizing antibody 3 N 4 -CoPdNPs-Ab 1 Preparation of the solution
(1) Porous g-C 3 N 4 Is prepared from
Takes melamine as raw material and SiO with the grain diameter of 10nm 2 The ball is used as a template, and the g-C with a porous structure is prepared by a thermal condensation method 3 N 4 2g melamine and 1g SiO are first reacted 2 Ball milling, mixing, transferring into crucible with cover, and standing at 550deg.C for 5 min −1 Heating 4h at a heating rate to obtain g-C with core-shell structure 3 N 4 @SiO 2 Next, g-C 3 N 4 @SiO 2 Soaking in 30mL 10% HF aqueous solution for 6h, washing with water for several times to remove SiO 2 Obtaining porous g-C 3 N 4 ；
(2) CoPd alloy nanoparticle modified porous g-C 3 N 4 Composite g-C 3 N 4 Preparation of CoPdNPs
0.35mmol of cobalt acetylacetonate and 0.30mmol of palladium dibromide are dissolved in 18mL of oily amine, the mixture is then stirred, heated to 60℃under nitrogen, then 0.5mL of tri-n-octylphosphine is added, the colour of the mixture changes from pink to dark green, the solution is stirred at 5℃for a period of minutes -1 Is heated to 260C and maintained at this temperature for 2h C. Subsequently, the solution was cooled to room temperature, 40mL ethanol was added, and centrifuged to give CoPdNPs, and in addition, 250mg porous g-C 3 N 4 Dissolving in 20mL ethanol, ultrasonic treating with the prepared CoPdNPs dispersion, and mixing with porous g-C 3 N 4 Mixing and stirring overnight, and finally at 7000r min -1 Centrifuging, washing with ethanol for several times, dispersing in 10mL ethanol, and preserving at 4deg.C;
(3) Porous g-C 3 N 4 Porous g-C as primary antibody label for binding-CoPdNPs to recognition antibody of neuroenolase 3 N 4 -CoPdNPs-Ab 1 Preparation of the solution
Will 10 mu g mL -1 The primary antibody of the neuroenolase-capturing antibody of (C) was activated at 4℃using EDC, NHS, and then reacted with porous g-C 3 N 4 CoPdNPs are hatched together with 6h to obtain a primary antibody marker porous g-C 3 N 4 -CoPdNPs-Ab 1 A solution.
3. Detection of a neuroenolase
(1) The Ag/AgCl is used as a reference electrode, the platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cartridge of a chemiluminescent detector, an electrochemical workstation is connected with the chemiluminescent detector, the high voltage of a photomultiplier is set to 600V, and the electrochemical luminescence sensor contains 20-100mmol L -1 The test was performed in PBS buffer solution with ph=7.4 of potassium persulfate;
(2) Detecting the standard solution of the nervonase by using an electrochemiluminescence method, wherein the voltage test range is-1.5-0V;
(3) And (3) observing the electrochemiluminescence intensity of the sensor before and after the addition of the nervonase, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the nervonase, and drawing a working curve.
(1) The inventor of the invention modifies the CoPd alloy nano particles into porous g-C 3 N 4 Composite g-C 3 N 4 The CoPdNPs are used as a substrate luminescent material, and the Fe MOFs is loaded with small-size CuO to be used as a quencher in the preparation of the electrochemical luminescence sensor. Porous g-C 3 N 4 Is greatly increased in g-C 3 N 4 Becomes a good carrier for loading CoPdNPs, and the CoPd alloy nano particles have good conductivity and promote the electron transfer on the surface of the electrode material. In addition, the CoPdNPs have good biocompatibility, can firmly combine with the capturing antibody of the nervonase, and promote the porous g-C 3 N 4 Is provided. The synthesized Fe MOFs can load small-size CuO in high quantity, has quenching effect and can increase the fixed quantity of the recognition antibody. The properties of these materials improve the sensitivity and stability of the sensor.
(2) The invention adopts a novel donor-acceptor pair for resonance energy transfer to construct a sandwich-type electrochemical luminescence sensor, and effectively detects the neuroenolase, and the method is simpler to operate.
(3) The electrochemical luminescence sensor prepared by the invention is used for detecting the nervonase, has high stability, good reproducibility, high sensitivity and wide linear range, and can realize simple, rapid, high-sensitivity and specific detection.
Description of the embodiments
Example 1 g-C based 3 N 4 Method for preparing electrochemical luminescence sensor of-CoPdNPs
(1) With Al 2 O 3 Polishing powder polishes a glassy carbon electrode with the diameter of 4mm, and cleaning the glassy carbon electrode with ultrapure water, wherein the volume of the glassy carbon electrode is 6 mu L and the volume of the glassy carbon electrode is 0.5 mg mL -1 Porous g-C of primary antibody marker of (2) 3 N 4 -CoPdNPs-Ab 1 Dropping the solution into electricityThe polar surface is dried to form a film at room temperature;
(2) 3 mu L of BSA solution with the mass fraction of 0.1% is dripped on the surface of the electrode, the electrode is washed by ultrapure water, and the electrode is dried at room temperature;
(3) Dripping 6 mu L and 0.00005~100 ng mL -1 A series of different concentrations of the neuroenolase antigen are added on the surface of the electrode, incubated 2h, flushed by ultrapure water and dried at room temperature;
(4) Small-size CuO loaded Fe MOFs-sCuO-Ab combined with secondary antibody marker of neuroenolase recognition antibody and dropwise added with 6 mu L Fe MOFs 2 Washing the solution with ultrapure water, and airing at room temperature to obtain the electrochemical luminescence sensor.
Example 2 g-C based 3 N 4 Method for preparing electrochemical luminescence sensor of-CoPdNPs
(1) With Al 2 O 3 Polishing powder polishes glassy carbon electrode with diameter of 4mm, and cleaning with ultrapure water to obtain a polishing powder with the concentration of 6 mu L and 1 mg mL -1 Porous g-C of primary antibody marker of (2) 3 N 4 -CoPdNPs-Ab 1 Dropwise adding the solution onto the surface of the electrode, and airing at room temperature to form a film;
(2) 3 mu L of BSA solution with the mass fraction of 0.1% is dripped on the surface of the electrode, the electrode is washed by ultrapure water, and the electrode is dried at room temperature;
(3) Dripping 6 mu L and 0.00005~100 ng mL -1 A series of different concentrations of the neuroenolase antigen are added on the surface of the electrode, incubated 2h, flushed by ultrapure water and dried at room temperature;
(4) Small-size CuO loaded Fe MOFs-sCuO-Ab combined with secondary antibody marker of neuroenolase recognition antibody and dropwise added with 6 mu L Fe MOFs 2 Washing the solution with ultrapure water, and airing at room temperature to obtain the electrochemical luminescence sensor.
Example 3 g-C based 3 N 4 Method for preparing electrochemical luminescence sensor of-CoPdNPs
(1) With Al 2 O 3 Polishing powder polishes glassy carbon electrode with diameter of 4mm, and cleaning with ultrapure water to obtain 6 mu L and 2 mg mL -1 Porous g-C of primary antibody marker of (2) 3 N 4 -CoPdNPs-Ab 1 Dropwise adding the solution onto the surface of the electrode, and airing at room temperature to form a film;
(2) 3 mu L of BSA solution with the mass fraction of 0.1% is dripped on the surface of the electrode, the electrode is washed by ultrapure water, and the electrode is dried at room temperature;
(3) Dripping 6 mu L and 0.00005~100 ng mL -1 A series of different concentrations of the neuroenolase antigen are added on the surface of the electrode, incubated 2h, flushed by ultrapure water and dried at room temperature;
(4) Small-size CuO loaded Fe MOFs-sCuO-Ab combined with secondary antibody marker of neuroenolase recognition antibody and dropwise added with 6 mu L Fe MOFs 2 Washing the solution with ultrapure water, and airing at room temperature to obtain the electrochemical luminescence sensor.
Example 4 g-C based 3 N 4 Method for preparing electrochemical luminescence sensor of-CoPdNPs
(1) With Al 2 O 3 Polishing powder polishes glassy carbon electrode with diameter of 4mm, and cleaning with ultrapure water to obtain a polishing powder with the concentration of 6 mu L and 3mg mL -1 Porous g-C of primary antibody marker of (2) 3 N 4 -CoPdNPs-Ab 1 Dropwise adding the solution onto the surface of the electrode, and airing at room temperature to form a film;
(2) 3 mu L of BSA solution with the mass fraction of 0.1% is dripped on the surface of the electrode, the electrode is washed by ultrapure water, and the electrode is dried at room temperature;
(3) Dripping 6 mu L and 0.00005~100 ng mL -1 A series of different concentrations of the neuroenolase antigen are added on the surface of the electrode, incubated 2h, flushed by ultrapure water and dried at room temperature;
(4) Small-size CuO loaded Fe MOFs-sCuO-Ab combined with secondary antibody marker of neuroenolase recognition antibody and dropwise added with 6 mu L Fe MOFs 2 Washing the solution with ultrapure water, and airing at room temperature to obtain the electrochemical luminescence sensor.
EXAMPLE 5 detection of a Neuroenolase
(1) The Ag/AgCl is used as a reference electrode, the platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cartridge of a chemiluminescent detector, an electrochemical workstation is connected with the chemiluminescent detector, and the electrochemical workstation and the chemiluminescent detector are connected togetherThe high voltage of the photomultiplier was set to 600V at a concentration of 20 mmol L -1 Testing in PBS buffer solution of potassium persulfate;
(2) Detecting the standard solution of the nervonase by using an electrochemiluminescence method, wherein the voltage test range is-1.5-0V;
(3) And (3) observing the electrochemiluminescence intensity of the sensor before and after the addition of the nervonase, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the nervonase, and drawing a working curve.
EXAMPLE 6 detection of a Neuroenolase
(1) The Ag/AgCl is used as a reference electrode, the platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cartridge of a chemiluminescent detector, an electrochemical workstation is connected with the chemiluminescent detector, the high voltage of a photomultiplier is set to 600V, and the electrochemical luminescence sensor contains 40 mmol L -1 Testing in PBS buffer solution of potassium persulfate;
(2) Detecting the standard solution of the nervonase by using an electrochemiluminescence method, wherein the voltage test range is-1.5-0V;
(3) And (3) observing the electrochemiluminescence intensity of the sensor before and after the addition of the nervonase, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the nervonase, and drawing a working curve.
EXAMPLE 7 detection of a Neuroenolase
(1) The Ag/AgCl is used as a reference electrode, the platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cartridge of a chemiluminescent detector, an electrochemical workstation is connected with the chemiluminescent detector, the high voltage of a photomultiplier is set to 600V, and the electrochemical luminescence sensor contains 60 mmol L -1 Testing in PBS buffer solution of potassium persulfate;
(2) Detecting the standard solution of the nervonase by using an electrochemiluminescence method, wherein the voltage test range is-1.5-0V;
(3) And (3) observing the electrochemiluminescence intensity of the sensor before and after the addition of the nervonase, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the nervonase, and drawing a working curve.
Example 8 detection of a Neuroenolase
(1) The Ag/AgCl is used as a reference electrode, the platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cartridge of a chemiluminescent detector, an electrochemical workstation is connected with the chemiluminescent detector, the high voltage of a photomultiplier is set to 600V, and the electrochemical luminescence sensor contains 80 mmol L -1 Testing in PBS buffer solution of potassium persulfate;
(2) Detecting the standard solution of the nervonase by using an electrochemiluminescence method, wherein the voltage test range is-1.5-0V;
(3) And (3) observing the electrochemiluminescence intensity of the sensor before and after the addition of the nervonase, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the nervonase, and drawing a working curve.
Example 9 detection of a Neuroenolase
(1) The Ag/AgCl is used as a reference electrode, the platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cartridge of a chemiluminescent detector, an electrochemical workstation is connected with the chemiluminescent detector, the high voltage of a photomultiplier is set to 600V, and the electrochemical luminescence sensor contains 100mmol L -1 Testing in PBS buffer solution of potassium persulfate;
(2) Detecting the standard solution of the nervonase by using an electrochemiluminescence method, wherein the voltage test range is-1.5-0V;
(3) And (3) observing the electrochemiluminescence intensity of the sensor before and after the addition of the nervonase, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the nervonase, and drawing a working curve.

Claims (3)

1. g-C based 3 N 4 -a method for preparing an electrochemical luminescence sensor of the CoPdNPs, characterized in that it comprises the following steps:
(1) With Al 2 O 3 Polishing powderGrinding the glassy carbon electrode with the diameter of 4mm, cleaning with ultrapure water, and cleaning with 6 mu L and 0.5-3 mg mL -1 Porous g-C modified by CoPd alloy nano particles 3 N 4 Composite g-C 3 N 4 Porous g-C as primary antibody label binding to CoPdNPs and to a neuroenolase-recognizing antibody 3 N 4 -CoPdNPs-Ab 1 Dropwise adding the solution onto the surface of the electrode, and airing at room temperature to form a film;
(2) Dropwise adding 3 mu L of BSA solution with the mass fraction of 0.1% to the surface of an electrode, washing with ultrapure water, and airing at room temperature;
(3) Dripping 6 mu L and 0.00005-100 ng mL -1 A series of different concentrations of the neuroenolase antigens are added on the surface of the electrode, incubated for 2 hours, flushed by ultrapure water and dried at room temperature;
(4) 6 mu L of Fe MOFs-sCuO-Ab as secondary antibody marker for loading small-size CuO and combining with neuroenolase recognition antibody is dripped 2 Washing the solution with ultrapure water, and airing at room temperature to prepare an electrochemical luminescence sensor;
the Fe MOFs loads small-size CuO and binds to a secondary antibody marker Fe MOFs-sCuO-Ab of a neuroenolase recognition antibody 2 The preparation steps of the solution are as follows:
(1) Preparation of Fe MOFs
0.126g of 2-amino terephthalic acid and 0.187g of FeCl 3 ·6H 2 O and 3.0. Mu.L of acetic acid were dissolved in 15mL of DMF, the mixed solution was transferred to a reaction vessel, and then heated at 120℃for 4 hours, the crude product obtained was purified at 8000r min -1 Centrifuging, washing 3 times with DMF, washing 1 time with ethanol, and drying overnight in a vacuum drying oven at 60deg.C to obtain Fe MOFs;
(2) Preparation of small-sized CuO (sCuO)
150mL of 0.02M Cu (CH) 3 COO) 2 ·H 2 The O aqueous solution was mixed with 0.5mL glacial acetic acid and placed in a round bottom flask with reflux apparatus, the solution was heated to boiling with vigorous stirring, and then 10mL of 0.04g mL was added rapidly -1 Immediately forming a large amount of black precipitate, centrifuging the precipitate, washing the precipitate with absolute ethyl alcohol three times, and drying the precipitate in a vacuum oven for 12 hours to obtain sCuO;
(3) Fe MOFs-sCuO-Ab loaded with small-sized CuO and combined with secondary antibody of neuroenolase recognition antibody 2 Preparation of the solution
Dissolving 40mg of small-size copper oxide and 20mg of Fe MOFs in 150mL of ethanol, uniformly dispersing by ultrasonic waves, stirring overnight, centrifuging, washing with absolute ethyl alcohol for three times, and drying in a vacuum oven at 60 ℃ overnight to obtain Fe MOFs-sCuO;
100 mu L of the mixture was concentrated to 10 mu g mL -1 Is a neuroenolase-recognizing antibody Ab 2 Added to 1mL with the concentration of 1.5mg mL -1 In Fe MOFs-sCuO aqueous solution, and shaking and hatching for 12 hours at 4 ℃, dispersing the obtained product in 1mL PBS with pH of 7.4 after centrifugation, thus obtaining the secondary antibody marker Fe MOFs-sCuO-Ab of Fe MOFs loaded with small-size CuO and combined with a neuroenolase recognition antibody 2 A solution.
2. A g-C based according to claim 1 3 N 4 The preparation method of the electrochemical luminescence sensor of the CoPdNPs is characterized in that the CoPd alloy nanoparticle modified porous g-C 3 N 4 Composite g-C 3 N 4 Porous g-C as primary antibody label binding to CoPdNPs and to a neuroenolase-recognizing antibody 3 N 4 -CoPdNPs-Ab 1 The preparation steps of the solution are as follows:
(1) Porous g-C 3 N 4 Is prepared from
Takes melamine as raw material and SiO with the grain diameter of 10nm 2 The ball is used as a template, and the g-C with a porous structure is prepared by a thermal condensation method 3 N 4 Firstly, 2g of melamine and 1g of SiO are added 2 Ball milling, mixing, transferring into crucible with cover, and standing at 550deg.C for 5 min -1 Heating for 4h at the heating rate of (2) to obtain g-C with core-shell structure 3 N 4 @SiO 2 Next, g-C 3 N 4 @SiO 2 Soaking in 30mL 10% HF aqueous solution for 6 hr, washing with water for several times, and removing SiO 2 Obtaining porous g-C 3 N 4 ；
(2) CoPd alloy nanoparticlesModified porous g-C 3 N 4 Composite g-C 3 N 4 Preparation of CoPdNTPs 0.35mmol of cobalt acetylacetonate and 0.30mmol of palladium dibromide were dissolved in 18mL of oleylamine, the mixture was then stirred, heated to 60℃under nitrogen, then 0.5mL of tri-n-octylphosphine was added, the color of the mixture changed from pink to dark green, and the solution was stirred at 5℃for min -1 Is heated to 260℃and maintained at this temperature for 2 hours, after which the solution is cooled to room temperature, 40mL of ethanol are added and centrifuged to give CoPdNPs, and in addition 250mg of porous g-C 3 N 4 Dissolving in 20mL ethanol, ultrasonic treating the prepared CoPdNPs dispersion liquid with porous g-C 3 N 4 Mixing and stirring overnight, and finally at 7000r min -1 Centrifuging, washing with ethanol for several times, dispersing in 10mL ethanol, and preserving at 4deg.C;
(3) Porous g-C 3 N 4 Porous g-C as primary antibody label for binding-CoPdNPs to recognition antibody of neuroenolase 3 N 4 -CoPdNPs-Ab 1 Preparation of the solution
10 μg mL -1 The primary antibody of the neuroenolase-capturing antibody of (C) was activated at 4℃using EDC, NHS, and then reacted with porous g-C 3 N 4 CoPdNPs are hatched for 6 hours together to obtain primary antibody marker porous g-C 3 N 4 -CoPdNPs-Ab 1 A solution.
3. Use of an electrochemiluminescence sensor for non-disease diagnosis and therapy purposes in the detection of a neuroenolase, characterized in that said electrochemiluminescence sensor is produced by the preparation method according to claim 1 or 2, the detection steps being as follows:
(1) The Ag/AgCl is used as a reference electrode, the platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cartridge of a chemiluminescent detector, an electrochemical workstation is connected with the chemiluminescent detector, the high voltage of a photomultiplier is set to 600V, and the electrochemical luminescence sensor contains 20-100mmol L -1 The test was performed in PBS buffer solution with ph=7.4 of potassium persulfate;
(2) Detecting the standard solution of the nervonase by using an electrochemiluminescence method, wherein the voltage test range is-1.5-0V;
(3) And (3) observing the electrochemiluminescence intensity of the sensor before and after the addition of the nervonase, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the nervonase, and drawing a working curve.
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