CN115219483A - Preparation method of electrochemical luminescence immunosensor based on cuprous oxide nanocube quenched iron-doped graphite carbon nitride - Google Patents
Preparation method of electrochemical luminescence immunosensor based on cuprous oxide nanocube quenched iron-doped graphite carbon nitride Download PDFInfo
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- CN115219483A CN115219483A CN202210865856.XA CN202210865856A CN115219483A CN 115219483 A CN115219483 A CN 115219483A CN 202210865856 A CN202210865856 A CN 202210865856A CN 115219483 A CN115219483 A CN 115219483A
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- carbon nitride
- cuprous oxide
- iron
- cytokeratin
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 66
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 title claims abstract description 63
- 229910002804 graphite Inorganic materials 0.000 title claims abstract description 62
- 239000010439 graphite Substances 0.000 title claims abstract description 62
- KRFJLUBVMFXRPN-UHFFFAOYSA-N cuprous oxide Chemical compound [O-2].[Cu+].[Cu+] KRFJLUBVMFXRPN-UHFFFAOYSA-N 0.000 title claims abstract description 61
- BERDEBHAJNAUOM-UHFFFAOYSA-N copper(I) oxide Inorganic materials [Cu]O[Cu] BERDEBHAJNAUOM-UHFFFAOYSA-N 0.000 title claims abstract description 46
- 229940112669 cuprous oxide Drugs 0.000 title claims abstract description 46
- 238000004020 luminiscence type Methods 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims description 47
- 102100033420 Keratin, type I cytoskeletal 19 Human genes 0.000 claims abstract description 69
- 108010066302 Keratin-19 Proteins 0.000 claims abstract description 69
- 239000012634 fragment Substances 0.000 claims abstract description 68
- 239000003550 marker Substances 0.000 claims abstract description 39
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 28
- 229910052742 iron Inorganic materials 0.000 claims abstract description 12
- 238000010791 quenching Methods 0.000 claims abstract description 12
- 230000000171 quenching effect Effects 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 68
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
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- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 10
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 10
- DGLRDKLJZLEJCY-UHFFFAOYSA-L disodium hydrogenphosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O DGLRDKLJZLEJCY-UHFFFAOYSA-L 0.000 claims description 10
- 229910021397 glassy carbon Inorganic materials 0.000 claims description 10
- 238000005498 polishing Methods 0.000 claims description 10
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- 102000036639 antigens Human genes 0.000 claims description 9
- 108091007433 antigens Proteins 0.000 claims description 9
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- 229920000877 Melamine resin Polymers 0.000 claims description 5
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 claims description 5
- 229960005070 ascorbic acid Drugs 0.000 claims description 5
- 235000010323 ascorbic acid Nutrition 0.000 claims description 5
- 239000011668 ascorbic acid Substances 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 238000001354 calcination Methods 0.000 claims description 5
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- OPQARKPSCNTWTJ-UHFFFAOYSA-L copper(ii) acetate Chemical compound [Cu+2].CC([O-])=O.CC([O-])=O OPQARKPSCNTWTJ-UHFFFAOYSA-L 0.000 claims description 5
- 238000001704 evaporation Methods 0.000 claims description 5
- 229940044631 ferric chloride hexahydrate Drugs 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- JDSHMPZPIAZGSV-UHFFFAOYSA-N melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- 239000012299 nitrogen atmosphere Substances 0.000 claims description 5
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 claims description 5
- 229910052573 porcelain Inorganic materials 0.000 claims description 5
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 5
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 5
- 239000002244 precipitate Substances 0.000 claims description 5
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- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
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- 238000001514 detection method Methods 0.000 abstract description 16
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- LELOWRISYMNNSU-UHFFFAOYSA-N hydrogen cyanide Chemical compound N#C LELOWRISYMNNSU-UHFFFAOYSA-N 0.000 abstract description 5
- 238000000295 emission spectrum Methods 0.000 abstract description 4
- 238000011896 sensitive detection Methods 0.000 abstract description 3
- 229910002558 Fe-Nx Inorganic materials 0.000 abstract description 2
- 229910002559 Fe−Nx Inorganic materials 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 2
- 125000004433 nitrogen atom Chemical group N* 0.000 abstract 1
- 108010036226 antigen CYFRA21.1 Proteins 0.000 description 13
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- 229910021607 Silver chloride Inorganic materials 0.000 description 4
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- 230000002401 inhibitory effect Effects 0.000 description 4
- 238000002796 luminescence method Methods 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 4
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- 230000006798 recombination Effects 0.000 description 4
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- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical group C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 3
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
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- 230000004048 modification Effects 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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Abstract
The invention relates to a method for quenching iron doping based on cuprous oxide nanocubesA method for preparing a graphite carbon nitride electrochemical luminescence immunosensor. Fe in the invention 3+ By reaction with g-C 3 N 4 The N atom of (A) coordinates to form a key active site Fe-Nx group coordinated to g-C 3 N 4 The iron-doped graphitic carbon nitride provides a strong and consistently stable electrochemiluminescence signal required by the sensor. The electrochemical luminescence emission spectrum of the iron-doped graphite carbon nitride is greatly overlapped with the ultraviolet absorption spectrum of the cuprous oxide, electrochemical luminescence-resonance energy transfer can be generated between the iron-doped graphite carbon nitride and the cuprous oxide, and the ECL emission of the iron-doped graphite carbon nitride can be effectively quenched by the cuprous oxide. The iron-doped graphite carbon nitride is combined with the primary antibody of the soluble fragment of the cytokeratin 19 to form a primary antibody marker, and the cuprous oxide is combined with the secondary antibody of the soluble fragment of the cytokeratin 19 to form a secondary antibody marker, so that the construction process of the sensor is simplified, the ultra-sensitive detection of the soluble fragment of the cytokeratin 19 is realized, and the detection limit is 15.53 fg/mL.
Description
Technical Field
The invention relates to a preparation method of a quenched iron-doped graphite carbon nitride electrochemical luminescence immunosensor based on cuprous oxide nanocubes, in particular to a preparation method of a quenched iron-doped graphite carbon nitride electrochemical luminescence immunosensor, wherein iron-doped graphite carbon nitride is combined with a primary antibody of a soluble fragment of cytokeratin 19 to form a primary antibody marker, a cuprous oxide nanocubes material is combined with a secondary antibody of the soluble fragment of cytokeratin 19 to form a secondary antibody marker, and a sandwich quenched electrochemical immunosensor for detecting the soluble fragment of cytokeratin 19 is prepared, and belongs to the technical field of novel functional materials and biosensing detection.
Background
The lung cancer is a clinical common tumor with high malignancy degree and rapid development, is mainly divided into small cell lung cancer and non-small cell lung cancer, and the incidence rate of the lung cancer is high in many countries, so that the lung cancer is particularly significant for early diagnosis, prevention and treatment of the lung cancer. In malignant lung cancer tissues, especially in lung squamous cell carcinoma, the soluble fraction of cytokeratin 19 (Cyfra 21-1) is high. Cyfra21-1 is currently considered a tumor marker, particularly for the diagnosis of non-small cell lung cancer. Therefore, the determination of the level of Cyfra21-1 is of great significance for monitoring the progress and therapeutic effect of non-small cell lung cancer in clinical diagnosis. The electrochemical luminescence analysis method has the advantages of low background, wide dynamic range, simple and convenient instruments and equipment, sensitive detection and the like, and is widely concerned in the fields of biological analysis, food safety analysis, environmental pollution monitoring and the like.
Graphitic carbon nitride (g-C) 3 N 4 ) The catalyst has the advantages of simple preparation, good electrochemical performance, large adsorption capacity, good photochemical performance, good thermal stability, good acid and alkali chemical corrosion resistance and the like, thereby becoming a hotspot of research in the scientific community. But due to the original g-C 3 N 4 Has the defects of high carrier recombination rate, low conductivity and no absorption above 460 nm, which hinders the further application of the compound, and is used for g-C 3 N 4 Modifications were made to optimize their activity. Doping is to introduce impurities into g-C 3 N 4 Efficient ways to change the electronic structure and band configuration in the matrix. Fe 3+ By reaction with g-C 3 N 4 Coordinate to form a key active site Fe-Nx group, thereby coordinating to g-C 3 N 4 Iron-doped graphitic carbon nitride (FeCN) is formed in the "nitrogen can" of (a). Fe 3+ The coordination doping can improve the transfer of photo-generated electrons, inhibit the recombination of photoelectrons and holes, reduce the energy band gap, passivate the carbon position of a triazine ring, inhibit the crystal growth of graphite carbon nitride, and increase the specific surface area, so that the graphene oxide has strong, continuous and stable electrochemical luminescence signals, and is favorable for electrochemical application.
Cuprous oxide (Cu) 2 O) is a p-type semiconductor with an energy gap of 2.1 eV, has the advantages of low cost, no toxicity, high abundance, easy synthesis and the like, and can realize multiple functions due to the physicochemical property. The ultraviolet absorption spectrum of the cuprous oxide is overlapped with the electrochemiluminescence emission spectrum of the iron-doped graphite carbon nitride, resonance energy transfer can occur between the ultraviolet absorption spectrum and the electrochemiluminescence emission spectrum, and the cuprous oxide can quench the electrochemiluminescence of the iron-doped graphite carbon nitride. The invention adopts the iron element doping mode to process g-C 3 N 4 Regulating and controlling the structure and the shape, improving and stabilizing the g-C 3 N 4 Electrochemical luminescence response, improved luminescence efficiency。Cu 2 The O nanocubes can be directly connected with the second antibody, and can quench FeCN, so that Cyfra21-1 can be sensitively detected.
Electrochemical immunosensors are a class of detection devices that determine the concentration of an analyte based on the electrical-to-optical signal transduction characteristics of a substance. Common construction processes require layer-by-layer modification, are complex and affect the biological activity of immune molecules.
According to the invention, in a manner of constructing the sandwich type electrochemical sensor, the iron-doped graphite carbon nitride is combined with the primary antibody of Cyfra21-1 to form the primary antibody marker, and the cuprous oxide nanocube material is combined with the secondary antibody of Cyfra21-1 to form the secondary antibody marker, so that the construction process of the sensor is simplified. Electrochemiluminescence emission Spectroscopy of FeCN and Cu 2 The ultraviolet absorption spectra of O have great overlap, and the two can generate electrochemical luminescence-resonance energy transfer. FeCN as energy transfer donor, cu 2 O as an acceptor for energy transfer, ECL emission of FeCN can be by Cu 2 And (3) efficient quenching of O. FeCN has amino and can be directly compounded with primary antibody of Cyfra21-1, thereby simplifying the construction process of the sensor. Mixing Cu 2 And the secondary antibody of the O used for marking Cyfra21-1 is used as a secondary antibody marker, so that the ultra-sensitive detection of Cyfra21-1 is realized, and the detection limit is 15.53 fg/mL. Compared with the conventional sensor, the electrochemical immunosensor constructed by the invention has the advantages that the number of constructed layers is reduced, and the process of constructing the sensor is simple and easy to implement. Based on the excellent performance of the material and a simplified detection method, the electrochemical immunosensor provided by the invention has the advantages of 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 purposes of the invention is to adopt flower-shaped iron-doped graphitic carbon nitride as a substrate luminescent material and original blocky g-C 3 N 4 Compared with the prior art, the method has the advantages of improving the transfer of photo-generated electrons, inhibiting the recombination of photoelectrons and holes, reducing the energy band gap, passivating the carbon position of the triazine ring, inhibiting the crystal growth of the graphite carbon nitride, increasing the specific surface area of the graphite carbon nitride, greatly shortening the electron transfer distance, having strong and continuous and stable electrochemical luminescence signals, and solving the problem of high efficiency and low costThe defects of weak electrochemical luminescence signal and unstable luminescence signal of the original graphite-like carbon nitride have important significance for the extended application of the graphite carbon nitride.
The second purpose of the invention is to use Cu 2 The O nanocubes are used as quenching materials to construct a sandwich quenching type electrochemical immunosensor. Cu 2 The ultraviolet absorption spectrum of O and the electrochemical luminescence emission spectrum of FeCN are greatly overlapped, efficient quenching can be realized based on the resonance energy transfer principle, and a new resonance energy transfer donor-acceptor pair is provided for constructing an electrochemical immunosensor.
The invention also aims to provide a preparation method of the novel sandwich type electrochemical immunosensor based on resonance energy transfer.
The fourth purpose of the invention is to realize the construction of the electrochemical immunosensor and sensitively detect Cyfra21-1, the detection limit is 15.53 fg/mL, and the purpose of the electrochemical luminescence sensor in detecting Cyfra21-1 is achieved.
Technical scheme of the invention
1. A preparation method of an immunosensor based on cuprous oxide nanocubes quenching iron-doped graphite carbon nitride electrochemiluminescence is characterized by comprising the following steps:
(1) Preparation of iron-doped graphite carbon nitride
Firstly, preparing graphite carbon nitride with different iron doping amounts: adding 40 mL of water into a 100 mL three-neck flask, and heating to 100 ℃; then adding 0.2-2 mL of concentrated hydrochloric acid under vigorous stirring, then adding 1-5 g of melamine and 0.02-0.2 g of ferric chloride hexahydrate, stirring for thirty minutes, evaporating and drying at 100 ℃ to remove water, finally putting the obtained powder into a porcelain boat wrapped by tinfoil paper, and calcining at 550 ℃ for 6 hours under the nitrogen atmosphere of a tube furnace to obtain graphite carbon nitride with different iron doping amounts;
(2) Preparation of PBS buffer solution
Taking 11.94 g of disodium hydrogen phosphate dodecahydrate, and dissolving the disodium hydrogen phosphate dodecahydrate in a 500 mL volumetric flask to prepare a 1/15M aqueous solution as a liquid A; taking 4.54 g of monopotassium phosphate, fixing the volume in a 500 mL volumetric flask to prepare an aqueous solution with the concentration of 1/15M as a solution B; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;
(3) Preparation of primary antibody marker of soluble fragment recognition antibody of iron-doped graphite carbon nitride connected cytokeratin 19
Dissolving 150-200 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 250-300 mg of N-hydroxysuccinimide in 5 mL of PBS buffer solution with pH 7.4, adding 5-30 mg of iron-doped graphite carbon nitride with the mass fraction of 3%, uniformly mixing by ultrasonic waves, adding 200-500 mu L of 10 mu g/mL primary antibody of the soluble fragment recognition antibody of the cytokeratin 19, and incubating the obtained solution at 4 ℃ overnight. Followed by centrifugation and redispersion in 5 mL of PBS buffer pH 7.4. Finally, storing the primary anti-marker of the soluble fragment recognition antibody of the iron-doped graphite carbon nitride-linked cytokeratin 19 at 4 ℃ for later use;
(4) Preparation of cuprous oxide nanocubes
Dissolving 0.1-1.5 mM copper acetate and 0.001-0.05 mM polyvinylpyrrolidone in 100 mL water to form a solution A; dissolving 1-10 mM sodium hydroxide in 20 mL of water to form a solution B; dissolving 0.1-1.2 mM ascorbic acid in 15 mL water to form a solution C; then dropwise adding the solution B into the solution A, and violently stirring at room temperature to form a blue suspension; solution C was then added dropwise to the above suspension at a rate of three seconds per drop with vigorous stirring. Finally, centrifuging the obtained orange suspension for fifteen minutes at the rotating speed of 6000 rpm, washing the orange suspension for three times by using water, and carrying out vacuum drying at the temperature of 60 ℃ for one night;
(5) Preparation of aminated cuprous oxide nanocubes
Dispersing 0.02-2 g of cuprous oxide in 10 mL of ethanol, adding 0.02-2 mL of aminopropyltriethoxysilane, refluxing for 1.5 h at 70 ℃, and centrifugally washing and drying to obtain an aminated cuprous oxide nanocube;
(6) Preparation of secondary antibody marker of soluble fragment recognition antibody of cuprous oxide nanocube-linked cytokeratin 19
50 to 300. Mu.L of a 10. Mu.g/mL secondary antibody against a soluble fragment recognition antibody for cytokeratin 19 was dispersed in 10 mL of a pH 7.4 PBS buffer solution, and 350 to 500 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 550 to 750 mg of N-hydroxysuccinimide were added thereto, and the mixture was stirred at 4 ℃ for 4 hours, followed by centrifugation at 15000 rpm at 4 ℃ for 20 minutes. Re-dispersing the obtained macromolecular precipitate into 10 mL of PBS buffer solution with pH 7.4, adding 10-50 mg of aminated cuprous oxide, continuously stirring for 6 hours at 4 ℃, centrifuging, dispersing the obtained secondary antibody marker of the soluble fragment recognition antibody of the cuprous oxide nanocube connecting cytokeratin 19 into 5 mL of PBS buffer solution with pH 7.4, and storing for later use at 4 ℃;
(7) Preparation of electrochemical immunosensor
1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning the glassy carbon electrode by using ultrapure water, dripping 6 mu L of primary-antibody marker solution of iron-doped graphite carbon nitride connecting soluble fragment recognition antibody of cytokeratin 19 with the concentration of 0.1-3.0 mg/mL on the surface of the electrode, and airing at room temperature;
2) Continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% and 3 muL to the surface of the electrode, airing at room temperature, and washing with PBS buffer solution;
3) Continuously dropwise adding 6 mu L of soluble fragment antigen of cytokeratin 19 of 0.0001-200 ng/mL to the surface of the electrode, airing at room temperature, and washing with PBS buffer solution;
4) And finally, continuously dropwise adding a solution of 6 mu L of a second antibody marker of the soluble fragment recognition antibody of the cuprous oxide nanocube connecting cytokeratin 19 to the surface of the electrode, drying the electrode at room temperature, and washing the electrode with PBS buffer solution to obtain the immunosensor based on the quenched iron-doped graphite carbon nitride electrochemiluminescence of the cuprous oxide nanocube.
2. The detection method of the electrochemical immunosensor comprises the following steps:
(1) Taking Ag/AgCl as a reference electrode, taking a platinum wire as a counter electrode, correctly connecting the prepared electrochemical luminescence sensor serving as a working electrode in a cassette of a chemiluminescence detector, connecting an electrochemical workstation with the chemiluminescence detector, setting the high voltage of a photomultiplier to be 500-800V, and testing in 10 mL of PBS buffer solution;
(2) Detecting a soluble fragment standard solution of the cytokeratin 19 by an electrochemical luminescence method, wherein the voltage test range is-1.6-0V;
(3) Observing the electrochemiluminescence intensity of the sensor before and after adding the soluble fragment of the cytokeratin 19, then recording the linear relation between the electrochemiluminescence intensity value and the concentration of the soluble fragment of the cytokeratin 19, and drawing a working curve;
(4) And (3) replacing Cyfra21-1 antigen standard solution with the soluble fragment antigen sample solution of the cytokeratin 19 to be detected for detection.
The linear range of the sensor for detecting the soluble fragment antigen of the cytokeratin 19 is 0.00005-100 ng/mL, and the detection limit is 15.53 fg/mL;
the chemicals required for the synthesis were all purchased at the local reagent store and were not reprocessed.
Advantageous results of the invention
(1) The invention adopts the iron-doped graphite carbon nitride as the substrate luminescent material, and has strong and stable electrochemical luminescence signals. With original g-C 3 N 4 Compared with the prior art, the method has the advantages of improving the transfer of photo-generated electrons, inhibiting the recombination of photoelectrons and holes, reducing the energy band gap, passivating the carbon position of the triazine ring, inhibiting the crystal growth of the graphite carbon nitride, increasing the specific surface area of the graphite carbon nitride, having good biocompatibility, stable electrochemical signals and excellent anti-interference capability, and having important significance for the extended application of the graphite carbon nitride.
(2) The invention provides a novel donor-acceptor pair for resonance energy transfer, which is characterized in that iron-doped graphitic carbon nitride nanoflowers are used as substrate luminescent materials, cuprous oxide is used as quenching materials, and a sandwich quenching type electrochemiluminescence immunosensor is constructed.
(3) The invention provides a novel method for constructing an electrochemical immunosensor. According to the method, the primary antibody and the secondary antibody of the substance to be detected are respectively marked by the substrate luminescent material and the quenching material, and then the antigen to be detected is identified, so that the preparation of the sensor is simpler and more convenient, and the prepared sensor has good stability, high sensitivity and good reproducibility.
(4) The electrochemical immunosensor prepared by the invention can be used for sensitively detecting Cyfra21-1, the detection range is 0.00005-100 ng/mL, the detection limit is 15.53 fg/mL, and the detection with simplicity, rapidness, high sensitivity, high specificity and high stability can be realized.
Detailed Description
EXAMPLE 1 preparation of electrochemical immunosensor
(1) Preparation of iron-doped graphite carbon nitride
Firstly, preparing graphite carbon nitride with different iron doping amounts: adding 40 mL of water into a 100 mL three-neck flask, and heating to 100 ℃; then adding 0.2 mL of concentrated hydrochloric acid under vigorous stirring, then adding 1 g of melamine and 0.02 g of ferric chloride hexahydrate, stirring for thirty minutes, evaporating and drying at 100 ℃ to remove water, finally putting the obtained powder into a porcelain boat wrapped by tinfoil paper, and calcining at 550 ℃ for 6 hours under the nitrogen atmosphere of a tube furnace to obtain graphite carbon nitride with different iron doping amounts;
(2) Preparation of PBS buffer solution
Taking 11.94 g of disodium hydrogen phosphate dodecahydrate, and dissolving the disodium hydrogen phosphate dodecahydrate in a 500 mL volumetric flask to prepare a 1/15M aqueous solution as a liquid A; taking 4.54 g of monopotassium phosphate, fixing the volume in a 500 mL volumetric flask to prepare an aqueous solution with the concentration of 1/15M as a solution B; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;
(3) Preparation of primary antibody marker of soluble fragment recognition antibody of iron-doped graphite carbon nitride connected cytokeratin 19
150 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 250 mg of N-hydroxysuccinimide were dissolved in 5 mL of a PBS buffer solution at pH 7.4, 5 mg of 3% by mass FeCN was added, and after uniform ultrasonic mixing, 200. Mu.L of 10. Mu.g/mL of a primary antibody against the soluble fragment recognition antibody for cytokeratin 19 was added, and then the resulting solution was incubated overnight at 4 ℃. Followed by centrifugation and redispersion in 5 mL of PBS buffer, pH 7.4; finally, storing the primary anti-marker of the soluble fragment recognition antibody of the iron-doped graphite carbon nitride-linked cytokeratin 19 at 4 ℃ for later use;
(4) Preparation of cuprous oxide nanocubes
Dissolving 0.1 mM copper acetate and 0.001 mM polyvinylpyrrolidone in 100 mL water to form solution A; dissolving 1 mM sodium hydroxide in 20 mL water to form solution B; dissolving 0.1 mM ascorbic acid in 15 mL water to form solution C; then dropwise adding the solution B into the solution A, and violently stirring at room temperature to form a blue suspension; then dropwise adding the solution C into the suspension at a speed of three seconds per drop under the condition of vigorous stirring; finally, centrifuging the obtained orange suspension for fifteen minutes at the rotating speed of 6000 rpm, washing the orange suspension for three times by using water, and carrying out vacuum drying at the temperature of 60 ℃ for one night;
(5) Preparation of aminated cuprous oxide nanocubes
Dispersing 0.02 g of cuprous oxide in 10 mL of ethanol, adding 0.02 mL of aminopropyltriethoxysilane, refluxing for 1.5 h at 70 ℃, and centrifugally washing and drying to obtain an aminated cuprous oxide nanocube;
(6) Preparation of secondary antibody marker of soluble fragment recognition antibody of cuprous oxide nanocube-linked cytokeratin 19
50. Mu.L of a secondary antibody against a soluble fragment recognition antibody for cytokeratin 19 of 10. Mu.g/mL was dispersed in 10 mL of a PBS buffer solution having pH 7.4, and 350 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 550 mg of N-hydroxysuccinimide were added thereto, followed by stirring at 4 ℃ for 4 hours, followed by centrifugation at 15000 rpm for 20 minutes at 4 ℃; the obtained macromolecular precipitate was redispersed in 10 mL of PBS buffer solution of pH 7.4, 10 mg of aminated cuprous oxide was added and continuously stirred at 4 ℃ for 6 hours, centrifuged, and the obtained secondary antibody label of the soluble fragment recognition antibody of cuprous oxide nanocubes linked to cytokeratin 19 was dispersed in 5 mL of PBS buffer solution of pH 7.4 and stored at 4 ℃ for further use.
(7) Preparation of electrochemical immunosensor
1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning the glassy carbon electrode by using ultrapure water, dripping 6 mu L of 0.1 mg/mL of primary anti-marker solution of soluble fragment recognition antibody of iron-doped graphite carbon nitride connected cytokeratin 19 on the surface of the electrode, and airing at room temperature;
2) Continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% and 3 muL to the surface of the electrode, airing at room temperature, and washing with PBS buffer solution;
3) Continuously dropwise adding 6 mu L of soluble fragment antigen of cytokeratin 19 of 0.0001-200 ng/mL to the surface of the electrode, airing at room temperature, and washing with PBS buffer solution;
4) And finally, continuously dropwise adding a second antibody marker solution of a soluble fragment recognition antibody of the cuprous oxide nanocube connecting cytokeratin 19 to the surface of the electrode, drying at room temperature, and washing with PBS buffer solution to obtain the immune sensor based on the quenched iron-doped graphite carbon nitride electrochemiluminescence of the cuprous oxide nanocube.
EXAMPLE 2 preparation of electrochemical immunosensor
(1) Preparation of iron-doped graphite carbon nitride
Firstly, preparing graphite carbon nitride with different iron doping amounts: adding 40 mL of water into a 100 mL three-neck flask, and heating to 100 ℃; then adding 1 mL of concentrated hydrochloric acid under vigorous stirring, then adding 3 g of melamine and 0.1 g of ferric chloride hexahydrate, stirring for thirty minutes, evaporating and drying at 100 ℃ to remove water, finally putting the obtained powder into a porcelain boat wrapped by tinfoil paper, and calcining at 550 ℃ for 6 hours in a nitrogen atmosphere of a tubular furnace to obtain graphite carbon nitride with different iron doping amounts;
(2) Preparation of PBS buffer solution
Taking 11.94 g of disodium hydrogen phosphate dodecahydrate, and putting the disodium hydrogen phosphate dodecahydrate into a 500 mL volumetric flask to prepare an aqueous solution with the concentration of 1/15M to serve as a liquid A; taking 4.54 g of monopotassium phosphate, fixing the volume in a 500 mL volumetric flask to prepare an aqueous solution with the concentration of 1/15M as a solution B; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;
(3) Preparation of primary anti-marker of soluble fragment recognition antibody of iron-doped graphite carbon nitride connected cytokeratin 19
Dissolving 180 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 280 mg of N-hydroxysuccinimide in 5 mL of PBS buffer solution with pH of 7.4, adding 5-30 mg of iron-doped graphite carbon nitride with the mass fraction of 3%, uniformly mixing by ultrasonic waves, adding 350 mu L of 10 mu g/mL of primary antibody of soluble fragment recognition antibody of cytokeratin 19, and then incubating the obtained solution at 4 ℃ overnight; followed by centrifugation and redispersion in 5 mL of PBS buffer pH 7.4. Finally, storing the primary anti-marker of the soluble fragment recognition antibody of the iron-doped graphite carbon nitride-linked cytokeratin 19 at 4 ℃ for later use;
(4) Preparation of cuprous oxide nanocubes
Dissolving 1.0 mM copper acetate and 0.02 mM polyvinylpyrrolidone in 100 mL water to form solution A; dissolving 5 mM sodium hydroxide in 20 mL water to form solution B; dissolving 0.7 mM ascorbic acid in 15 mL water to form solution C; then dropwise adding the solution B into the solution A, and violently stirring at room temperature to form a blue suspension; solution C was then added dropwise to the above suspension at a rate of three seconds per drop with vigorous stirring. Finally, centrifuging the obtained orange suspension for fifteen minutes at the rotating speed of 6000 rpm, washing the orange suspension for three times by using water, and carrying out vacuum drying at the temperature of 60 ℃ for one night;
(5) Preparation of aminated cuprous oxide nanocubes
Dispersing 1 g of cuprous oxide in 10 mL of ethanol, adding 1 mL of aminopropyltriethoxysilane, refluxing for 1.5 h at 70 ℃, and centrifugally washing and drying to obtain an aminated cuprous oxide nanocube;
(6) Preparation of secondary antibody marker of soluble fragment recognition antibody of cuprous oxide nanocubes connected with cytokeratin 19
mu.L of 10. Mu.g/mL secondary antibody against the soluble fragment recognition antibody for cytokeratin 19 was dispersed in 10 mL of PBS buffer solution having pH 7.4, and 400 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 650 mg of N-hydroxysuccinimide were added thereto, followed by stirring at 4 ℃ for 4 hours, followed by centrifugation at 15000 rpm for 20 minutes at 4 ℃; redispersing the obtained macromolecular precipitate in 10 mL PBS buffer solution with pH 7.4, adding 30 mg of aminated cuprous oxide, continuously stirring for 6 h at 4 ℃, centrifuging, dispersing the obtained secondary antibody marker of the soluble fragment recognition antibody of the cuprous oxide nanocubes connected with the cytokeratin 19 in 5 mL PBS buffer solution with pH 7.4, and storing at 4 ℃ for later use;
(7) Preparation of electrochemical immunosensor
1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning the glassy carbon electrode by using ultrapure water, dripping 6 mu L of primary anti-marker solution of a soluble fragment recognition antibody of iron-doped graphite carbon nitride-linked cytokeratin 19 with the concentration of 1.5 mg/mL on the surface of the electrode, and airing at room temperature;
2) Continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% and 3 muL to the surface of the electrode, airing at room temperature, and washing with PBS buffer solution;
3) Continuously dropwise adding 6 mu L of soluble fragment antigen of cytokeratin 19 of 0.0001-200 ng/mL to the surface of the electrode, airing at room temperature, and washing with PBS buffer solution;
4) And finally, continuously dropwise adding a second antibody marker solution of a soluble fragment recognition antibody of the cuprous oxide nanocube connection cytokeratin 19, which is 6 mu L and 1.5 mg/mL, to the surface of the electrode, airing at room temperature, and washing with a PBS buffer solution to prepare the quenched iron-doped graphite carbon nitride electrochemiluminescence immunosensor based on the cuprous oxide nanocube.
EXAMPLE 3 preparation of electrochemical immunosensor
(1) Preparation of iron-doped graphite carbon nitride
Firstly, preparing graphite carbon nitride with different iron doping amounts: adding 40 mL of water into a 100 mL three-neck flask, and heating to 100 ℃; then adding 2 mL of concentrated hydrochloric acid under vigorous stirring, then adding 5 g of melamine and 0.2 g of ferric chloride hexahydrate, stirring for thirty minutes, evaporating and drying at 100 ℃ to remove water, finally putting the obtained powder into a porcelain boat wrapped by tinfoil paper, and calcining for 6 hours at 550 ℃ in a tubular furnace nitrogen atmosphere to obtain graphite carbon nitride with different iron doping amounts;
(2) Preparation of PBS buffer solution
Taking 11.94 g of disodium hydrogen phosphate dodecahydrate, and putting the disodium hydrogen phosphate dodecahydrate into a 500 mL volumetric flask to prepare an aqueous solution with the concentration of 1/15M to serve as a liquid A; taking 4.54 g of monopotassium phosphate, fixing the volume in a 500 mL volumetric flask to prepare an aqueous solution with the concentration of 1/15M as a solution B; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;
(3) Preparation of primary anti-marker of soluble fragment recognition antibody of iron-doped graphite carbon nitride connected cytokeratin 19
Dissolving 200 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 300 mg of N-hydroxysuccinimide in 5 mL of PBS buffer solution with pH 7.4, adding 30 mg of iron-doped graphite carbon nitride with the mass fraction of 3%, uniformly mixing by ultrasonic waves, adding 500 mu L of 10 mu g/mL of primary antibody of the soluble fragment recognition antibody of the cytokeratin 19, and then incubating the obtained solution at 4 ℃ overnight; followed by centrifugation and redispersion in 5 mL of PBS buffer, pH 7.4; finally, storing the primary anti-marker of the soluble fragment recognition antibody of the iron-doped graphite carbon nitride connected cytokeratin 19 at 4 ℃ for later use;
(4) Preparation of cuprous oxide nanocubes
Dissolving 1.5 mM copper acetate and 0.05 mM polyvinylpyrrolidone in 100 mL water to form solution A; dissolving 10 mM sodium hydroxide in 20 mL water to form solution B; dissolving 1.2 mM ascorbic acid in 15 mL water to form solution C; then dropwise adding the solution B into the solution A, and violently stirring at room temperature to form a blue suspension; then dropwise adding the solution C into the suspension at a speed of three seconds per drop under the condition of vigorous stirring; finally, centrifuging the obtained orange suspension for fifteen minutes at the rotating speed of 6000 rpm, washing the orange suspension for three times by using water, and carrying out vacuum drying at the temperature of 60 ℃ for one night;
(5) Preparation of aminated cuprous oxide nanocube
Dispersing 2 g of cuprous oxide in 10 mL of ethanol, adding 2 mL of aminopropyltriethoxysilane, refluxing for 1.5 h at 70 ℃, centrifuging, washing and drying to obtain an aminated cuprous oxide nanocube;
(6) Preparation of secondary antibody marker of soluble fragment recognition antibody of cuprous oxide nanocubes connected with cytokeratin 19
300. Mu.L of a secondary antibody against a soluble fragment recognition antibody for cytokeratin 19 of 10. Mu.g/mL was dispersed in 10 mL of a PBS buffer solution having pH 7.4, and 500 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 750 mg of N-hydroxysuccinimide were added thereto, followed by stirring at 4 ℃ for 4 hours, followed by centrifugation at 15000 rpm for 20 minutes at 4 ℃; re-dispersing the obtained macromolecular precipitate in 10 mL of PBS buffer solution with pH 7.4, adding 50 mg of aminated cuprous oxide, continuously stirring for 6 hours at 4 ℃, centrifuging, dispersing the obtained secondary antibody marker of the soluble fragment recognition antibody of the cuprous oxide nanocubes connected with the cytokeratin 19 in 5 mL of PBS buffer solution with pH 7.4, and storing for later use at 4 ℃;
(7) Preparation of electrochemical immunosensor
1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning the glassy carbon electrode by using ultrapure water, dripping 6 mu L of primary-antibody marker solution of iron-doped graphite carbon nitride connecting soluble fragment recognition antibody of cytokeratin 19 with the concentration of 3.0 mg/mL on the surface of the electrode, and airing at room temperature;
2) Continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% and 3 muL to the surface of the electrode, airing at room temperature, and washing with PBS buffer solution;
3) Continuously dropwise adding 6 mu L of soluble fragment antigen of cytokeratin 19 of 0.0001-200 ng/mL to the surface of the electrode, airing at room temperature, and washing with PBS buffer solution;
4) And finally, continuously dropwise adding 6 mu L of 3.0 mg/mL solution of a second antibody marker of the cuprous oxide nanocube connecting cytokeratin 19 soluble fragment recognition antibody to the surface of the electrode, airing at room temperature, and washing with PBS buffer solution to prepare the immunosensor based on the quenched iron-doped graphite carbon nitride electrochemiluminescence of the cuprous oxide nanocube.
Example 4 detection of soluble fragments of cytokeratin 19
(1) Taking Ag/AgCl as a reference electrode, taking a platinum wire as a counter electrode, correctly connecting the prepared electrochemical luminescence sensor serving as a working electrode in a cassette of a chemiluminescence detector, connecting an electrochemical workstation and the chemiluminescence detector together, setting the high voltage of a photomultiplier to be 600V, and testing in PBS (phosphate buffered saline) buffer solution containing 60 mM potassium persulfate;
(2) Detecting a soluble fragment standard solution of the cytokeratin 19 by an electrochemical luminescence method, wherein the voltage test range is-1.6-0V;
(3) And observing the electrochemiluminescence intensity of the sensor before and after the soluble fragment of the cytokeratin 19 is added, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the soluble fragment of the cytokeratin 19, and drawing a working curve.
Example 5 detection of soluble fragments of cytokeratin 19
(1) Taking Ag/AgCl as a reference electrode, a platinum wire as a counter electrode, correctly connecting the prepared electrochemical luminescence sensor as a working electrode in a cassette of a chemiluminescence detector, connecting an electrochemical workstation and the chemiluminescence detector together, setting the high voltage of a photomultiplier to be 700V, and testing in PBS (phosphate buffered saline) containing 80 mM potassium persulfate;
(2) Detecting the soluble fragment standard solution of the cytokeratin 19 by an electrochemical luminescence method, wherein the voltage test range is-1.5-0V;
(3) And observing the electrochemiluminescence intensity of the sensor before and after the soluble fragment of the cytokeratin 19 is added, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the soluble fragment of the cytokeratin 19, and drawing a working curve.
Example 6 detection of soluble fragments of cytokeratin 19
(1) Taking Ag/AgCl as a reference electrode, a platinum wire as a counter electrode, correctly connecting the prepared electrochemical luminescence sensor as a working electrode in a cassette of a chemiluminescence detector, connecting an electrochemical workstation and the chemiluminescence detector together, setting the high voltage of a photomultiplier to be 800V, and testing in PBS (phosphate buffered saline) containing 100 mM potassium persulfate;
(2) Detecting a soluble fragment standard solution of the cytokeratin 19 by an electrochemical luminescence method, wherein the voltage test range is-1.4-0V;
(3) And observing the electrochemiluminescence intensity of the sensor before and after the soluble fragment of the cytokeratin 19 is added, recording the linear relation between the electrochemiluminescence intensity value and the concentration of the soluble fragment of the cytokeratin 19, and drawing a working curve.
Claims (1)
1. A preparation method of an electrochemical luminescence immunosensor based on cuprous oxide nanocubes quenching iron-doped graphite carbon nitride is characterized by comprising the following steps:
(1) Preparation of iron-doped graphite carbon nitride
Firstly, preparing graphite carbon nitride with different iron doping amounts: adding 40 mL of water into a 100 mL three-neck flask, heating to 100 ℃, then adding 0.2-2 mL of concentrated hydrochloric acid under vigorous stirring, then adding 1-5 g of melamine and 0.02-0.2 g of ferric chloride hexahydrate, stirring for 30 minutes, evaporating and drying at 100 ℃ to remove water, finally putting the obtained powder into a porcelain boat wrapped by tinfoil paper, and calcining at 550 ℃ for 6 hours in a tubular furnace nitrogen atmosphere to obtain graphite carbon nitride with different iron doping amounts;
(2) Preparation of PBS buffer solution
Taking 11.94 g of disodium hydrogen phosphate dodecahydrate, and dissolving the disodium hydrogen phosphate dodecahydrate in a 500 mL volumetric flask to prepare a 1/15M aqueous solution as a liquid A; taking 4.54 g of monopotassium phosphate, fixing the volume in a 500 mL volumetric flask to prepare an aqueous solution with the concentration of 1/15M as a solution B; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;
(3) Preparation of primary anti-marker of soluble fragment recognition antibody of iron-doped graphite carbon nitride connected cytokeratin 19
Dissolving 150-200 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 250-300 mg of N-hydroxysuccinimide in 5 mL of PBS buffer solution with pH 7.4, adding 5-30 mg of iron-doped graphite carbon nitride with the mass fraction of 3%, ultrasonically mixing uniformly, adding 200-500 mu L of primary antibody of soluble fragment recognition antibody of cytokeratin 19 with 10 mu g/mL, incubating the obtained solution at 4 ℃ overnight, centrifuging, re-dispersing in 5 mL of PBS buffer solution with pH 7.4, and storing the obtained primary antibody marker of soluble fragment recognition antibody of iron-doped graphite carbon nitride-linked cytokeratin 19 at 4 ℃ for later use;
(4) Preparation of cuprous oxide nanocubes
Dissolving 0.1-1.5 mM copper acetate and 0.001-0.05 mM PVP in 100 mL water to form a solution A; dissolving 1-10 mM sodium hydroxide in 20 mL of water to form a solution B; dissolving 0.1-1.2 mM ascorbic acid in 15 mL water to form a solution C, then dropwise adding the solution B into the solution A, vigorously stirring at room temperature to form a blue suspension, then dropwise adding the solution C into the suspension at a speed of three seconds per drop under vigorous stirring, finally centrifuging the obtained orange yellow suspension at a rotating speed of 6000 rpm for fifteen minutes, washing with water for three times, and carrying out vacuum drying at 60 ℃ for one night;
(5) Preparation of aminated cuprous oxide nanocubes
Dispersing 0.02-2 g of cuprous oxide in 10 mL of ethanol, adding 0.02-2 mL of aminopropyltriethoxysilane, refluxing for 1.5 h at 70 ℃, and centrifugally washing and drying to obtain an aminated cuprous oxide nanocube;
(6) Preparation of secondary antibody marker of soluble fragment recognition antibody of cuprous oxide nanocubes connected with cytokeratin 19
Dispersing 50-300 mu L of 10 mu g/mL secondary antibody of the soluble fragment recognition antibody of the cytokeratin 19 in 10 mL PBS buffer solution with pH 7.4, adding 350-500 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 550-750 mg of N-hydroxysuccinimide, stirring for 4 h at 4 ℃, then centrifuging for twenty minutes at the rotating speed of 15000 rpm at 4 ℃, redispersing the obtained macromolecular precipitate in 10 mL PBS buffer solution with pH 7.4, adding 10-50 mg of aminated cuprous oxide, continuously stirring for 6 h at 4 ℃, centrifuging, dispersing the obtained secondary antibody marker of the soluble fragment recognition antibody of the cuprous oxide nanocubube connected with the cytokeratin 19 in 5 mL PBS buffer solution with pH 7.4, and storing for later use at 4 ℃;
(7) Preparation of electrochemical immunosensor
1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning the glassy carbon electrode by using ultrapure water, dripping 6 mu L of 0.1-3.0 mg/mL of primary anti-marker solution of soluble fragment recognition antibody of iron-doped graphite carbon nitride connected cytokeratin 19 on the surface of the electrode, and airing at room temperature;
2) Continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% and 3 muL to the surface of the electrode, airing at room temperature, and washing with PBS buffer solution;
3) Continuously dropwise adding 6 mu L of soluble fragment antigen of cytokeratin 19 of 0.0001-200 ng/mL to the surface of the electrode, airing at room temperature, and washing with PBS buffer solution;
4) And finally, continuously dropwise adding a second antibody marker solution of a soluble fragment recognition antibody of the cuprous oxide nanocubes connecting cytokeratin 19 to the surface of the electrode, wherein the volume of the second antibody marker solution is 6 mu L and 0.1-3.0 mg/mL, drying the second antibody marker solution at room temperature, and washing the dried second antibody marker solution by using a PBS buffer solution to prepare the immune sensor based on electrochemical luminescence of quenched iron doped graphite carbon nitride of the cuprous oxide nanocubes.
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