CN110346438B - Based on PbS/Co3O4Preparation method of composite signal attenuation type photoelectrochemical immunosensor - Google Patents
Based on PbS/Co3O4Preparation method of composite signal attenuation type photoelectrochemical immunosensor Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 20
- 239000002131 composite material Substances 0.000 title description 5
- 108010048233 Procalcitonin Proteins 0.000 claims abstract description 57
- CWCXERYKLSEGEZ-KDKHKZEGSA-N procalcitonin Chemical compound C([C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCSC)NC(=O)[C@H]1NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@@H](N)CSSC1)[C@@H](C)O)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 CWCXERYKLSEGEZ-KDKHKZEGSA-N 0.000 claims abstract description 57
- UBEWDCMIDFGDOO-UHFFFAOYSA-N cobalt(II,III) oxide Inorganic materials [O-2].[O-2].[O-2].[O-2].[Co+2].[Co+3].[Co+3] UBEWDCMIDFGDOO-UHFFFAOYSA-N 0.000 claims abstract description 55
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims abstract description 54
- 238000002360 preparation method Methods 0.000 claims abstract description 42
- 150000001875 compounds Chemical class 0.000 claims abstract description 25
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- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000004408 titanium dioxide Substances 0.000 claims abstract description 10
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 28
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 16
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- 238000001035 drying Methods 0.000 claims description 14
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- 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 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 8
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 8
- 229940098773 bovine serum albumin Drugs 0.000 claims description 8
- UFMZWBIQTDUYBN-UHFFFAOYSA-N cobalt dinitrate Chemical compound [Co+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O UFMZWBIQTDUYBN-UHFFFAOYSA-N 0.000 claims description 8
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- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 5
- 239000012279 sodium borohydride Substances 0.000 claims description 5
- ZFBOVYJITDWWBB-UHFFFAOYSA-N 3-triethoxysilylpropane-1,1,1-triamine Chemical compound CCO[Si](OCC)(OCC)CCC(N)(N)N ZFBOVYJITDWWBB-UHFFFAOYSA-N 0.000 claims description 4
- 239000002211 L-ascorbic acid Substances 0.000 claims description 4
- 235000000069 L-ascorbic acid Nutrition 0.000 claims description 4
- 238000005576 amination reaction Methods 0.000 claims description 4
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- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 claims description 4
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- 239000012299 nitrogen atmosphere Substances 0.000 claims description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 4
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 4
- 229940038773 trisodium citrate Drugs 0.000 claims description 4
- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 claims description 4
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- CBACFHTXHGHTMH-UHFFFAOYSA-N 2-piperidin-1-ylethyl 2-phenyl-2-piperidin-1-ylacetate;dihydrochloride Chemical compound Cl.Cl.C1CCCCN1C(C=1C=CC=CC=1)C(=O)OCCN1CCCCC1 CBACFHTXHGHTMH-UHFFFAOYSA-N 0.000 abstract description 2
- 238000013399 early diagnosis Methods 0.000 abstract description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 abstract description 2
- 229910052737 gold Inorganic materials 0.000 abstract description 2
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- 238000011896 sensitive detection Methods 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 abstract 1
- AMGQUBHHOARCQH-UHFFFAOYSA-N indium;oxotin Chemical compound [In].[Sn]=O AMGQUBHHOARCQH-UHFFFAOYSA-N 0.000 abstract 1
- 208000027866 inflammatory disease Diseases 0.000 abstract 1
- 230000031700 light absorption Effects 0.000 abstract 1
- 238000012544 monitoring process Methods 0.000 abstract 1
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- OCKGFTQIICXDQW-ZEQRLZLVSA-N 5-[(1r)-1-hydroxy-2-[4-[(2r)-2-hydroxy-2-(4-methyl-1-oxo-3h-2-benzofuran-5-yl)ethyl]piperazin-1-yl]ethyl]-4-methyl-3h-2-benzofuran-1-one Chemical compound C1=C2C(=O)OCC2=C(C)C([C@@H](O)CN2CCN(CC2)C[C@H](O)C2=CC=C3C(=O)OCC3=C2C)=C1 OCKGFTQIICXDQW-ZEQRLZLVSA-N 0.000 description 1
- 210000003771 C cell Anatomy 0.000 description 1
- 102000055006 Calcitonin Human genes 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 201000005008 bacterial sepsis Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
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- 238000003018 immunoassay Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- XCAUINMIESBTBL-UHFFFAOYSA-N lead(ii) sulfide Chemical compound [Pb]=S XCAUINMIESBTBL-UHFFFAOYSA-N 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Abstract
The invention relates to a lead sulfide-cobaltosic oxide-based compound (PbS/Co)3O4) The preparation method and the application of the photoelectrochemistry procalcitonin sensor. The invention is particularly directed to Indium Tin Oxide (ITO) conductorsBlack titanium dioxide nanoparticles (B-TiO) on electric glass2NPs), bismuth oxyiodide nanosheets (BiOI NSs) and gold nanoparticles (Au NPs) are compounded to serve as a substrate material, and the formed sensitized structure can increase the light absorption range, promote the separation efficiency of photo-generated electron holes and be used for supporting antibodies. Prepared PbS/Co3O4The compound and a substrate material compete for a light source and a cavity sacrificial agent, a signal-weakening-type-based photoelectrochemical immunosensor is constructed, sensitive detection of procalcitonin is achieved, and the method has important significance for early diagnosis and monitoring of bacterial inflammatory disease infection.
Description
Technical Field
The invention belongs to the technical field of nano functional materials, immunoassay and biosensing, and particularly relates to a method for preparing a nano material by using black titanium dioxide nanoparticles, bismuth oxyiodide nanosheets and gold nanoparticles (B-TiO)2NPs/BiOI NSs/Au NPs) as substrate material, PbS/Co3O4The photoelectrochemical immunosensor for detecting procalcitonin is constructed as a signal antibody marker.
Background
Procalcitonin (PCT), a natural precursor of calcitonin, produced by thyroid C cells and proteolysis, is a promising disease marker for the diagnosis of bacterial infections, and is more sensitive to the diagnosis of sepsis than other known disease markers. The level of procalcitonin in normal humans is below 0.1 ng/mL, but when bacterial infection or sepsis occurs, the level of procalcitonin increases rapidly, the increase depending on the extent of the infection. Therefore, the early diagnosis of procalcitonin has important clinical significance for treating diseases such as bacterial infection and the like. At present, there are many methods for detecting procalcitonin, such as enzyme-linked immunoassay, radioimmunoassay, fluorescence or surface plasmon resonance, but the methods have the disadvantages of complex operation, expensive instrument and the like, so the detection method developed by the invention has important significance in simple operation, low price and high sensitivity. The photoelectrochemical sensor constructed by the invention is a device for determining the concentration of an analyte by utilizing the photoelectric conversion property of a photoelectric active substance, and has the advantages of low background signal, high sensitivity, simple and convenient preparation and low cost because the detection signal is an electric signal and the excitation signal is a light source, thereby realizing the separation of the excitation signal and the detection signal, and having wide application in the fields of food analysis, environment detection, water quality analysis, biological analysis and the like.
Photoactive materials are key components of photoelectrochemical sensors. As a photoelectric conversion semiconductor material with excellent performance, titanium dioxide is widely used in the fields of photocatalysis, fuel cells and the like, but the wide band gap (3.2 eV) of titanium dioxide only can absorb ultraviolet light, so that the application of titanium dioxide is limited. Therefore, in the invention, on the basis of titanium dioxide, sodium borohydride is used for reduction to obtain B-TiO2NPs, the creation of oxygen vacancies, can increase the utilization of visible light. In addition, BiOI NSs and B-TiO are added2The NPs are compounded to form a band gap matching structure to promote the transfer of electrons. Au NPs are dripped on the surface of the electrode to form a sensitized structure, so that the conductivity of the electrode is increased, and the photocurrent response is improved. And PbS/Co3O4The compound is used as a signal marker, and the competitive absorption of the compound to light and a hole sacrificial agent is utilized to improve the sensitivity of the photoelectrochemical sensor.
Disclosure of Invention
One of the objects of the present invention is to synthesize B-TiO, respectively2NPs, BiOI NSs and Au NPs to form B-TiO2NPs/BiOI NSs/Au NPs multilayer composite sensitization structure using B-TiO2The band-gap matching structure between the NPs and the BiOI NSs and the good conductivity of the Au NPs promote the transfer of photo-generated electrons.
Another object of the present invention is to synthesize PbS/Co3O4The complex is combined with a signal antibody to form PbS/Co3O4-Ab2BioconjugationA compound (I) is provided.
The third object of the present invention is to provide B-TiO2NPs/BiOI NSs/Au NPs multilayer composite sensitized structure is used as a photoactive substrate material, and PbS/Co3O4The compound is used as a signal marker to construct a sandwich type photoelectrochemical immunosensor and is used for rapid and sensitive detection of procalcitonin.
The technical scheme of the invention is as follows:
1. based on PbS/Co3O4The preparation method of the compound signal weakening type photoelectrochemical immunosensor is characterized by comprising the following steps of:
1) cutting a large piece of ITO conductive glass into 2.0 cm multiplied by 0.8 cm, respectively ultrasonically cleaning the large piece of ITO conductive glass for 30 min by using acetone, ethanol and ultrapure water in sequence, and drying the large piece of ITO conductive glass by using nitrogen;
2) dropwise adding 10 mu L of B-TiO of 5-8 mg/mL2NPs uniformly disperse the suspension to the conductive surface of the ITO conductive glass, and air-drying at room temperature to obtain B-TiO2An NPs electrode;
3) mixing B-TiO2NPs electrodes are at 5-10 mM Bi (NO)3Dipping the mixture in 5-10 mM KI solution for 10 s, washing with ultrapure water, and repeating the above circulation for 20-30 times to obtain B-TiO2An NPs/BiOI NSs electrode;
4) dropwise adding 8 mu L Au NPs solution to B-TiO2Preparing B-TiO on the surface of the NPs/BiOI NSs electrode2NPs/BiOI NSs/Au NPs electrode;
5) dropwise adding 5 muL and 8-12 mug/mL procalcitonin capture antibody solution to the surface of the modified electrode, airing in a refrigerator at 4 ℃, and washing with ultrapure water;
6) dropwise adding 3 muL of bovine serum albumin solution with the mass fraction of 1.0% to the surface of the modified electrode to seal the nonspecific active sites on the surface of the electrode, drying in a refrigerator at 4 ℃, and washing with ultrapure water;
7) dropwise adding 5 muL and 0.1 pg/mL-50 ng/mL of procalcitonin antigen standard solution to the surface of the modified electrode, airing in a refrigerator at 4 ℃, and washing with ultrapure water;
8) dropwise adding 5 mu L and 3.0-5.0 mg/mL PbS/Co3O4Placing procalcitonin detection antibody compound solution on the surface of an electrode, and incubating in a refrigerator at 4 DEG CAnd washing with ultrapure water for 60 min to prepare a completely modified electrode, namely the electrochemical immunosensor for detecting the procalcitonin signal attenuation type.
A PbS/Co based polymer according to claim 13O4Preparation method of compound signal-weakening type photoelectrochemical immunosensor, B-TiO2Preparation of NPs, the procedure was as follows:
4.0-5.0 g P25 g of titanium dioxide nanoparticles and 1.5-2.0 g of NaBH4Mixing and grinding for 30 min, calcining in a tubular furnace at 300 ℃ for 50-60 min under argon atmosphere, cooling to room temperature, respectively centrifugally cleaning the obtained product with ultrapure water and ethanol for 3 times, and drying in a vacuum drying oven at 35 ℃ for 12 h to obtain B-TiO2 NPs。
A PbS/Co based polymer according to claim 13O4The preparation method of the compound signal weakening type photoelectrochemical immunosensor comprises the following steps of:
will coat with B-TiO2ITO conductive glasses of NPs are respectively 5-10 mM Bi (NO)3And 5-10 mM KI solution for 10 s, rinsing with ultrapure water, and repeating the above cycle 20-30 times to obtain BiOI NSs.
A PbS/Co based polymer according to claim 13O4The preparation method of the compound signal attenuation type photoelectrochemical immunosensor comprises the following steps of:
50-55 mL HAuCl with mass fraction of 0.01%4Boiling the solution in an oil bath at 120 ℃, adding 2.5-5.0 mL of trisodium citrate solution with the mass fraction of 1% into the solution, and boiling for 30 min under stirring until the solution turns purple red to prepare the Au NPs solution.
A PbS/Co based polymer according to claim 13O4A preparation method of a compound signal-weakening type photoelectrochemical immunosensor, and PbS/Co3O4The preparation method of the procalcitonin detection antibody complex solution comprises the following steps:
1)Co3O4preparation of polyhedra and amination thereof:
3.0-6.0 g Co(NO3)2·6H2o and 13-16 g of 2-methylimidazole were dissolved in 250 mL of methanol, respectively, and the 2-methylimidazole solution was added to Co (NO)3)2Aging in the solution at room temperature for 24 h, centrifuging and washing with methanol for 3 times, drying the obtained solid in a vacuum drying oven, and calcining in a tubular furnace at 350 deg.C for 120 min to obtain Co3O4Polyhedron, 0.05-0.1 g Co3O4Dispersing polyhedron in 10 mL ethanol solution containing 0.15 mL triaminopropyltriethoxysilane, ultrasonic dispersing for 30 min, refluxing at 70 deg.C for 24 h, centrifuging and washing with anhydrous ethanol for 3 times, and vacuum drying at 35 deg.C for 12 h to obtain aminated Co3O4A polyhedron;
2) preparation of PbS QDs:
adding 7-21 μ L thioglycolic acid to 25 mL of 1-7 mmol/L Pb (NO)3)2Bubbling for 20 min under nitrogen, adjusting the pH value to 5-12 by using 1 mol/L sodium hydroxide solution, and adding 2 mL of 0.01-0.015 mol/L Na after bubbling for 20 min2Stirring the S solution for 4 hours under the nitrogen atmosphere to obtain a PbS QDs solution;
3)PbS/Co3O4the preparation of (1):
5-10 mg aminated Co3O4Polyhedron, 0.8 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 0.1 g of N-hydroxysuccinimide are dissolved in 15 mL of PbS QDs solution, the mixed solution is stirred for 2 hours at room temperature, and is washed for 3 times by ultrapure water, and then the product is dispersed in 2 mL of ultrapure water;
4)PbS/Co3O4preparation of procalcitonin detection antibody complex solution:
2 mL of a procalcitonin detection antibody solution of 10 mug/mL and 10 mug L of 5 mg/mL of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 10 mug L of 1 mg/mL of N-hydroxysuccinimide oscillating at 4 ℃ for 30 min, 6 mg of PbS/Co3O4Adding the solution, shaking at 4 ℃ for 6 h, injecting 100 microliter bovine serum albumin solution with mass fraction of 1% into the solution, shaking at 4 ℃ for 12 h, centrifugally washing for 3 times by using phosphate buffer solution with pH of 7.4, and dispersing in 2 mL phosphate buffer solution with pH of 7.4.
A compound prepared by the process of claim 1Based on PbS/Co3O4The compound signal weakening type photoelectrochemical immunosensor is used for detecting procalcitonin, and comprises the following detection steps:
(1) the test is carried out by using an electrochemical workstation in a three-electrode system, the ITO modified electrode prepared according to claim 1 is used as a working electrode, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, and the test is carried out in 10 mL of phosphate buffer solution containing 0.1 mol/L ascorbic acid and having the pH value of 7.4;
(2) detecting the analyte by using a time-current method, setting the voltage to be 0V, operating the time to be 100 s, and irradiating the LED lamp with the wavelength of 400-450 nm;
(3) when the background current tends to be stable, turning on the lamp every 10 s for continuously irradiating for 10 s, then recording the photocurrent, and drawing a working curve;
(4) and (3) replacing the procalcitonin standard solution with the procalcitonin sample solution to be detected for detection, and checking the detection result through a working curve.
Advantageous results of the invention
1. B-TiO synthesized by the invention2 NPs/BiOI NSs/Au NPs,B-TiO2The NPs can improve the roughness of the ITO surface, which is beneficial to the growth of BiOI NSs, so that the capability of capturing a light source is improved, meanwhile, the staggered nanosheet structure has a large specific surface area, the loading capacity of a capture antibody can be increased, the good conductivity and the surface plasma resonance effect of the Au NPs can accelerate the electron transfer rate on the surface of the electrode, and the photoelectric conversion efficiency is obviously improved.
2. The invention synthesizes the nano composite material PbS/Co3O4And constructing a signal-attenuated photoelectrochemical immunosensor as a detection antibody marker. The PbS quantum dot is a semiconductor with a narrower band gap, can absorb visible and infrared light of 400-1300 nm, and can react with B-TiO2NP/BiOI NSs/Au NPs compete for absorption light source and electron donor; co3O4Is a polyhedron, P-type semiconductor structure, has larger specific surface area, can be used for loading PbS quantum dots, has larger steric hindrance, blocks the transmission of electron and hole sacrificial agents, and has the structure with the PbS quantum dotsThe synergistic effect is achieved, so that the photocurrent response is reduced, and the photoelectrochemistry sandwich type immunosensor with a reduced signal is formed.
3. The invention utilizes the nano composite material PbS/Co3O4The enzyme-free immunosensor is directly combined with a marker detection antibody to construct an enzyme-free immunosensor, so that detection errors caused by inactivation or leakage of enzyme are avoided. Simultaneous use of PbS/Co3O4The composite material realizes signal attenuation, greatly improves the detection sensitivity of the photoelectrochemical sensor, and has important scientific significance and application value.
4. The signal attenuation type photoelectrochemical immunosensor prepared by the invention is used for detecting procalcitonin, has short response time and good stability, and can realize simple, quick, high-sensitivity and specific detection of the procalcitonin. The detection range of the sensor prepared by the invention on procalcitonin is 0.1 pg/mL-200 ng/mL, and the detection limit is 0.02 pg/mL.
Detailed Description
The present invention will now be further illustrated by, but not limited to, specific embodiments thereof.
Example 1A catalyst based on PbS/Co3O4The preparation method of the photoelectrochemistry procalcitonin sensor of the compound comprises the following steps:
1) cutting a large piece of ITO conductive glass into 2.0 cm multiplied by 0.8 cm, respectively ultrasonically cleaning the large piece of ITO conductive glass for 30 min by using acetone, ethanol and ultrapure water in sequence, and drying the large piece of ITO conductive glass by using nitrogen;
2) dropwise adding 10 mu L of 5 mg/mL B-TiO2NPs uniformly disperse the suspension to the conductive surface of the ITO conductive glass, and air-drying at room temperature to obtain B-TiO2An NPs electrode;
3) mixing B-TiO2NPs electrodes are at 5 mM Bi (NO)3Dipping the mixture in 5 mM KI solution for 10 s, washing with ultrapure water, and repeating the cycle for 20 times to obtain B-TiO2An NPs/BiOI NSs electrode;
4) dropwise adding 8 mu L Au NPs solution to B-TiO2Preparing B-TiO on the surface of the NPs/BiOI NSs electrode2NPs/BiOI NSs/Au NPs electrode;
5) dropwise adding a procalcitonin capture antibody solution of 5 muL and 8 mug/mL to the surface of the modified electrode, airing in a refrigerator at 4 ℃, and washing with ultrapure water;
6) dropwise adding 3 muL of bovine serum albumin solution with the mass fraction of 1.0% to the surface of the modified electrode to seal the nonspecific active sites on the surface of the electrode, drying in a refrigerator at 4 ℃, and washing with ultrapure water;
7) dropwise adding 5 muL and 0.1 pg/mL-50 ng/mL of procalcitonin antigen standard solution to the surface of the modified electrode, airing in a refrigerator at 4 ℃, and washing with ultrapure water;
8) dropwise adding 5 mu L and 3.0 mg/mL PbS/Co3O4And (3) adding the procalcitonin detection antibody compound solution to the surface of the electrode, incubating in a refrigerator at 4 ℃ for 60 min, washing with ultrapure water, and preparing the completely modified electrode, namely the procalcitonin signal detection weakening type photoelectrochemical immunosensor.
Example 2A catalyst based on PbS/Co3O4The preparation method of the photoelectrochemistry procalcitonin sensor of the compound comprises the following steps:
1) cutting a large piece of ITO conductive glass into 2.0 cm multiplied by 0.8 cm, respectively ultrasonically cleaning the large piece of ITO conductive glass for 30 min by using acetone, ethanol and ultrapure water in sequence, and drying the large piece of ITO conductive glass by using nitrogen;
2) dropwise adding 10 mu L of B-TiO of 7 mg/mL2NPs uniformly disperse the suspension to the conductive surface of the ITO conductive glass, and air-drying at room temperature to obtain B-TiO2An NPs electrode;
3) mixing B-TiO2NPs electrodes are at 8 mM Bi (NO)3Dipping in 8 mM KI solution for 10 s, rinsing with ultrapure water, and repeating the cycle for 25 times to obtain B-TiO2An NPs/BiOI NSs electrode;
4) dropwise adding 8 mu L Au NPs solution to B-TiO2Preparing B-TiO on the surface of the NPs/BiOI NSs electrode2NPs/BiOI NSs/Au NPs electrode;
5) dropwise adding a procalcitonin capture antibody solution of 5 muL and 10 mug/mL to the surface of the modified electrode, airing in a refrigerator at 4 ℃, and washing with ultrapure water;
6) dropwise adding 3 muL of bovine serum albumin solution with the mass fraction of 1.0% to the surface of the modified electrode to seal the nonspecific active sites on the surface of the electrode, drying in a refrigerator at 4 ℃, and washing with ultrapure water;
7) dropwise adding 5 muL and 0.1 pg/mL-50 ng/mL of procalcitonin antigen standard solution to the surface of the modified electrode, airing in a refrigerator at 4 ℃, and washing with ultrapure water;
8) dropwise adding 5 mu L and 5.0 mg/mL PbS/Co3O4And (3) adding the procalcitonin detection antibody compound solution to the surface of the electrode, incubating in a refrigerator at 4 ℃ for 60 min, washing with ultrapure water, and preparing the completely modified electrode, namely the procalcitonin signal detection weakening type photoelectrochemical immunosensor.
Example 3B-TiO2Preparation of NPs, the procedure was as follows:
4.0 g P25 titanium dioxide nanoparticles and 1.5 g NaBH4Mixing and grinding for 30 min, calcining at 300 ℃ for 50 min in a tubular furnace in argon atmosphere, cooling to room temperature, respectively centrifugally cleaning the obtained product for 3 times by using ultrapure water and ethanol, and performing centrifugal cleaning in a vacuum drying oven at 35 ℃ for 12 h to obtain B-TiO2 NPs。
Example 4B-TiO2Preparation of NPs, the procedure was as follows:
5.0 g P25 titanium dioxide nanoparticles and 2.0 g NaBH4Mixing and grinding for 30 min, calcining at 300 ℃ for 60 min in a tubular furnace in argon atmosphere, cooling to room temperature, respectively centrifugally cleaning the obtained product for 3 times by using ultrapure water and ethanol, and performing centrifugal cleaning in a vacuum drying oven at 35 ℃ for 12 h to obtain B-TiO2 NPs。
Example 5 preparation of BiOI NSs, the procedure was as follows:
will coat with B-TiO2ITO conductive glasses of NPs are respectively 5 mM Bi (NO)3And 5 mM KI solution for 10 seconds, rinsed with ultra pure water, and the above cycle was repeated 20 times to obtain BiOI NSs.
Example 6 preparation of BiOI NSs, the procedure was as follows:
will coat with B-TiO2ITO conductive glasses of NPs are respectively 8 mM Bi (NO)3And 8 mM KI solution for 10 seconds, rinsed with ultra pure water, and the above cycle was repeated 30 times to obtain BiOI NSs.
Example 7 preparation of Au NPs, procedure was as follows:
50 mL HAuCl with mass fraction of 0.01%4The solution is boiled in oil bath at 120 ℃, and the mass fraction of 2.5 mL is 1% trisodium citrate solution is added into the solution, and the solution is boiled for 30 min under stirring until the solution turns purple red to prepare Au NPs solution.
Example 8 preparation of Au NPs, procedure was as follows:
55 mL HAuCl with the mass fraction of 0.01%4Boiling the solution in an oil bath at 120 ℃, adding 5.0 mL of trisodium citrate solution with the mass fraction of 1% into the solution, and continuously boiling for 30 min under stirring until the solution turns purple red to prepare the Au NPs solution.
Example 9 PbS/Co3O4The preparation method of the procalcitonin detection antibody complex solution comprises the following steps:
1)Co3O4preparation of polyhedra and amination thereof:
3.0 g Co(NO3)2·6H2o and 13 g of 2-methylimidazole were dissolved in 250 mL of methanol, respectively, and the 2-methylimidazole solution was added to Co (NO)3)2Aging in the solution at room temperature for 24 h, centrifuging and washing with methanol for 3 times, drying the obtained solid in a vacuum drying oven, and calcining in a tubular furnace at 350 deg.C for 120 min to obtain Co3O4Polyhedron, 0.05 g Co3O4Dispersing polyhedron in 10 mL ethanol solution containing 0.15 mL triaminopropyltriethoxysilane, ultrasonic dispersing for 30 min, refluxing at 70 deg.C for 24 h, centrifuging and washing with anhydrous ethanol for 3 times, and vacuum drying at 35 deg.C for 12 h to obtain aminated Co3O4A polyhedron;
2) preparation of PbS QDs:
mu.L of thioglycolic acid to 25 mL of 1 mmol/L Pb (NO)3)2After the solution was bubbled with nitrogen for 20 min and adjusted to pH 8 with 1 mol/L sodium hydroxide solution, 2 mL of 0.01 mol/L Na was added after bubbling for 20 min2Stirring the S solution for 4 hours under the nitrogen atmosphere to obtain a PbS QDs solution;
3)PbS/Co3O4the preparation of (1):
5 mg of aminated Co3O4Polyhedron, 0.8 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 0.1 g of N-hydroxysuccinimide dissolved in 15 mL of PbS QDs solution, and the mixture was stirred at room temperatureStirring for 2 h, washing with ultrapure water for 3 times, and then dispersing the product in 2 mL of ultrapure water;
4)PbS/Co3O4preparation of procalcitonin detection antibody complex solution:
2 mL of procalcitonin detection antibody solution of 10 mug/mL, 10 mug L of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide of 5 mg/mL and 10 mug L of N-hydroxysuccinimide of 1 mg/mL, shaking at 4 ℃ for 30 min, and 6 mg of PbS/Co3O4Adding the solution, shaking at 4 ℃ for 6 h, injecting 100 microliter bovine serum albumin solution with mass fraction of 1% into the solution, shaking at 4 ℃ for 12 h, centrifugally washing for 3 times by using phosphate buffer solution with pH of 7.4, and dispersing in 2 mL phosphate buffer solution with pH of 7.4.
Example 10 PbS/Co3O4The preparation method of the procalcitonin detection antibody complex solution comprises the following steps:
1)Co3O4preparation of polyhedra and amination thereof:
6.0 g Co(NO3)2·6H2o and 16 g of 2-methylimidazole were dissolved in 250 mL of methanol, respectively, and the 2-methylimidazole solution was added to Co (NO)3)2Aging in the solution at room temperature for 24 h, centrifuging and washing with methanol for 3 times, drying the obtained solid in a vacuum drying oven, and calcining in a tubular furnace at 350 deg.C for 120 min to obtain Co3O4Polyhedron, 0.1 g Co3O4Dispersing polyhedron in 10 mL ethanol solution containing 0.15 mL triaminopropyltriethoxysilane, ultrasonic dispersing for 30 min, refluxing at 70 deg.C for 24 h, centrifuging and washing with anhydrous ethanol for 3 times, and vacuum drying at 35 deg.C for 12 h to obtain aminated Co3O4A polyhedron;
2) preparation of PbS QDs:
mu.L of thioglycolic acid to 25 mL of 7 mmol/L Pb (NO)3)2After the solution was bubbled with nitrogen for 20 min and adjusted to pH 11 with 1 mol/L sodium hydroxide solution, 2 mL of 0.015 mol/L Na was added after bubbling for 20 min2Stirring the S solution for 4 hours under the nitrogen atmosphere to obtain a PbS QDs solution;
3)PbS/Co3O4the preparation of (1):
8 mg of aminated Co3O4Polyhedron, 0.8 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 0.1 g of N-hydroxysuccinimide are dissolved in 15 mL of PbS QDs solution, the mixed solution is stirred for 2 hours at room temperature, and is washed for 3 times by ultrapure water, and then the product is dispersed in 2 mL of ultrapure water;
4)PbS/Co3O4preparation of procalcitonin detection antibody complex solution:
2 mL of procalcitonin detection antibody solution of 10 mug/mL, 10 mug L of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide of 5 mg/mL and 10 mug L of N-hydroxysuccinimide of 1 mg/mL, shaking at 4 ℃ for 30 min, and 6 mg of PbS/Co3O4Adding the solution, shaking at 4 ℃ for 6 h, injecting 100 microliter bovine serum albumin solution with mass fraction of 1% into the solution, shaking at 4 ℃ for 12 h, centrifugally washing for 3 times by using phosphate buffer solution with pH of 7.4, and dispersing in 2 mL phosphate buffer solution with pH of 7.4.
Example 11A PbS/Co based formulation3O4The compound signal weakening type photoelectrochemical immunosensor is used for detecting procalcitonin, and comprises the following detection steps:
(1) testing by using an electrochemical workstation and a three-electrode system, wherein the prepared ITO modified electrode is a working electrode, a saturated calomel electrode is a reference electrode, a platinum wire electrode is an auxiliary electrode, and the testing is carried out in 10 mL of phosphate buffer solution with pH of 7.4 and containing 0.1 mol/L ascorbic acid;
(2) detecting the analyte by using a time-current method, setting the voltage to be 0V, operating the time to be 100 s, and irradiating the LED lamp with the wavelength of 400-450 nm;
(3) when the background current tends to be stable, turning on the lamp every 10 s for continuously irradiating for 10 s, then recording the photocurrent, and drawing a working curve;
(4) and (3) replacing the procalcitonin standard solution with the procalcitonin sample solution to be detected for detection, and checking the detection result through a working curve.
Example 12A PbS/Co based formulation3O4A photoelectrochemical immunosensor with reduced complex signal for reducing procalcitoninAnd (3) detection, which comprises the following steps:
(1) testing by using an electrochemical workstation and a three-electrode system, wherein the prepared ITO modified electrode is a working electrode, a saturated calomel electrode is a reference electrode, a platinum wire electrode is an auxiliary electrode, and the testing is carried out in 10 mL of phosphate buffer solution with pH of 7.4 and containing 0.1 mol/L ascorbic acid;
(2) detecting the analyte by using a time-current method, setting the voltage to be 0V, operating the time to be 100 s, and irradiating the LED lamp with the wavelength of 400-450 nm;
(3) when the background current tends to be stable, turning on the lamp every 10 s for continuously irradiating for 10 s, then recording the photocurrent, and drawing a working curve;
(4) and (3) replacing the procalcitonin standard solution with the procalcitonin sample solution to be detected for detection, and checking the detection result through a working curve.
Example 13 the sensor constructed in example 1 and example 2 was used to detect procalcitonin standard solution according to the detection methods of examples 11 and 12, and the linear detection range of the sensor was found to be 0.1 pg/mL-50 ng/mL, and the detection limit was found to be 0.02 ng/mL.
Claims (5)
1. Based on PbS/Co3O4The preparation method of the compound signal weakening type photoelectrochemical immunosensor is characterized by comprising the following steps of:
1) cutting a large piece of ITO conductive glass into 2.0 cm multiplied by 0.8 cm, respectively ultrasonically cleaning the large piece of ITO conductive glass for 30 min by using acetone, ethanol and ultrapure water in sequence, and drying the large piece of ITO conductive glass by using nitrogen;
2) dropwise adding B-TiO uniformly dispersed by 10 mu L and 5-8 mg/mL2NPs suspension is dried at room temperature to the conductive surface of the ITO conductive glass to prepare the B-TiO2An NPs electrode;
3) mixing B-TiO2NPs electrodes are at 5-10 mM Bi (NO)3Dipping the mixture in 5-10 mM KI solution for 10 s, washing the mixture with ultrapure water, and repeating the steps for 20-30 times to obtain the B-TiO2An NPs/BiOI NSs electrode;
4) dropwise adding 8 mu L Au NPs solution to B-TiO2Preparing B-TiO on the surface of the NPs/BiOI NSs electrode2 NPsa/BiOI NSs/Au NPs electrode;
5) dropwise adding 5 muL and 8-12 mug/mL procalcitonin capture antibody solution to the surface of the modified electrode, airing in a refrigerator at 4 ℃, and washing with ultrapure water;
6) dropwise adding 3 muL of bovine serum albumin solution with the mass fraction of 1.0% to the surface of the modified electrode to seal the nonspecific active sites on the surface of the electrode, drying in a refrigerator at 4 ℃, and washing with ultrapure water;
7) dropwise adding 5 muL and 0.1 pg/mL-50 ng/mL of procalcitonin antigen standard solution to the surface of the modified electrode, airing in a refrigerator at 4 ℃, and washing with ultrapure water;
8) dropwise adding 5 mu L and 3.0-5.0 mg/mL PbS/Co3O4And (3) adding the procalcitonin detection antibody compound solution to the surface of the electrode, incubating in a refrigerator at 4 ℃ for 60 min, washing with ultrapure water, and preparing the completely modified electrode, namely the procalcitonin signal detection weakening type photoelectrochemical immunosensor.
2. A PbS/Co based polymer according to claim 13O4Preparation method of compound signal-weakening type photoelectrochemical immunosensor, B-TiO2Preparation of NPs, the procedure was as follows:
4.0-5.0 g P25 g of titanium dioxide nanoparticles and 1.5-2.0 g of NaBH4Mixing and grinding for 30 min, calcining in a tubular furnace at 300 ℃ for 50-60 min under argon atmosphere, cooling to room temperature, respectively centrifugally cleaning the obtained product with ultrapure water and ethanol for 3 times, and drying in a vacuum drying oven at 35 ℃ for 12 h to obtain B-TiO2 NPs。
3. A PbS/Co based polymer according to claim 13O4The preparation method of the compound signal attenuation type photoelectrochemical immunosensor comprises the following steps of:
50-55 mL HAuCl with mass fraction of 0.01%4Boiling the solution in an oil bath at 120 ℃, adding 2.5-5.0 mL of trisodium citrate solution with the mass fraction of 1% into the solution, and boiling for 30 min under stirring until the solution turns purple red to prepare the Au NPs solution.
4. A PbS/Co based polymer according to claim 13O4A preparation method of a compound signal-weakening type photoelectrochemical immunosensor, and PbS/Co3O4The preparation method of the procalcitonin detection antibody complex solution comprises the following steps:
1)Co3O4preparation of polyhedron:
3.0-6.0 g Co(NO3)2·6H2o and 13-16 g of 2-methylimidazole were dissolved in 250 mL of methanol, respectively, and the 2-methylimidazole solution was added to Co (NO)3)2Aging in the solution at room temperature for 24 h, centrifuging and washing with methanol for 3 times, drying the obtained solid in a vacuum drying oven, and placing in a tubular furnace at 350 deg.C for 120 min to obtain Co3O4A polyhedron;
2) amination of Co3O4Preparation of polyhedron:
0.05-0.1 g Co3O4dispersing polyhedron in 10 mL ethanol solution containing 0.15 mL triaminopropyltriethoxysilane, ultrasonic dispersing for 30 min, refluxing at 70 deg.C for 24 h, centrifuging and washing with anhydrous ethanol for 3 times, and vacuum drying at 35 deg.C for 12 h to obtain aminated Co3O4A polyhedron;
3) preparation of PbS QDs:
adding 7-21 μ L thioglycolic acid to 25 mL of 1-7 mmol/L Pb (NO)3)2After the solution is bubbled for 20 min under nitrogen, 1 mol/L sodium hydroxide solution is used for adjusting the pH value to 5-12, and after the solution is bubbled for 20 min, 2 mL of 0.01-0.015 mol/L Na is added2Stirring the S solution for 4 hours under the nitrogen atmosphere to obtain a PbS QDs solution;
4)PbS/Co3O4the preparation of (1):
5-10 mg aminated Co3O4Polyhedron, 0.8 g 1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide and 0.1 g N-hydroxysuccinimide are dissolved in 15 mL PbS QDs solution, the mixed solution is stirred for 2 h at room temperature, washed for 3 times by ultrapure water, and then the product is dispersed in 2 mL ultrapure water;
5)PbS/Co3O4preparation of procalcitonin detection antibody complex solution:
2 mL of procalcitonin detection antibody solution of 10 mug/mL, 10 mug L of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide of 5 mg/mL and 10 mug L of N-hydroxysuccinimide of 1 mg/mL, shaking at 4 ℃ for 30 min, and 6 mg of PbS/Co3O4Adding the solution, shaking at 4 ℃ for 6 h, injecting 100 microliter bovine serum albumin solution with mass fraction of 1% into the solution, shaking at 4 ℃ for 12 h, centrifugally washing for 3 times by using phosphate buffer solution with pH of 7.4, and dispersing in 2 mL phosphate buffer solution with pH of 7.4.
5. A PbS/Co based composition prepared by the method of claim 13O4The compound signal weakening type photoelectrochemical immunosensor is characterized by being used for detecting procalcitonin, and the detection steps are as follows:
(1) testing by using an electrochemical workstation and a three-electrode system, wherein the prepared procalcitonin signal detection weakening type photoelectrochemical immunosensor is used as a working electrode, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, and the testing is carried out in 10 mL of phosphate buffer solution with pH of 7.4 and containing 0.1 mol/L ascorbic acid;
(2) detecting the analyte by using a time-current method, setting the voltage to be 0V, operating the time to be 100 s, and irradiating the LED lamp with the wavelength of 400-450 nm;
(3) when the background current tends to be stable, turning on the lamp every 10 s for continuously irradiating for 10 s, then recording the photocurrent, and drawing a working curve by using procalcitonin standard solutions with different concentrations as recognition antigens;
(4) and (3) replacing the procalcitonin standard solution with the procalcitonin sample solution to be detected for detection, and checking the detection result through a working curve.
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