CN110530949A - A kind of preparation method and application based on copper nano-cluster-Resonance energy transfer system construction immunosensor - Google Patents
A kind of preparation method and application based on copper nano-cluster-Resonance energy transfer system construction immunosensor Download PDFInfo
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- 239000010949 copper Substances 0.000 title claims abstract description 56
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 title claims abstract description 26
- 229910052802 copper Inorganic materials 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 238000002165 resonance energy transfer Methods 0.000 title claims abstract description 17
- 238000010276 construction Methods 0.000 title claims abstract description 14
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 230000000694 effects Effects 0.000 claims abstract description 7
- 239000002086 nanomaterial Substances 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 54
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 14
- 239000000427 antigen Substances 0.000 claims description 12
- 102000036639 antigens Human genes 0.000 claims description 12
- 108091007433 antigens Proteins 0.000 claims description 12
- 229910052799 carbon Inorganic materials 0.000 claims description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 108010048233 Procalcitonin Proteins 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 10
- 238000011010 flushing procedure Methods 0.000 claims description 10
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 10
- 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 description 10
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 7
- 239000012888 bovine serum Substances 0.000 claims description 7
- 230000003287 optical effect Effects 0.000 claims description 7
- 150000003839 salts Chemical class 0.000 claims description 7
- 210000002966 serum Anatomy 0.000 claims description 7
- SJUCACGNNJFHLB-UHFFFAOYSA-N O=C1N[ClH](=O)NC2=C1NC(=O)N2 Chemical compound O=C1N[ClH](=O)NC2=C1NC(=O)N2 SJUCACGNNJFHLB-UHFFFAOYSA-N 0.000 claims description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 5
- 229910021607 Silver chloride Inorganic materials 0.000 claims description 5
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims description 5
- 230000000890 antigenic effect Effects 0.000 claims description 5
- 238000005253 cladding Methods 0.000 claims description 5
- 229910052593 corundum Inorganic materials 0.000 claims description 5
- 238000002484 cyclic voltammetry Methods 0.000 claims description 5
- 239000008367 deionised water Substances 0.000 claims description 5
- 229910021641 deionized water Inorganic materials 0.000 claims description 5
- UNXNGGMLCSMSLH-UHFFFAOYSA-N dihydrogen phosphate;triethylazanium Chemical compound OP(O)(O)=O.CCN(CC)CC UNXNGGMLCSMSLH-UHFFFAOYSA-N 0.000 claims description 5
- 238000001548 drop coating Methods 0.000 claims description 5
- 238000005401 electroluminescence Methods 0.000 claims description 5
- 235000019441 ethanol Nutrition 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 239000008363 phosphate buffer Substances 0.000 claims description 5
- 239000008055 phosphate buffer solution Substances 0.000 claims description 5
- 229910052697 platinum Inorganic materials 0.000 claims description 5
- 238000005498 polishing Methods 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 5
- 239000012086 standard solution Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000010408 sweeping Methods 0.000 claims description 5
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000003643 water by type Substances 0.000 claims description 5
- 229910001845 yogo sapphire Inorganic materials 0.000 claims description 5
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims description 4
- 229910001431 copper ion Inorganic materials 0.000 claims description 4
- 208000035143 Bacterial infection Diseases 0.000 claims description 2
- 230000001154 acute effect Effects 0.000 claims description 2
- 208000022362 bacterial infectious disease Diseases 0.000 claims description 2
- 239000003550 marker Substances 0.000 claims description 2
- 238000002866 fluorescence resonance energy transfer Methods 0.000 abstract description 5
- 238000010791 quenching Methods 0.000 abstract description 4
- 230000000171 quenching effect Effects 0.000 abstract description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 abstract description 3
- 229940098773 bovine serum albumin Drugs 0.000 abstract description 3
- 238000001228 spectrum Methods 0.000 abstract description 2
- 239000010931 gold Substances 0.000 abstract 3
- 239000000090 biomarker Substances 0.000 abstract 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 abstract 1
- 229910052737 gold Inorganic materials 0.000 abstract 1
- 238000011065 in-situ storage Methods 0.000 abstract 1
- 239000002105 nanoparticle Substances 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 8
- 229910021397 glassy carbon Inorganic materials 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- GDSOZVZXVXTJMI-SNAWJCMRSA-N (e)-1-methylbut-1-ene-1,2,4-tricarboxylic acid Chemical compound OC(=O)C(/C)=C(C(O)=O)\CCC(O)=O GDSOZVZXVXTJMI-SNAWJCMRSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Natural products OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- -1 aglucon Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000004454 trace mineral analysis Methods 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
Classifications
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- 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
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Hematology (AREA)
- Electrochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Nanotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
A kind of preparation method and application based on copper nano-cluster-Resonance energy transfer system construction immunosensor, the invention belong to nano material and the field Resonance energy transfer FRET；The present invention is using the copper nanocluster Cu NCs that bovine serum albumin BSA is wrapped up as energy donor, and on its surface using L-AA growth in situ gold nanoparticle Au NPs as energy acceptor, obtain Cu NCs-Au NPs three-dimensional resonance structure, energy is transmitted for the non-radiative activity mode between receptor pair by dipole-dipole, six powers of its delivery rate and distance are in inverse ratio, the height ratio overlapping of spectrum between the two and super close matching are adjusted the distance, the ECL for significantly having quenched Cu NCs emits and controls luminous intensity in the reasonable scope, a kind of new ECL-FRET energy transfer new model is provided for the trace detection of biomarker, it is probed into for the quenching type ECL mechanism for sensing system and provides a kind of completely new thinking.
Description
Technical field
The invention belongs to nano sciences and immunoassay field.
Background technique
Copper nanocluster is that nano science develops a kind of novel nano-material for being risen, good optical stability,
Big stoke shift, high quantum yield and good biocompatibility are that its application in bio-sensing field is established
Solid foundation.Copper nanocluster can be synthesized in alkaline environment by a variety of micro- reducing agents such as nucleic acid, aglucon, protein,
In the electroluminescent optical signal of shorter wavelength is shown under low potential using bovine serum albumin as the copper nanocluster of template, meanwhile,
Its surface coated bovine serum albumin exposes a large amount of functional group's active site, provides greatly for the modification of biomolecule
It is convenient, there is advantageous advantage in immunoassay compared to electroluminors such as semiconductor-quantum-points.However, it is higher
Electroluminescent efficiency also allow sensing platform signal be difficult to control, be based on this it is proposed that using Resonance energy transfer system come
The needs of object trace analysis can be met by controlling its luminous intensity, it is intended to the mechanism of system is sensed for quenching type ECL
It probes into and provides a kind of completely new thinking.
Summary of the invention
Technical assignment of the invention first is that in order to make up the deficiency of ECL-FRET model evidence at this stage, a kind of Cu is provided
The preparation method of NCs-Au NPs three-dimensional resonance structure.This method is to realize that three conditions that Resonance energy transfer to be met are to grab
Hand, gets rid of the unilateral behavior that traditional ECL-FRET sensing model emphasizes merely spectra overlapping, and emphasis probes into verifying quenching efficiency
The relationship of distance between co-receptor probes into for the quenching type ECL mechanism for sensing system and provides a kind of completely new thinking.
Technical assignment two of the invention is to provide the ECL-FRET model in the purposes in bio-sensing field, is based on the mould
The biosensor of type research and development can quickly detect Procalcitonin, and high sensitivity, detection limit is low, favorable reproducibility, in clinical detection
In have broad application prospects.
To achieve the above object, The technical solution adopted by the invention is as follows:
1. a kind of preparation method based on copper nano-cluster-Resonance energy transfer system construction immunosensor, feature exist
In, comprising the following steps:
(1) glass-carbon electrode is cleaned by ultrasonic 30 s in ethyl alcohol and deionized water respectively, successively with 1.0 μm, 0.3 μm and
0.05 μm of Al2O3Polishing powder, which polishes it, is allowed to smooth as similar mirror surface, with being dried with nitrogen；
(2) 6 μ L are uniformly dispersed, Cu NCs-Au NPs solution drop coating that concentration is 1 ~ 3 mg/mL is to glass-carbon electrode table
Face, at room temperature naturally dry；
(3) the above-mentioned glass-carbon electrode dried is inserted into the recombinant protein A solution of 5 ~ 10 μ g/mL, in 4oHatch 30 under C
Min, ultrapure water are cleaned, at room temperature naturally dry；
(4) bovine serum albumen solution that 6 μ L mass fractions are 0.1% is added dropwise, is allowed to close nonspecific activity site, uses pH
7.4 phosphate buffer solution rinses electrode surface, at room temperature naturally dry；
(5) the antibody standard solution that 6 μ L concentration are 1 ~ 2 μ g/mL is added dropwise, in 4oHatch 1 h under C, with the phosphorus of pH 7.4
Hydrochlorate buffer solution flushing electrode surface, 4oC dries；
(6) antigenic solution corresponding to antibody of 6 μ L unknown concentrations is added dropwise, in 4oHatch 1 h under C, with the phosphoric acid of pH 7.4
Salt buffer solution flushing electrode surface, 4oC dries, and sensor building finishes.
A kind of preparation method based on copper nano-cluster-Resonance energy transfer system construction immunosensor described in 2.,
The Cu NCs-Au NPs solution, prepares according to the following steps:
It at room temperature, is 1 ~ 2 mg/mL's by 1 mL, the copper-bath that concentration is 5 ~ 10 mmol/L and 1 mL, concentration
Bovine serum albumen solution is sufficiently mixed, and after being vigorously stirred 2 ~ 5 min, 0.1 ~ 1 mL is added, concentration is 1 ~ 2 mol/L's
Then the mixed liquor is placed in 65 by sodium hydroxide solutiono12 h in C water-bath, until solution colour becomes brown from light blue,
At this point, copper ion is successfully restored by BSA, the copper nano-cluster Cu NCs of BSA cladding is obtained.It is with concentration by the Cu NCs of preparation
The L-AA solution of 10 mmol/L mixes, and 5 mL deionized waters are then added and are diluted, then, in Xiang Shangshu solution
The chlorauric acid solution of 50 ~ 150 uL, mass fraction 2% is added, obtains the Cu NCs-Au NPs of atropurpureus after persistently stirring 2 h
Three-dimensional resonance nanostructure.
A kind of preparation method based on copper nano-cluster-Resonance energy transfer system construction immunosensor described in 3.,
It is characterized in that, the antigen is human body acute bacterial infection and pyemic marker Procalcitonin.
A kind of preparation method system based on copper nano-cluster-Resonance energy transfer system construction immunosensor described in 4.
Standby sensor is used to detect the concentration of Procalcitonin.
The concentration of detection Procalcitonin described in 5., which is characterized in that detecting step is as follows:
(1) use the three-electrode system of electrochemical workstation as excitaton source, Ag/AgCl electrode is as reference electrode, platinum electrode
As to electrode, prepared electroluminescent sensor is as working electrode, by electrochemical workstation and ultraweak optical detector
Combination, photomultiplier tube high pressure are set as 800 V, and cyclic voltammetry scan current potential is 0 ~ 1.5 V, and sweeping speed is 200 mV/s；
(2) in 50 ~ 80 mmol/L triethylamine phosphate buffers, pass through electroluminescent containing concentration in 10 mL, pH 7.4
System detects the electroluminescence signal intensity in a series of determinand Modified antigen state lower sensor of various concentrations, draws
Working curve；
(3) determined antigen is replaced to detect practical blood serum sample.
Beneficial achievement of the invention
(1) three-dimensional resonance ECL sensing model Cu NCs-Au NPs is prepared for based on copper nanocluster for the first time, which passes through confession
The non-radiative activity mode of dipole-dipole, which transmits energy, between receptor pair can effectively control the ECL intensity of copper nano-cluster；
(2) present invention is based on three-dimensional resonance structure C u NCs-Au NPs for the first time, constructs a kind of ECL sensing platform for dropping calcium
Plain former actual sample detection, the transducer sensitivity is high, and detection range is wide, and detection limit is down to 2.9 fg/mL.
Specific embodiment:
Present invention will be further explained below with reference to specific examples, it should be appreciated that these embodiments be merely to illustrate the present invention without
For limiting the scope of the invention.
A kind of preparation method based on copper nano-cluster-Resonance energy transfer system construction immunosensor of embodiment 1.,
Characterized by comprising the following steps:
(1) glass-carbon electrode is cleaned by ultrasonic 30 s in ethyl alcohol and deionized water respectively, successively with 1.0 μm, 0.3 μm and
0.05 μm of Al2O3Polishing powder, which polishes it, is allowed to smooth as similar mirror surface, with being dried with nitrogen；
(2) 6 μ L are uniformly dispersed, Cu NCs-Au NPs solution drop coating that concentration is 1 mg/mL is to glassy carbon electrode surface, room
The lower naturally dry of temperature；
(3) the above-mentioned glass-carbon electrode dried is inserted into the recombinant protein A solution of 5 μ g/mL, in 4oHatch 30 min under C,
Ultrapure water is cleaned, at room temperature naturally dry；
(4) bovine serum albumen solution that 6 μ L mass fractions are 0.1% is added dropwise, is allowed to close nonspecific activity site, uses pH
7.4 phosphate buffer solution rinses electrode surface, at room temperature naturally dry；
(5) the antibody standard solution that 6 μ L concentration are 1 μ g/mL is added dropwise, in 4oHatch 1 h under C, with the phosphate of pH 7.4
Buffer solution flushing electrode surface, 4oC dries；
(6) antigenic solution corresponding to antibody of 6 μ L unknown concentrations is added dropwise, in 4oHatch 1 h under C, with the phosphoric acid of pH 7.4
Salt buffer solution flushing electrode surface, 4oC dries, and sensor building finishes.
A kind of preparation method based on copper nano-cluster-Resonance energy transfer system construction immunosensor of embodiment 2.,
Characterized by comprising the following steps:
(1) glass-carbon electrode is cleaned by ultrasonic 30 s in ethyl alcohol and deionized water respectively, successively with 1.0 μm, 0.3 μm and
0.05 μm of Al2O3Polishing powder, which polishes it, is allowed to smooth as similar mirror surface, with being dried with nitrogen；
(2) 6 μ L are uniformly dispersed, Cu NCs-Au NPs solution drop coating that concentration is 2 mg/mL is to glassy carbon electrode surface, room
The lower naturally dry of temperature；
(3) the above-mentioned glass-carbon electrode dried is inserted into the recombinant protein A solution of 7.5 μ g/mL, in 4oHatch 30 under C
Min, ultrapure water are cleaned, at room temperature naturally dry；
(4) bovine serum albumen solution that 6 μ L mass fractions are 0.1% is added dropwise, is allowed to close nonspecific activity site, uses pH
7.4 phosphate buffer solution rinses electrode surface, at room temperature naturally dry；
(5) the antibody standard solution that 6 μ L concentration are 1.5 μ g/mL is added dropwise, in 4oHatch 1 h under C, with the phosphoric acid of pH 7.4
Salt buffer solution flushing electrode surface, 4oC dries；
(6) antigenic solution corresponding to antibody of 6 μ L unknown concentrations is added dropwise, in 4oHatch 1 h under C, with the phosphoric acid of pH 7.4
Salt buffer solution flushing electrode surface, 4oC dries, and sensor building finishes.
A kind of preparation method based on copper nano-cluster-Resonance energy transfer system construction immunosensor of embodiment 3.,
Characterized by comprising the following steps:
(1) glass-carbon electrode is cleaned by ultrasonic 30 s in ethyl alcohol and deionized water respectively, successively with 1.0 μm, 0.3 μm and
0.05 μm of Al2O3Polishing powder, which polishes it, is allowed to smooth as similar mirror surface, with being dried with nitrogen；
(2) 6 μ L are uniformly dispersed, Cu NCs-Au NPs solution drop coating that concentration is 3 mg/mL is to glassy carbon electrode surface, room
The lower naturally dry of temperature；
(3) the above-mentioned glass-carbon electrode dried is inserted into the recombinant protein A solution of 10 μ g/mL, in 4oHatch 30 min under C,
Ultrapure water is cleaned, at room temperature naturally dry；
(4) bovine serum albumen solution that 6 μ L mass fractions are 0.1% is added dropwise, is allowed to close nonspecific activity site, uses pH
7.4 phosphate buffer solution rinses electrode surface, at room temperature naturally dry；
(5) the antibody standard solution that 6 μ L concentration are 2 μ g/mL is added dropwise, in 4oHatch 1 h under C, with the phosphate of pH 7.4
Buffer solution flushing electrode surface, 4oC dries；
(6) antigenic solution corresponding to antibody of 6 μ L unknown concentrations is added dropwise, in 4oHatch 1 h under C, with the phosphoric acid of pH 7.4
Salt buffer solution flushing electrode surface, 4oC dries, and sensor building finishes.
4. Cu NCs-Au NPs solution of embodiment, prepares according to the following steps:
At room temperature, the cow's serum egg for being 1 mg/mL by copper-bath and 1 mL, concentration that 1 mL, concentration are 5 mmol/L
White solution is sufficiently mixed, and after being vigorously stirred 2 min, 0.1 mL is added, the sodium hydroxide solution that concentration is 1 mol/L, then will
The mixed liquor is placed in 65o12 h in C water-bath, until solution colour becomes brown from light blue, at this point, copper ion is by BSA
Success restores, and obtains the copper nano-cluster Cu NCs of BSA cladding.The Cu NCs of preparation is anti-bad for the L- of 10 mmol/L with concentration
The mixing of hematic acid solution is then added 5 mL deionized waters and is diluted, then, 50 uL, mass fraction is added in Xiang Shangshu solution
2% chlorauric acid solution obtains the Cu NCs-Au NPs three-dimensional resonance nanostructure of atropurpureus after persistently stirring 2 h.
5. Cu NCs-Au NPs solution of embodiment, prepares according to the following steps:
At room temperature, by the copper-bath that 1 mL, concentration are 7.5 mmol/L and the ox blood that 1 mL, concentration are 1.5 mg/mL
Albumin soln is sufficiently mixed, and after being vigorously stirred 3.5 min, it is molten for the sodium hydroxide of 1.5 mol/L that 0.5 mL, concentration is added
Then the mixed liquor is placed in 65 by liquido12 h in C water-bath, until solution colour from light blue becomes brown, at this point, copper from
Son is successfully restored by BSA, obtains the copper nano-cluster Cu NCs of BSA cladding.It is 10 mmol/L by the Cu NCs of preparation and concentration
L-AA solution mixing, then be added 5 mL deionized waters be diluted, then, be added 100 in Xiang Shangshu solution
The chlorauric acid solution of uL, mass fraction 2% obtain the Cu NCs-Au NPs three-dimensional resonance nanometer of atropurpureus after persistently stirring 2 h
Structure.
6. Cu NCs-Au NPs solution of embodiment, prepares according to the following steps:
At room temperature, the cow's serum egg for being 2 mg/mL by copper-bath and 1 mL, concentration that 1 mL, concentration are 10 mmol/L
White solution is sufficiently mixed, and after being vigorously stirred 5 min, 1 mL is added, the sodium hydroxide solution that concentration is 2 mol/L, then should
Mixed liquor is placed in 65o12 h in C water-bath, until solution colour from light blue becomes brown, at this point, copper ion by BSA at
Function reduction, obtains the copper nano-cluster Cu NCs of BSA cladding.The L- Vitamin C for being 10 mmol/L by the Cu NCs of preparation and concentration
Acid solution mixing is then added 5 mL deionized waters and is diluted, then, 150 uL, mass fraction is added in Xiang Shangshu solution
2% chlorauric acid solution obtains the Cu NCs-Au NPs three-dimensional resonance nanostructure of atropurpureus after persistently stirring 2 h.
Embodiment 7. detects Procalcitonin concentration
(1) use the three-electrode system of electrochemical workstation as excitaton source, Ag/AgCl electrode is as reference electrode, platinum electrode
As to electrode, prepared electroluminescent sensor is as working electrode, by electrochemical workstation and ultraweak optical detector
Combination, photomultiplier tube high pressure are set as 800 V, and cyclic voltammetry scan current potential is 0 ~ 1.5 V, and sweeping speed is 200 mV/s；
(2) it is in 50 mmol/L triethylamine phosphate buffers containing concentration in 10 mL, pH 7.4, by electro-luminescent systems,
The electroluminescence signal intensity in a series of determinand Modified antigen state lower sensor of various concentrations is detected, it is bent to draw work
Line；
(3) determined antigen is replaced to detect practical blood serum sample.
Embodiment 8. detects Procalcitonin concentration
(1) use the three-electrode system of electrochemical workstation as excitaton source, Ag/AgCl electrode is as reference electrode, platinum electrode
As to electrode, prepared electroluminescent sensor is as working electrode, by electrochemical workstation and ultraweak optical detector
Combination, photomultiplier tube high pressure are set as 800 V, and cyclic voltammetry scan current potential is 0 ~ 1.5 V, and sweeping speed is 200 mV/s；
(2) it is in 65 mmol/L triethylamine phosphate buffers containing concentration in 10 mL, pH 7.4, by electro-luminescent systems,
The electroluminescence signal intensity in a series of determinand Modified antigen state lower sensor of various concentrations is detected, it is bent to draw work
Line；
(3) determined antigen is replaced to detect practical blood serum sample.
Embodiment 9 detects Procalcitonin concentration
(1) use the three-electrode system of electrochemical workstation as excitaton source, Ag/AgCl electrode is as reference electrode, platinum electrode
As to electrode, prepared electroluminescent sensor is as working electrode, by electrochemical workstation and ultraweak optical detector
Combination, photomultiplier tube high pressure are set as 800 V, and cyclic voltammetry scan current potential is 0 ~ 1.5 V, and sweeping speed is 200 mV/s；
(2) it is in 80 mmol/L triethylamine phosphate buffers containing concentration in 10 mL, pH 7.4, by electro-luminescent systems,
The electroluminescence signal intensity in a series of determinand Modified antigen state lower sensor of various concentrations is detected, it is bent to draw work
Line；
(3) determined antigen is replaced to detect practical blood serum sample.
Claims (5)
1. a kind of preparation method based on copper nano-cluster-Resonance energy transfer system construction immunosensor, which is characterized in that
The following steps are included:
(1) glass-carbon electrode is cleaned by ultrasonic 30 s in ethyl alcohol and deionized water respectively, successively with 1.0 μm, 0.3 μm and
0.05 μm of Al2O3Polishing powder, which polishes it, is allowed to smooth as similar mirror surface, with being dried with nitrogen；
(2) 6 μ L are uniformly dispersed, Cu NCs-Au NPs solution drop coating that concentration is 1 ~ 3 mg/mL is to glass-carbon electrode table
Face, at room temperature naturally dry；
(3) the above-mentioned glass-carbon electrode dried is inserted into the recombinant protein A solution of 5 ~ 10 μ g/mL, in 4oHatch 30 under C
Min, ultrapure water are cleaned, at room temperature naturally dry；
(4) bovine serum albumen solution that 6 μ L mass fractions are 0.1% is added dropwise, is allowed to close nonspecific activity site, uses pH
7.4 phosphate buffer solution rinses electrode surface, at room temperature naturally dry；
(5) the antibody standard solution that 6 μ L concentration are 1 ~ 2 μ g/mL is added dropwise, in 4oHatch 1 h under C, with the phosphoric acid of pH 7.4
Salt buffer solution flushing electrode surface, 4oC dries；
(6) antigenic solution corresponding to antibody of 6 μ L unknown concentrations is added dropwise, in 4oHatch 1 h under C, with the phosphoric acid of pH 7.4
Salt buffer solution flushing electrode surface, 4oC dries, and sensor building finishes.
2. a kind of system based on copper nano-cluster-Resonance energy transfer system construction immunosensor as described in claim 1
Preparation Method, the Cu NCs-Au NPs solution, prepares according to the following steps:
It at room temperature, is 1 ~ 2 mg/mL's by 1 mL, the copper-bath that concentration is 5 ~ 10 mmol/L and 1 mL, concentration
Bovine serum albumen solution is sufficiently mixed, and after being vigorously stirred 2 ~ 5 min, 0.1 ~ 1 mL is added, concentration is 1 ~ 2 mol/L's
Then the mixed liquor is placed in 65 by sodium hydroxide solutiono12 h in C water-bath, until solution colour becomes brown from light blue,
At this point, copper ion is successfully restored by BSA, the copper nano-cluster Cu NCs of BSA cladding is obtained；
The Cu NCs of preparation is mixed with the L-AA solution that concentration is 10 mmol/L, 5 mL deionized waters are then added
It is diluted, then, the chlorauric acid solution of 50 ~ 150 uL, mass fraction 2% is added in Xiang Shangshu solution, persistently stirs 2 h
The Cu NCs-Au NPs three-dimensional resonance nanostructure of atropurpureus is obtained afterwards.
3. a kind of system based on copper nano-cluster-Resonance energy transfer system construction immunosensor as described in claim 1
Preparation Method, which is characterized in that the antigen is human body acute bacterial infection and pyemic marker Procalcitonin.
4. a kind of system based on copper nano-cluster-Resonance energy transfer system construction immunosensor as described in claim 1
The sensor of Preparation Method preparation is used to detect the concentration of Procalcitonin.
5. the concentration of detection Procalcitonin as claimed in claim 4, which is characterized in that detecting step is as follows:
(1) use the three-electrode system of electrochemical workstation as excitaton source, Ag/AgCl electrode is as reference electrode, platinum electrode
As to electrode, prepared electroluminescent sensor is as working electrode, by electrochemical workstation and ultraweak optical detector
Combination, photomultiplier tube high pressure are set as 800 V, and cyclic voltammetry scan current potential is 0 ~ 1.5 V, and sweeping speed is 200 mV/s；
(2) in 50 ~ 80 mmol/L triethylamine phosphate buffers, pass through electroluminescent containing concentration in 10 mL, pH 7.4
System detects the electroluminescence signal intensity in a series of determinand Modified antigen state lower sensor of various concentrations, draws
Working curve；
(3) determined antigen is replaced to detect practical blood serum sample.
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