CN107543851B - A kind of preparation method and application of the electrochemical luminescence sensor based on silver oxalate bridging tris (bipyridine) ruthenium nano-complex - Google Patents
A kind of preparation method and application of the electrochemical luminescence sensor based on silver oxalate bridging tris (bipyridine) ruthenium nano-complex Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method and applications of electrochemical luminescence sensor based on silver oxalate bridging tris (bipyridine) ruthenium nano-complex, belong to nano material and electrochemical analysis techniques field.Electrochemical luminescence sensor is prepared using golden functional graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite and ferroso-ferric oxide@poly-dopamine core-shell composite material, the Sensitive Detection for N-terminal B-type natriuretic peptide.
Description
Technical field
The present invention relates to a kind of electrochemical luminescence sensors based on silver oxalate bridging tris (bipyridine) ruthenium nano-complex
Preparation method and application belongs to nano material and analytical chemistry field.
Background technique
N-terminal B-type natriuretic peptide is the more chemokines of Recent study, screening, curative effect evaluation in heart failure
With in Index for diagnosis have important value.The index most sensitive and most special as cardiac disorder, N-terminal B-typeNatriuretic Peptide
Original has important clinical indices meaning.It is the more sensitive cardiac nerve hormone of a kind of pair of volume load, mainly by ventricle
Cardiac muscle cell secreted when vascular capacitance increases and pressure load increases, the left indoor pressure of reflection and wall tension variation.
The patient of Diastolic heart failure, even if the corresponding clinical symptoms without cardiac insufficiency, Plasma N-Terminal B-typeNatriuretic Peptide original
It can increase, can be used as the New Set of early diagnosis diastolic heart failure, how efficiently and accurately realizes N-terminal Type B benefit sodium
The detection of peptide former is the urgent problem to be solved faced now.
In recent years, electrochemical luminescence method is widely paid close attention to due to its advantage outstanding, and electrochemical luminescence is electrification
It learns and two methods of the combination of chemiluminescence, the advantages of being integrated with two methods: first, it shines not needing to apply and external swash
Light emitting source, background interference is small, and the range of linearity is wide, therefore is shown higher by electrogenerated chemiluminescence to analyte detection to be measured
Sensitivity.Second, electrogenerated chemiluminescence can improve it by selecting different coreagent or adjusting high pressure to shine to control it
Selectivity, some coreagents can be regenerated, be participated in reaction again, react and release energy with excessive luminescent substance, be produced
Hair tonic optical phenomenon, regeneration coreagent is cost-saved and simplifies experimental provision.In consideration of it, inventing a kind of electrochemical luminescence detection
The sensor of N-terminal B-type natriuretic peptide, has important practical significance.
The present invention is prepared for a kind of silver oxalate bridging tris (bipyridine) ruthenium electrochemical luminescence sensor, specific using golden functionalization
Graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite is as sensor platform, ferroso-ferric oxide@poly-dopamine core
Shell composite material constructs a kind of interlayer type electrochemical luminescence sensor as N-terminal B-type natriuretic peptide antibody marker, and
Sensitive Detection for N-terminal B-type natriuretic peptide.Tris (bipyridine) ruthenium is as electrochemical luminescence signals source, tris (bipyridine) ruthenium molecule
Water-soluble preferable, fixing means is always a stubborn problem.The patent of CN1752745 discloses one kind in electrochemical electrode
The method of surface fixing tripyridyl ruthenium.The water for the noble metal nano particles that tris (bipyridine) ruthenium and citrate anion are protected
Solution mixes according to a certain percentage, has obtained tris (bipyridine) ruthenium-noble metal nano particles aggregation liquid suspension, then this is assembled
Liquid suspension is fixed on the electrode surface of surface sulfhydrylation.Bard group is by tris (bipyridine) ruthenium or its derivative to be formed
The mode of Langmuir-Blodgett film or self-assembled film be fixed on electrode surface (J. Phys. Chem. B, 1998,92,
5566).Tris (bipyridine) ruthenium is fixed on electrode surface using LBL self-assembly method by Dong group, forms silica/tri-
Pyridine ruthenium multilayer film (Anal. Chem. 2004,76,184), while the group also discloses a kind of will contain carbon nanotube
With the composite membrane of Nafion on glass-carbon electrode fixing tripyridyl ruthenium new method (Anal. Chem. 2004,26,
2683).But there are pass through electrostatic adsorption power between some defects, such as tris (bipyridine) ruthenium and other substances for the above method
Not strong, the mass transfer of Nafion membrane is slow, and fixed amount is less, or needs by cumbersome layer assembly process etc..In order to
The defect of the above tris (bipyridine) ruthenium fixing means is made up, the present invention prepares silver oxalate and tris (bipyridine) ruthenium compound by one-step method
Nanosphere, oxalate denominationby and tris (bipyridine) ruthenium form stable chemical bonds by coordination, not only make tris (bipyridine) ruthenium
It can be more stably fixed to electrode surface, the silver oxalate and tris (bipyridine) ruthenium compound nanosphere of formation also include a large amount of three
Bipyridyl ruthenium molecule is realized so that electrochemical luminescence sensor shows stable and stronger electrochemical luminescence signals to N-terminal
The Sensitive Detection of B-typeNatriuretic Peptide original.
Summary of the invention
An object of the present invention is to propose one kind in electrode surface fixing tripyridyl ruthenium Ru (bpy)3 2+Method.?
Containing in graphene oxide-carbonic acid argentum nano composite material solution, while oxalic acid and tris (bipyridine) ruthenium is added, is with oxalate
Ligand, while silver carbonate is converted into silver oxalate, oxalate ligand bridging tris (bipyridine) ruthenium formation graphene oxide-silver oxalate/
A large amount of tris (bipyridine) ruthenium molecules effectively can be immobilized on electrode surface by tris (bipyridine) ruthenium nanocomposite.
The second object of the present invention is prepared for ferroso-ferric oxide@poly-dopamine core-shell composite material, the quinone in poly-dopamine
Energy transfer occurs for formula structure and tris (bipyridine) ruthenium, can effectively quench the electrochemical luminescence of tris (bipyridine) ruthenium.Poly-dopamine simultaneously
It is upper to contain functional group abundant, it can be directly connected to N-terminal B-type natriuretic peptide antibody, do not need to simplify reality using other bridging agents
Test operation.
The third object of the present invention is using tris (bipyridine) ruthenium/potassium peroxydisulfate system acquisition tris (bipyridine) ruthenium electrochemical cathode hair
Optical signal.In the system, there are two the excitation state that approach generates tris (bipyridine) ruthenium respectively: (1) Ru (bpy)3 2+Reduzate
Ru(bpy)3 +Ru (bpy) can be formed directly in by electronics transfer with the reduzate sulfate anion free radical of over cure acid group3 2+
Excitation state Ru (bpy)3 2+*；(2) Ru (bpy)3 2+Directly with the reduzate sulfate anion radical reaction of over cure acid group
It generates Ru (bpy)3 3+, and then Ru (bpy)3 3+Electronics is obtained under negative potential, is formed Ru (bpy)3 2+Excitation state Ru
(bpy)3 2+*, two approach produce the excitation state of tris (bipyridine) ruthenium simultaneously, and then return to ground state, can emit stronger electrochemistry
Luminous signal is conducive to the N-terminal B-type natriuretic peptide for detecting higher concentration, widens the model to N-terminal B-type natriuretic peptide detection
It encloses.
The fourth object of the present invention is prepared for a kind of signal reduction type electrochemical luminescence sensor, can increase to N-terminal Type B
The Monitoring lower-cut of natriuretic peptide original realizes the detection of wider range concentration, improves the sensitivity of detection.
Technical scheme is as follows:
1. a kind of preparation method of the electrochemical luminescence sensor based on silver oxalate bridging tris (bipyridine) ruthenium nano-complex
(1) preparation of golden functional graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite
By 60 ~ 100mg silver nitrate, 0.5 ~ 2g polyvinylpyrrolidone and 1 ~ 10mg graphene oxide are dissolved in 20mL ultrapure water
In, 10 ~ 30mL is added dropwise into solution, the sodium bicarbonate solution that concentration is 0.05mol/L, persistently stirs 1h at room temperature, from
Heart separation, is washed 3 times with water and ethyl alcohol respectively, and graphene oxide-carbonic acid argentum nano composite material is made；
It disperses 10 ~ 70mg graphene oxide-carbonic acid argentum nano composite material in 20mL ethylene glycol, ultrasonic 4h is obtained
Solution A；50 ~ 300mg oxalic acid and 1 ~ 5mL, the terpyridyl ruthenium solution that concentration is 1mmol/L are dissolved in 20mL ethylene glycol, obtained
To solution B；Solution A is added dropwise to solution B, persistently stirs 1h, is centrifugally separating to obtain product, respectively with ultrapure water and
Ethyl alcohol is washed product 3 times, and graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite is made；
It disperses 50 ~ 200mg graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite in 20mL ultrapure water,
Ultrasonic 4h, obtains solution C, by 1 ~ 7mL, the chlorauric acid solution that mass fraction is 2%, 1 ~ 10mg polyvinylpyrrolidone and 1 ~
3mL, the sodium citrate solution that mass fraction is 5% mix, and obtain solution D, solution D is added dropwise in solution C under stirring and is obtained
To mixed solution, 1 ~ 5mg sodium borohydride is added into mixed solution, mixed solution becomes kermesinus, 3 ~ 10h is persistently stirred, from
Gold functional graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite is made in heart separation；
(2) system of the N-terminal B-type natriuretic peptide antibody-solutions of ferroso-ferric oxide@poly-dopamine core-shell composite material label
It is standby
50 ~ 200mg ferriferrous oxide nano-particle and 100 ~ 300mg dopamine are added to three that 120mL, pH are 8.8
In the mixed solution of hydroxymethyl aminomethane buffer and 50 ~ 200mL isopropanol, 20 ~ 50h is persistently stirred, solution becomes black,
Black product is placed in drying in 35oC vacuum oven, it is poly- that ferroso-ferric oxide@is made by centrifuge separation with milli-Q water 5 times
Dopamine core-shell composite material；
The N-terminal B-type natriuretic peptide that 0.5 ~ 2mL concentration is 10 μ g/mL is detected into antibody-solutions and is added to 1 ~ 3mL, concentration
For 10mg/mL ferroso-ferric oxide@poly-dopamine core-shell composite material solution, 48h is stirred under 4oC, is centrifugated, by solid product
Again it is scattered in the phosphate buffer solution that 1mL, pH are 7.4, ferroso-ferric oxide@poly-dopamine core-shell composite material mark is made
The N-terminal B-type natriuretic peptide antibody-solutions of note；
(3) preparation of electrochemical luminescence sensor
1) glass-carbon electrode is through Al2O3Powder is polishing to mirror surface, is thoroughly cleaned up with ultrapure water；
2) 6 μ L, concentration is nano combined for golden functional graphene oxide-silver oxalate/tris (bipyridine) ruthenium of 3 ~ 10mg/mL
Material solution is added drop-wise to electrode surface, drying under 4oC；
3) N-terminal B-type natriuretic peptide for continuing 6 μ L to be added dropwise, concentration is 6 ~ 12 μ g/mL captures antibody-solutions in modified electrode
Surface, 4oC are saved to drying；
4) continue the bovine serum albumin solution for 3 μ L being added dropwise, mass fraction is 0.5 ~ 1%, with the non-spy on enclosed-electrode surface
Electrode surface is rinsed with the PBS solution that pH is 7.4 in specific activities site, and 4oC dries；
5) the N-terminal B-type natriuretic peptide antigen that 6 μ L various concentrations are added dropwise respectively hatches 20 under modified electrode surface, 4oC
~ 40min rinses electrode surface with the phosphate buffer solution that pH is 7.4, and 4oC dries；
6) the N-terminal B for marking 6 μ L, the ferroso-ferric oxide@poly-dopamine core-shell composite material that concentration is 5 ~ 15mg/mL
Type natriuretic peptide original antibody solution is modified on the electrode, hatches 30 ~ 60min at room temperature, is rushed with the phosphate buffer solution that pH is 7.4
Electrode surface is washed, 4oC dries, and a kind of interlayer type electrochemical luminescence sensor is made.
2. in gold medal functional graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite, silver oxalate/terpyridyl
Ruthenium is made by silver oxalate and tris (bipyridine) ruthenium coordination.
3. the electrochemical luminescence sensor based on silver oxalate bridging tris (bipyridine) ruthenium nano-complex is as N-terminal Type B benefit
The application of sodium peptide former detection.
4. the application that electrochemical luminescence sensor is detected as N-terminal B-type natriuretic peptide, detecting step are as follows:
It (1) is working electrode by electrochemical luminescence sensor, for Ag/AgCl electrode as reference electrode, platinum electrode is pair
Electrode, the electrolyte solution containing 40 ~ 140mmol/L potassium persulfate solution for the use of 10mL, pH being 6.0 ~ 8.4 prepare electrochemistry work
It stands, and itself and chemiluminescence detector is linked together and tested；
(2) chemiluminescence detector parameter is set as, and the high pressure of photomultiplier tube is set as 900V, and sweep speed is
0.1V/s；
(3) electrochemical workstation parameter is set as, and cyclic voltammetry scan potential range is -1.6V ~ 0V, and sweep speed is
0.1V/s；
(4) by N-terminal B-type natriuretic peptide sample solution to be measured replace N-terminal B-type natriuretic peptide antigen standard solution into
Row detection, the electrochemical luminescence sensor of the invention based on silver oxalate bridging tris (bipyridine) ruthenium nano-complex are used for N-terminal B
The detection of type natriuretic peptide original, detection range 0.0005-100.0ng/mL, detection are limited to 2.8pg/mL.
The graphene oxide, the preparation method is as follows: 0.3g graphite powder and 1.8g potassium permanganate are put into three mouthfuls of 500mL
In flask, the 36mL concentrated sulfuric acid and 4mL phosphoric acid mixed liquor is added, three-necked flask is placed in oil bath, 12h is reacted under 50oC, reacts
After, solution is poured on ice cube made from 40mL ultrapure water, the hydrogen peroxide of 300 μ L is slowly added under stirring, is continued
30min is stirred, obtained mixed solution is centrifuged 30min under the revolving speed of 8000r/min, is then with 30mL, concentration
The hydrochloric acid centrifuge washing of 0.2mol/L 3 times, then with dehydrated alcohol centrifuge washing 3 times, ether centrifuge washing is finally used, by what is obtained
Solid sample is placed in 35oC vacuum oven dry, obtained graphene oxide.
The ferriferrous oxide nano-particle, partial size is 250 ± 10nm, the preparation method is as follows: 2.7g ferric trichloride is molten
In 100mL ethylene glycol, it is added with stirring 7.2g sodium acetate and 2.0g polyethylene glycol, mixed solution is placed under 50oC and is reacted
12h is transferred in stainless steel cauldron, and 8h is reacted under 200oC, and Magnetic Isolation uses water and ethanol washing 3 times respectively, black is produced
Object is placed in 80oC vacuum oven dry, obtained ferriferrous oxide nano-particle.
The tris (bipyridine) ruthenium is purchased from company, lark prestige Science and Technology Ltd., and other raw materials are in Chinese medicines group chemical reagent
Co., Ltd buys.
Beneficial achievement of the invention
(1) present invention realizes the efficient fixation to tris (bipyridine) ruthenium by silver oxalate bridging tris (bipyridine) ruthenium.
(2) present invention, which utilizes, can occur resonance energy transfer mechanism, structure between polycaprolactam polyamine quinoid structure and tris (bipyridine) ruthenium
Built a kind of quenching type electrochemical luminescence sensor, it can be achieved that the wide range of concentrations to N-terminal B-type natriuretic peptide detection,
(3) present invention is prepared for a kind of ferroso-ferric oxide@poly-dopamine core-shell composite material, good using ferroso-ferric oxide
Magnetism, be conducive to the separation of material, poly-dopamine can also be directly connected to N-terminal B-type natriuretic peptide antibody, not need other companies
Agent is connect, sensor is constructed using ferroso-ferric oxide@poly-dopamine core-shell composite material, simplifies experimental implementation.
(4) present invention is prepared for a kind of silver oxalate bridging tris (bipyridine) ruthenium electrochemical luminescence sensor and for N-terminal Type B
The detection of natriuretic peptide original.Easy to operate, the response time is short, and signal response range is wide, and the detection range of linearity is 0.0005-
100.0ng/mL, detection are limited to 2.8pg/mL, simple, quick, highly sensitive and specific detection may be implemented.
Specific embodiment
Embodiment 1
1. a kind of preparation method of the electrochemical luminescence sensor based on silver oxalate bridging tris (bipyridine) ruthenium nano-complex
(1) preparation of golden functional graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite
By 60mg silver nitrate, 0.5g polyvinylpyrrolidone and 1mg graphene oxide are dissolved in 20mL ultrapure water, to solution
In be added dropwise 10mL, concentration be 0.05mol/L sodium bicarbonate solution, persistently stir 1h at room temperature, be centrifugated, use respectively
Water and ethyl alcohol are washed 3 times, and graphene oxide-carbonic acid argentum nano composite material is made；
It disperses 10mg graphene oxide-carbonic acid argentum nano composite material in 20mL ethylene glycol, ultrasonic 4h obtains solution
A；50mg oxalic acid and 1mL, the terpyridyl ruthenium solution that concentration is 1mmol/L are dissolved in 20mL ethylene glycol, solution B is obtained；It will
Solution A is added dropwise to solution B, persistently stirs 1h, is centrifugally separating to obtain product, washes product with ultrapure water and ethyl alcohol respectively
3 times, graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite is made；
It disperses 50mg graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite in 20mL ultrapure water, ultrasound
4h obtains solution C, and by 1mL, the chlorauric acid solution that mass fraction is 2%, 1mg polyvinylpyrrolidone and 1mL, mass fraction are
5% sodium citrate solution mixing, obtains solution D, solution D is added dropwise in solution C under stirring and obtains mixed solution, to
1mg sodium borohydride is added in mixed solution, mixed solution becomes kermesinus, persistently stirs 3h, is centrifugated, and gold functionalization is made
Graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite；
(2) system of the N-terminal B-type natriuretic peptide antibody-solutions of ferroso-ferric oxide@poly-dopamine core-shell composite material label
It is standby
50mg ferriferrous oxide nano-particle and 100mg dopamine are added to the trihydroxy methyl ammonia that 120mL, pH are 8.8
In the mixed solution of methylmethane buffer and 50mL isopropanol, 20h is persistently stirred, solution becomes black, centrifuge separation, and use is ultrapure
Water washing 5 times, black product is placed in drying in 35oC vacuum oven, it is compound that ferroso-ferric oxide@poly-dopamine nucleocapsid is made
Material；
By the detection of N-terminal B-type natriuretic peptide that 0.5mL concentration is 10 μ g/mL, antibody-solutions are added to 1mL, concentration is
10mg/mL ferroso-ferric oxide@poly-dopamine core-shell composite material solution stirs 48h under 4oC, is centrifugated, by solid product weight
It is newly scattered in the phosphate buffer solution that 1mL, pH are 7.4, ferroso-ferric oxide@poly-dopamine core-shell composite material label is made
N-terminal B-type natriuretic peptide antibody-solutions；
(3) preparation of electrochemical luminescence sensor
1) glass-carbon electrode is through Al2O3Powder is polishing to mirror surface, is thoroughly cleaned up with ultrapure water；
2) by 6 μ L, golden functional graphene oxide-nano combined material of silver oxalate/tris (bipyridine) ruthenium that concentration is 3mg/mL
Material solution is added drop-wise to electrode surface, drying under 4oC；
3) N-terminal B-type natriuretic peptide for continuing 6 μ L to be added dropwise, concentration is 6 μ g/mL captures antibody-solutions in modified electrode table
Face, 4oC are saved to drying；
4) continue the bovine serum albumin solution for 3 μ L being added dropwise, mass fraction is 0.5%, with the non-specific of enclosed-electrode surface
Property active site, rinse electrode surface with the PBS solution that pH is 7.4,4oC dries；
5) the N-terminal B-type natriuretic peptide antigen that 6 μ L various concentrations are added dropwise respectively is hatched under modified electrode surface, 4oC
20min rinses electrode surface with the phosphate buffer solution that pH is 7.4, and 4oC dries；
6) the N-terminal Type B benefit for marking 6 μ L, the ferroso-ferric oxide@poly-dopamine core-shell composite material that concentration is 5mg/mL
Sodium peptide former antibody-solutions are modified on the electrode, hatch 30min at room temperature, rinse electrode with the phosphate buffer solution that pH is 7.4
Surface, 4oC dry, and a kind of interlayer type electrochemical luminescence sensor is made.
2. electrochemical luminescence sensor described in is detected for N-terminal B-type natriuretic peptide
It (1) is working electrode by electrochemical luminescence sensor, for Ag/AgCl electrode as reference electrode, platinum electrode is pair
Electrode, the electrolyte solution containing 40 ~ 140mmol/L potassium persulfate solution for the use of 10mL, pH being 6.0 ~ 8.4 prepare electrochemistry work
It stands, and itself and chemiluminescence detector is linked together and tested；
(2) chemiluminescence detector parameter is set as, and the high pressure of photomultiplier tube is set as 900V, and sweep speed is
0.1V/s；
(3) electrochemical workstation parameter is set as, and cyclic voltammetry scan potential range is -1.6V ~ 0V, and sweep speed is
0.1V/s；
(4) by N-terminal B-type natriuretic peptide sample solution to be measured replace N-terminal B-type natriuretic peptide antigen standard solution into
Row detection.
Embodiment 2
1. a kind of preparation method of the electrochemical luminescence sensor based on silver oxalate bridging tris (bipyridine) ruthenium nano-complex
(1) preparation of golden functional graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite
By 100mg silver nitrate, 2g polyvinylpyrrolidone and 10mg graphene oxide are dissolved in 20mL ultrapure water, to solution
In be added dropwise 30mL, concentration be 0.05mol/L sodium bicarbonate solution, persistently stir 1h at room temperature, be centrifugated, use respectively
Water and ethyl alcohol are washed 3 times, and graphene oxide-carbonic acid argentum nano composite material is made；
It disperses 70mg graphene oxide-carbonic acid argentum nano composite material in 20mL ethylene glycol, ultrasonic 4h obtains solution
A；300mg oxalic acid and 5mL, the terpyridyl ruthenium solution that concentration is 1mmol/L are dissolved in 20mL ethylene glycol, solution B is obtained；It will
Solution A is added dropwise to solution B, persistently stirs 1h, is centrifugally separating to obtain product, washes product with ultrapure water and ethyl alcohol respectively
3 times, graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite is made；
It disperses 200mg graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite in 20mL ultrapure water, surpasses
Sound 4h, obtains solution C, by 7mL, the chlorauric acid solution that mass fraction is 2%, 10mg polyvinylpyrrolidone and 3mL, quality point
Number mixes for 5% sodium citrate solutions, obtains solution D, solution D is added dropwise in solution C under stirring obtain mixing it is molten
5mg sodium borohydride is added into mixed solution for liquid, and mixed solution becomes kermesinus, persistently stirs 10h, is centrifugated, and gold is made
Functional graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite；
(2) system of the N-terminal B-type natriuretic peptide antibody-solutions of ferroso-ferric oxide@poly-dopamine core-shell composite material label
It is standby
200mg ferriferrous oxide nano-particle and 300mg dopamine are added to the trihydroxy methyl ammonia that 120mL, pH are 8.8
In the mixed solution of methylmethane buffer and 200mL isopropanol, 50h is persistently stirred, solution becomes black, centrifuge separation, with super
Pure water 5 times, black product is placed in drying in 35oC vacuum oven, it is multiple that ferroso-ferric oxide@poly-dopamine nucleocapsid is made
Condensation material；
The N-terminal B-type natriuretic peptide detection antibody-solutions that 2mL concentration is 10 μ g/mL are added to 3mL, concentration 10mg/
ML ferroso-ferric oxide@poly-dopamine core-shell composite material solution stirs 48h under 4oC, is centrifugated, solid product is divided again
It dissipates in the phosphate buffer solution that 1mL, pH are 7.4, the N of ferroso-ferric oxide@poly-dopamine core-shell composite material label is made
End B-typeNatriuretic Peptide original antibody solution；
(3) preparation of electrochemical luminescence sensor
1) glass-carbon electrode is through Al2O3Powder is polishing to mirror surface, is thoroughly cleaned up with ultrapure water；
2) by 6 μ L, golden functional graphene oxide-nano combined material of silver oxalate/tris (bipyridine) ruthenium that concentration is 10mg/mL
Material solution is added drop-wise to electrode surface, drying under 4oC；
3) N-terminal B-type natriuretic peptide for continuing 6 μ L to be added dropwise, concentration is 12 μ g/mL captures antibody-solutions in modified electrode table
Face, 4oC are saved to drying；
4) continue the bovine serum albumin solution for 3 μ L being added dropwise, mass fraction is 1%, with the non-specificity on enclosed-electrode surface
Active site rinses electrode surface with the PBS solution that pH is 7.4, and 4oC dries；
5) the N-terminal B-type natriuretic peptide antigen that 6 μ L various concentrations are added dropwise respectively is hatched under modified electrode surface, 4oC
40min rinses electrode surface with the phosphate buffer solution that pH is 7.4, and 4oC dries；
6) the N-terminal Type B for marking 6 μ L, the ferroso-ferric oxide@poly-dopamine core-shell composite material that concentration is 15mg/mL
Natriuretic peptide original antibody solution is modified on the electrode, hatches 60min at room temperature, rinses electricity with the phosphate buffer solution that pH is 7.4
Pole surface, 4oC dry, and a kind of interlayer type electrochemical luminescence sensor is made.
2. electrochemical luminescence sensor described in is detected for N-terminal B-type natriuretic peptide
It (1) is working electrode by electrochemical luminescence sensor, for Ag/AgCl electrode as reference electrode, platinum electrode is pair
Electrode, the electrolyte solution containing 40 ~ 140mmol/L potassium persulfate solution for the use of 10mL, pH being 6.0 ~ 8.4 prepare electrochemistry work
It stands, and itself and chemiluminescence detector is linked together and tested；
(2) chemiluminescence detector parameter is set as, and the high pressure of photomultiplier tube is set as 900V, and sweep speed is
0.1V/s；
(3) electrochemical workstation parameter is set as, and cyclic voltammetry scan potential range is -1.6V ~ 0V, and sweep speed is
0.1V/s；
(4) by N-terminal B-type natriuretic peptide sample solution to be measured replace N-terminal B-type natriuretic peptide antigen standard solution into
Row detection.
Embodiment 3
1. a kind of preparation method of the electrochemical luminescence sensor based on silver oxalate bridging tris (bipyridine) ruthenium nano-complex
(1) preparation of golden functional graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite
By 80mg silver nitrate, 1.0g polyvinylpyrrolidone and 5mg graphene oxide are dissolved in 20mL ultrapure water, to solution
In be added dropwise 20mL, concentration be 0.05mol/L sodium bicarbonate solution, persistently stir 1h at room temperature, be centrifugated, use respectively
Water and ethyl alcohol are washed 3 times, and graphene oxide-carbonic acid argentum nano composite material is made；
It disperses 50mg graphene oxide-carbonic acid argentum nano composite material in 20mL ethylene glycol, ultrasonic 4h obtains solution
A；200mg oxalic acid and 3mL, the terpyridyl ruthenium solution that concentration is 1mmol/L are dissolved in 20mL ethylene glycol, solution B is obtained；It will
Solution A is added dropwise to solution B, persistently stirs 1h, is centrifugally separating to obtain product, washes product with ultrapure water and ethyl alcohol respectively
3 times, graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite is made；
It disperses 100mg graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite in 20mL ultrapure water, surpasses
Sound 4h, obtains solution C, by 5mL, the chlorauric acid solution that mass fraction is 2%, 5mg polyvinylpyrrolidone and 2mL, mass fraction
It is mixed for 5% sodium citrate solution, obtains solution D, solution D is added dropwise in solution C under stirring and obtains mixed solution,
3mg sodium borohydride is added into mixed solution, mixed solution becomes kermesinus, persistently stirs 7h, is centrifugated, and gold function is made
Change graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite；
(2) system of the N-terminal B-type natriuretic peptide antibody-solutions of ferroso-ferric oxide@poly-dopamine core-shell composite material label
It is standby
100mg ferriferrous oxide nano-particle and 200mg dopamine are added to the trihydroxy methyl ammonia that 120mL, pH are 8.8
In the mixed solution of methylmethane buffer and 100mL isopropanol, 30h is persistently stirred, solution becomes black, centrifuge separation, with super
Pure water 5 times, black product is placed in drying in 35oC vacuum oven, it is multiple that ferroso-ferric oxide@poly-dopamine nucleocapsid is made
Condensation material；
The N-terminal B-type natriuretic peptide detection antibody-solutions that 1mL concentration is 10 μ g/mL are added to 2mL, concentration 10mg/
ML ferroso-ferric oxide@poly-dopamine core-shell composite material solution stirs 48h under 4oC, is centrifugated, solid product is divided again
It dissipates in the phosphate buffer solution that 1mL, pH are 7.4, the N of ferroso-ferric oxide@poly-dopamine core-shell composite material label is made
End B-typeNatriuretic Peptide original antibody solution；
(3) preparation of electrochemical luminescence sensor
1) glass-carbon electrode is through Al2O3Powder is polishing to mirror surface, is thoroughly cleaned up with ultrapure water；
2) by 6 μ L, golden functional graphene oxide-nano combined material of silver oxalate/tris (bipyridine) ruthenium that concentration is 6mg/mL
Material solution is added drop-wise to electrode surface, drying under 4oC；
3) N-terminal B-type natriuretic peptide for continuing 6 μ L to be added dropwise, concentration is 10 μ g/mL captures antibody-solutions in modified electrode table
Face, 4oC are saved to drying；
4) continue the bovine serum albumin solution for 3 μ L being added dropwise, mass fraction is 0.7%, with the non-specific of enclosed-electrode surface
Property active site, rinse electrode surface with the PBS solution that pH is 7.4,4oC dries；
5) the N-terminal B-type natriuretic peptide antigen that 6 μ L various concentrations are added dropwise respectively is hatched under modified electrode surface, 4oC
30min rinses electrode surface with the phosphate buffer solution that pH is 7.4, and 4oC dries；
6) the N-terminal Type B for marking 6 μ L, the ferroso-ferric oxide@poly-dopamine core-shell composite material that concentration is 10mg/mL
Natriuretic peptide original antibody solution is modified on the electrode, hatches 50min at room temperature, rinses electricity with the phosphate buffer solution that pH is 7.4
Pole surface, 4oC dry, and a kind of interlayer type electrochemical luminescence sensor is made.
2. electrochemical luminescence sensor described in is detected for N-terminal B-type natriuretic peptide
It (1) is working electrode by electrochemical luminescence sensor, for Ag/AgCl electrode as reference electrode, platinum electrode is pair
Electrode, the electrolyte solution containing 40 ~ 140mmol/L potassium persulfate solution for the use of 10mL, pH being 6.0 ~ 8.4 prepare electrochemistry work
It stands, and itself and chemiluminescence detector is linked together and tested；
(2) chemiluminescence detector parameter is set as, and the high pressure of photomultiplier tube is set as 900V, and sweep speed is
0.1V/s；
(3) electrochemical workstation parameter is set as, and cyclic voltammetry scan potential range is -1.6V ~ 0V, and sweep speed is
0.1V/s；
(4) by N-terminal B-type natriuretic peptide sample solution to be measured replace N-terminal B-type natriuretic peptide antigen standard solution into
Row detection.
Embodiment 4
Graphene oxide described in embodiment 1-3, the preparation method is as follows: 0.3g graphite powder and 1.8g potassium permanganate are put into
In the three-necked flask of 500mL, the 36mL concentrated sulfuric acid and 4mL phosphoric acid mixed liquor is added, three-necked flask is placed in oil bath, it is anti-under 50oC
12h is answered, after reaction, solution is poured on ice cube made from 40mL ultrapure water, the peroxide of 300 μ L is slowly added under stirring
Change hydrogen, continues to stir 30min, obtained mixed solution is centrifuged 30min under the revolving speed of 8000r/min, then with 30mL, dense
Degree is hydrochloric acid centrifuge washing 3 times of 0.2mol/L, then with dehydrated alcohol centrifuge washing 3 times, finally uses ether centrifuge washing, will
To solid sample be placed in 35oC vacuum oven dry, graphene oxide is made；
The ferriferrous oxide nano-particle, partial size is 250 ± 10nm, the preparation method is as follows: 2.7g ferric trichloride is molten
In 100mL ethylene glycol, it is added with stirring 7.2g sodium acetate and 2.0g polyethylene glycol, mixed solution is placed under 50oC and is reacted
12h is transferred in stainless steel cauldron, and 8h is reacted under 200oC, and Magnetic Isolation uses water and ethanol washing 3 times respectively, black is produced
Object is placed in 80oC vacuum oven dry, obtained ferriferrous oxide nano-particle；
The tris (bipyridine) ruthenium is purchased from company, lark prestige Science and Technology Ltd., and other raw materials are in Chinese medicines group chemical reagent
Co., Ltd buys.
Embodiment 5
Silver oxalate bridging tris (bipyridine) ruthenium electrochemical luminescence sensor described in embodiment 1-3 is simultaneously used for N-terminal Type B benefit sodium
The detection of peptide former, the detection range of linearity are 0.0005-100.0ng/mL, and detection is limited to 2.8pg/mL, may be implemented simple, fast
Fast, highly sensitive and specific detection.
Claims (4)
1. a kind of preparation method of the electrochemical luminescence sensor based on silver oxalate bridging tris (bipyridine) ruthenium nano-complex, special
Sign is that preparation step is as follows:
(1) preparation of golden functional graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite
By 60 ~ 100mg silver nitrate, 0.5 ~ 2g polyvinylpyrrolidone and 1 ~ 10mg graphene oxide are dissolved in 20mL ultrapure water,
The sodium bicarbonate solution that 10 ~ 30mL concentration is 0.05mol/L is added dropwise into solution, persistently stirs 1h, centrifugation point at room temperature
From, washed 3 times with water and ethyl alcohol respectively, be made graphene oxide-carbonic acid argentum nano composite material；
It disperses 10 ~ 70mg graphene oxide-carbonic acid argentum nano composite material in 20mL ethylene glycol, ultrasonic 4h obtains solution
The terpyridyl ruthenium solution that 50 ~ 300mg oxalic acid and 1 ~ 5mL concentration are 1mmol/L is dissolved in 20mL ethylene glycol, obtains solution by A
Solution A is added dropwise to solution B, persistently stirs 1h, is centrifugally separating to obtain product, washed respectively with ultrapure water and ethyl alcohol by B
3 times, graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite is made；
It disperses 50 ~ 200mg graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite in 20mL ultrapure water, ultrasound
4h obtains solution C, the chlorauric acid solution for being 2% by 1 ~ 7mL mass fraction, 1 ~ 10mg polyvinylpyrrolidone and 1 ~ 3mL mass
The sodium citrate solution that score is 5% mixes, and obtains solution D, solution D is added dropwise in solution C under stirring, then to mixed
It closes and 1 ~ 5mg sodium borohydride is added in solution, mixed solution becomes kermesinus, persistently stirs 3 ~ 10h, and gold function is made in centrifuge separation
Graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite can be changed；
(2) preparation of the N-terminal B-type natriuretic peptide antibody-solutions of ferroso-ferric oxide@poly-dopamine core-shell composite material label
50 ~ 200mg ferriferrous oxide nano-particle and 100 ~ 300mg dopamine are added to the three hydroxyl first that 120mL, pH are 8.8
In the mixed solution of base aminomethane buffer solution and 50 ~ 200mL isopropanol, 20 ~ 50h is persistently stirred, solution becomes black, is centrifuged
Black product is placed in 35oC vacuum oven dry, the obtained poly- DOPA of ferroso-ferric oxide@with milli-Q water 5 times by separation
Amine core-shell composite material；
The N-terminal B-type natriuretic peptide detection antibody-solutions that 0.5 ~ 2mL concentration is 10 μ g/mL, which are added to 1 ~ 3mL concentration, is
10mg/mL ferroso-ferric oxide@poly-dopamine core-shell composite material solution stirs 48h under 4oC, is centrifugated, by solid product weight
It is newly scattered in the phosphate buffer solution that 1mL, pH are 7.4, ferroso-ferric oxide@poly-dopamine core-shell composite material label is made
N-terminal B-type natriuretic peptide antibody-solutions；
(3) preparation of electrochemical luminescence sensor
1) glass-carbon electrode is through Al2O3Powder is polishing to mirror surface, is thoroughly cleaned up with ultrapure water；
2) the golden functional graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite for being 3 ~ 10mg/mL by 6 μ L concentration
Solution is added drop-wise to electrode surface, drying under 4oC；
3) continue to be added dropwise the N-terminal B-type natriuretic peptide that 6 μ L concentration are 6 ~ 12 μ g/mL and capture antibody-solutions in modified electrode surface,
4oC is saved to drying；
4) continue that the bovine serum albumin solution that 3 μ L mass fractions are 0.5 ~ 1% is added dropwise, with the non-specificity on enclosed-electrode surface
Active site rinses electrode surface with the PBS solution that pH is 7.4, and 4oC dries；
5) the N-terminal B-type natriuretic peptide antigen that 6 μ L various concentrations are added dropwise respectively hatches 20 under modified electrode surface, 4oC ~
40min rinses electrode surface with the phosphate buffer solution that pH is 7.4, and 4oC dries；
6) the N-terminal Type B benefit sodium for marking the ferroso-ferric oxide@poly-dopamine core-shell composite material that 6 μ L concentration are 5 ~ 15mg/mL
Peptide former antibody-solutions are modified on the electrode, hatch 30 ~ 60min at room temperature, rinse electrode with the phosphate buffer solution that pH is 7.4
Surface, 4oC dry, and a kind of interlayer type electrochemical luminescence sensor is made.
2. the electrochemical luminescence sensor according to claim 1 based on silver oxalate bridging tris (bipyridine) ruthenium nano-complex
Preparation method, which is characterized in that it is described gold functional graphene oxide-silver oxalate/tris (bipyridine) ruthenium nanocomposite, grass
Sour silver/tris (bipyridine) ruthenium is made by silver oxalate and tris (bipyridine) ruthenium coordination.
3. one kind of preparation method preparation according to claim 1 is based on silver oxalate bridging tris (bipyridine) ruthenium nano-complex
The application that is detected as N-terminal B-type natriuretic peptide of electrochemical luminescence sensor.
4. the application that electrochemical luminescence sensor according to claim 3 is detected as N-terminal B-type natriuretic peptide, special
Sign is that detecting step is as follows:
(1) be working electrode by electrochemical luminescence sensor, Ag/AgCl electrode is used as reference electrode, platinum electrode for electrode,
The electrolyte solution containing 40 ~ 140mmol/L potassium persulfate solution for the use of 10mL, pH being 6.0 ~ 8.4, prepares electrochemical workstation,
And itself and chemiluminescence detector are linked together and tested；
(2) chemiluminescence detector parameter setting: the high pressure of photomultiplier tube is set as 900V, sweep speed 0.1V/s；
(3) electrochemical workstation parameter setting: cyclic voltammetry scan potential range is -1.6V ~ 0V, sweep speed 0.1V/s；
(4) N-terminal B-type natriuretic peptide antigen is replaced to detect testing sample solution.
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