CN108918855A - A kind of preparation method and application based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator - Google Patents
A kind of preparation method and application based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator Download PDFInfo
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- CN108918855A CN108918855A CN201810856758.3A CN201810856758A CN108918855A CN 108918855 A CN108918855 A CN 108918855A CN 201810856758 A CN201810856758 A CN 201810856758A CN 108918855 A CN108918855 A CN 108918855A
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- 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
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
Abstract
The present invention relates to a kind of preparation method and applications based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator, and the invention belongs to new function materials and biosensor technique field.It is specifically a kind of with silver nanoparticle group (AgNCs) be electrogenerated chemiluminescence sensing platform, thiocarbamide (Sem) is wrapped in the surface AgNCs as coreagent promotor, and load upper gold nanoparticle (AuNPs), increase electrochemiluminescence signal with this compound AgNCs-Sem-AuNPs, forms the mode of signal "ON"；In addition, metal-organic framework compound (MOF) material NH2- MIL-125 generates Resonance energy transfer as secondary antibody marker and AgNCs, causes signal " closing ", the interlayer type Electrochemiluminescsensor sensor constructed with this, for brain natriuretic peptide (NT-Pro-BNP) before sensitive and accurate detection N-terminal.
Description
Technical field
The present invention relates to a kind of preparation and application of electrochemiluminescimmunosensor immunosensor, and specifically one kind is with AgNCs-
Sem-AuNPs is sensing platform, with NH2- MIL-125 is signal " on-off " type immunosensor of secondary antibody marker, the present invention
Belong to new function material, biosensor technique field.
Background technique
Brain natriuretic peptide (NT-Pro-BNP) is the natural hormone with biological activity synthesized by cardiac muscle cell before N-terminal, main
It to express, while be existed in brain tissue in ventricle.NT-Pro-BNP not only reflects that left room is received as heart failure quantitative marker
Contracting dysfunction also reflects Left ventricular dilatation dysfunction, valve disturbances and right ventricular function obstacle situation.Clinic shows NT-
Pro-BNP is pin-point accuracy for diagnosis of heart failure, is by monitoring NT-Pro-BNP content and observation patient symptom
The best method of clinic decompensation is determined, in addition, NT-Pro-BNP is also the most powerful of patients with acute coronary syndrome death
Predict object.Therefore, the diseases such as the content for monitoring NT-Pro-BNP can be effectively predicted, diagnosis of heart failure, acute coronary syndrome.
Electrogenerated chemiluminescence (ECL) is a kind of research method for combining electrochemical means with chemiluminescence, closely
Several years in terms of the widely concern that is subject to.ECL sensor has high sensitivity, highly selective, high specificity
The advantages such as low with detection limit can fast and accurately detect the content of determinand.
In recent years, metal nanometre cluster had obtained many concerns due to its optics/electrochemical properties, feature and semiconductor
Quantum dot is similar.Due to their excellent photostability, good biocompatibility and water solubility, they are more and more extensive
Apply in the fields such as cell imaging and biomolecule detection.AgNCs is applied to as electrogenerated chemiluminescence body in the present invention
In the detection of NT-Pro-BNP, by being combined with each other with coreagent promotor Sem and AuNPs, high-intensitive, high stable is generated
The electrochemiluminescence signal of property, improves the sensitivity and accuracy of detection.
Metal-organic framework compound (MOF) is a kind of coordination polymer quickly grown nearly ten years, has three-dimensional
Pore structure, generally using metal ion as tie point, the support of organic ligand position constitutes space 3D and extends, be zeolite and carbon nanotube it
The important novel porous materials of outer another class, are being catalyzed, sensing, are all widely used in energy storage and separation.It will in the present invention
NH2- MIL-125 forms Resonance energy transfer as secondary antibody marker and AgNCs, causes signal " closing ", the electricity constructed with this
Cause chemiluminescence sensor that can effectively detect the content of NT-Pro-BNP.
Summary of the invention
An object of the present invention is to prepare one kind using AgNCs-Sem-AuNPs as sensing platform, with NH2- MIL-125 is
Signal " on-off " type electrochemiluminescimmunosensor immunosensor of secondary antibody marker.
The second object of the present invention is to the sensor is used for highly sensitive, the specific detection of NT-Pro-BNP.
Technical scheme is as follows：
1. a kind of preparation method based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator is as follows：
（1）Glass-carbon electrode is polished with polishing powder, deionized water cleaning is reused, it is molten that electrode is placed in the 5 mmol/L potassium ferricyanides
It in liquid, and is scanned under -0.2 ~ 0.6 V current potential, makes the difference of spike potential less than 110 mV；
（2）By 8 μ L, 5 ~ 8 mg/mL AgNCs-Sem-AuNPs complex solutions are added dropwise on the electrode, dry at room temperature；
（3）By 8 μ L, 1 ~ 2 μ g/mL NT-Pro-BNP antibody is added dropwise on the electrode, clear with PBS after drying at room temperature
It washes, removes Excess antibody, dry at room temperature；
（4）By 3 μ L, mass fraction is that 1 ~ 2 % BSA solution is added dropwise on electrode, to close nonspecific binding site,
Extra BSA is washed away using PBS after drying, is dried at room temperature；
（5）By 8 μ L, 0.00001 ~ 100 ng mL-1A series of NT-Pro-BNP antigen standard solution of various concentrations drips
It is added on electrode, after drying at room temperature, is cleaned with PBS, remove extra antigen, dry at room temperature；
（6）By 8 μ L, 5 ~ 8 mg/mL are by NH2The secondary antibody of-MIL-125 label is added dropwise on electrode, after drying at room temperature,
It is cleaned with PBS, removes extra secondary antibody, dry at room temperature.
2. a kind of AgNCs-Sem- based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator
The preparation step of AuNPs material is as follows：
(1) preparation of AgNCs
0.15 ~ 0.18 g polyvinylpyrrolidone (PVP), 20 ~ 25 mL ethylene glycol (EG) are dissolved, then by 3 ~ 5
The AgNO of mL3The FeCl of (1 mM, be dissolved in EG) and 2 ~ 5 mL3(0.6 mM, be dissolved in EG) sequentially adds PVP solution
In, it then pours the mixture into the autoclave of 50 mL, 140 C react 1 h, and centrifuge washing for several times, is placed in 60 C vacuum
Dry 12 h, obtain AgNCs in drying box；
(2) preparation of AuNPs
Gold chloride (the HAuCl for being first 0.01 ~ 0.03% by 100 mL mass fractions4) the extremely boiling of 130 C oil bath heatings,
Then the sodium citrate that 2 ~ 3 mL mass fractions are 1 ~ 3% is added dropwise under agitation in above-mentioned solution, solution
Color becomes rufous, as AuNPs from faint yellow, is kept in dark place after being cooled to room temperature in case using；
(3) preparation of AgNCs-Sem-AuNPs
200 ~ 300 μ L semicarbazides (Sem, 10 mM) solution is added to 300 ~ 500 μ L of above-mentioned synthesis under stiring
In AuNCs solution (1 mg/mL), 12 hours are stirred to obtain AgNCs-Sem, after being washed with deionized twice, collects precipitating
And disperse in deionized water, then, it is molten that AgNCs-Sem is added in the AuNPs solution (1 mg/mL) of 200 ~ 400 μ L preparation
It in liquid, stirs 12 hours, is then centrifuged for washing for several times, be placed in 60 C vacuum ovens dry 12 h, obtain AgNCs-
Sem-AuNPs。
3. a kind of secondary antibody marker based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator
NH2The preparation step of-MIL-125 is as follows：
By 550 ~ 600 mg 2- amino terephthalic acid (TPA) (NH2- BDC) and the glacial acetic acid of 3.0 ~ 4.0 mL be dissolved in N, N-
The volume ratio of dimethylformamide (DMF) and methanol is 9:In 1 mixed solution, DMF and methanol total volume are 20 ~ 40 mL,
0.5 ~ 0.7 mL butyl titanate (TBOT) is then added, then pours the mixture into reaction kettle, 150 C heat treatment 24
H, then use DMF and methanol centrifuge washing for several times respectively, finally obtain yellow powder, as NH2-MIL-125。
4. the inspection of NT-Pro-BNP, steps are as follows：
（1）It is tested using the three-electrode system of electrochemical workstation, Ag/AgCl electrode is as reference electrode, platinum electrode
For to electrode, prepared electrochemiluminescimmunosensor immunosensor is working electrode, electrochemical workstation and chemiluminescence are examined
Survey instrument, which links together, sets 700 V for the high pressure of photomultiplier tube, and cyclic voltammetry scan potential range is 0 ~ 1.3 V,
Sweep speed is 0.1 V/s；
（2）What it is in 10 mL, pH 6.0 ~ 8.5 is the PBS buffer solution of the mmol/L tripropyl amine (TPA) of 1 mmol/L ~ 12 containing concentration
In, by electrochemical luminescence method, detect the electrochemical luminescence signals generated to the NT-Pro-BNP standard solution of various concentration
Intensity draws working curve；
（3）NT-Pro-BNP standard solution is replaced to be measured NT-Pro-BNP sample solution to be measured.
Beneficial achievement of the invention
（1）The present invention, as luminescent material, is wrapped in AgNCs for Sem as coreagent promotor, effectively using AgNCs
Increase and stabilize luminous signal, meanwhile, the AuNPs for being carried on the surface Sem also further improves signal, in addition, AgNCs-
The height of Sem-AuNPs composite material generation and stable luminous signal, effectively increase the sensitivity of detection NT-Pro-BNP.
（2）MOF material NH2- MIL-125 generates Resonance energy transfer as secondary antibody marker and AgNCs, effectively quenches
Luminous signal causes signal " closing ", in working sensor Drawing of Curve, with NT-Pro-BNP antigen concentration of standard solution
Increase, NH2The quenching effect of-MIL-125 is more obvious, so as to the content of accurate, special detection NT-Pro-BNP.
（3）The present invention realizes that signal is opened and amplification, NH as sensing platform using AgNCs-Sem-AuNPs2-MIL-
125 as secondary antibody marker realization signal " closing ", and the hypersensitive electrochemiluminescimmunosensor immunosensor constructed with this is applied to
The clinical detection of NT-Pro-BNP, have it is easy to operate, detection quickly, linearly range is wide（0.01 pg/mL ~ 100
ng/mL）It is low with detection limit（3.23 fg/mL）The advantages of.
Specific embodiment
Embodiment 1 is a kind of based on AgNCs be illuminator signal " on-off " type Electrochemiluminescsensor sensor
The preparation step of AgNCs-Sem-AuNPs material is as follows：
(1) preparation of AgNCs
0.15 g polyvinylpyrrolidone (PVP), 20 mL ethylene glycol (EG) are dissolved, then by the AgNO of 4 mL3(1 mM,
Be dissolved in EG) and 2 mL FeCl3(0.6 mM, be dissolved in EG) is sequentially added in PVP solution, then falls mixture
In the autoclave for entering 50 mL, 140 C react 1 h, and centrifuge washing for several times, is placed in 60 C vacuum ovens dry 12
H obtains AgNCs；
(2) preparation of AuNPs
Gold chloride (the HAuCl for being first 0.02% by 100 mL mass fractions4) 130 C oil bath heatings are to boiling, then by 2
The sodium citrate that mL mass fraction is 1.5% is added dropwise under agitation in above-mentioned solution, and solution colour is by yellowish discoloration
For rufous, as AuNPs, it is kept in dark place after being cooled to room temperature in case using；
(3) preparation of AgNCs-Sem-AuNPs
250 μ L semicarbazides (Sem, 10 mM) solution is added to 300 μ L AuNCs solution (1 of above-mentioned synthesis under stiring
Mg/mL in), stir 12 hours to obtain AgNCs-Sem, after being washed with deionized twice, collect precipitate and be dispersed in from
In sub- water, then, the AuNPs solution (1 mg/mL) of 250 μ L preparation is added in AgNCs-Sem solution, stirring 12 hours, so
Centrifuge washing for several times, is placed in 60 C vacuum ovens dry 12 h, obtains AgNCs-Sem-AuNPs afterwards.
Embodiment 2 is a kind of based on AgNCs be illuminator signal " on-off " type Electrochemiluminescsensor sensor
The preparation step of AgNCs-Sem-AuNPs material is as follows：
(1) preparation of AgNCs
0.16 g polyvinylpyrrolidone (PVP), 22 mL ethylene glycol (EG) are dissolved, then by the AgNO of 3 mL3(1 mM,
Be dissolved in EG) and 2.5 mL FeCl3(0.6 mM, be dissolved in EG) is sequentially added in PVP solution, then by mixture
It pours into the autoclave of 50 mL, 140 C react 1 h, and centrifuge washing for several times, is placed in 60 C vacuum ovens dry 12
H obtains AgNCs；
(2) preparation of AuNPs
Gold chloride (the HAuCl for being first 0.01% by 100 mL mass fractions4) the extremely boiling of 130 C oil bath heatings, then will
The sodium citrate that 2.5 mL mass fractions are 1% is added dropwise under agitation in above-mentioned solution, and solution colour is by faint yellow
Become rufous, as AuNPs, is kept in dark place after being cooled to room temperature in case using；
(3) preparation of AgNCs-Sem-AuNPs
200 μ L semicarbazides (Sem, 10 mM) solution is added to 500 μ L AuNCs solution (1 of above-mentioned synthesis under stiring
Mg/mL in), stir 12 hours to obtain AgNCs-Sem, after being washed with deionized twice, collect precipitate and be dispersed in from
In sub- water, then, the AuNPs solution (1 mg/mL) of 200 μ L preparation is added in AgNCs-Sem solution, stirring 12 hours, so
Centrifuge washing for several times, is placed in 60 C vacuum ovens dry 12 h, obtains AgNCs-Sem-AuNPs afterwards.
Embodiment 3 is a kind of based on AgNCs be illuminator signal " on-off " type Electrochemiluminescsensor sensor
The preparation step of AgNCs-Sem-AuNPs material is as follows：
(1) preparation of AgNCs
0.18 g polyvinylpyrrolidone (PVP), 25 mL ethylene glycol (EG) are dissolved, then by the AgNO of 5 mL3(1 mM,
Be dissolved in EG) and 5 mL FeCl3(0.6 mM, be dissolved in EG) is sequentially added in PVP solution, then falls mixture
In the autoclave for entering 50 mL, 140 C react 1 h, and centrifuge washing for several times, is placed in 60 C vacuum ovens dry 12
H obtains AgNCs；
(2) preparation of AuNPs
Gold chloride (the HAuCl for being first 0.03% by 100 mL mass fractions4) 130 C oil bath heatings are to boiling, then by 3
The sodium citrate that mL mass fraction is 3% is added dropwise under agitation in above-mentioned solution, and solution colour is become from faint yellow
Rufous, as AuNPs are kept in dark place in case using after being cooled to room temperature；
(3) preparation of AgNCs-Sem-AuNPs
300 μ L semicarbazides (Sem, 10 mM) solution is added to 400 μ L AuNCs solution (1 of above-mentioned synthesis under stiring
Mg/mL in), stir 12 hours to obtain AgNCs-Sem, after being washed with deionized twice, collect precipitate and be dispersed in from
In sub- water, then, the AuNPs solution (1 mg/mL) of 400 μ L preparation is added in AgNCs-Sem solution, stirring 12 hours, so
Centrifuge washing for several times, is placed in 60 C vacuum ovens dry 12 h, obtains AgNCs-Sem-AuNPs afterwards.
A kind of secondary antibody based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator of embodiment 4
Marker NH2The preparation step of-MIL-125 is as follows：
By 550 mg 2- amino terephthalic acid (TPA) (NH2- BDC) and the glacial acetic acid of 3.0 mL be dissolved in N,N-dimethylformamide
(DMF) and the volume ratio of methanol is 9:In 1 mixed solution, DMF and methanol total volume are 20 mL, and 0.5 mL titanium is then added
Sour four butyl esters (TBOT), then pour the mixture into reaction kettle, and 150 C are heat-treated 24 h, then respectively with DMF and methanol from
The heart washs for several times, finally obtains yellow powder, as NH2-MIL-125。
A kind of secondary antibody based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator of embodiment 5
Marker NH2The preparation step of-MIL-125 is as follows：
By 560 mg 2- amino terephthalic acid (TPA) (NH2- BDC) and the glacial acetic acid of 3.2 mL be dissolved in N,N-dimethylformamide
(DMF) and the volume ratio of methanol is 9:In 1 mixed solution, DMF and methanol total volume are 40 mL, and 0.68 mL titanium is then added
Sour four butyl esters (TBOT), then pour the mixture into reaction kettle, and 150 C are heat-treated 24 h, then respectively with DMF and methanol from
The heart washs for several times, finally obtains yellow powder, as NH2-MIL-125。
A kind of secondary antibody based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator of embodiment 6
Marker NH2The preparation step of-MIL-125 is as follows：
By 600 mg 2- amino terephthalic acid (TPA) (NH2- BDC) and the glacial acetic acid of 4.0 mL be dissolved in N,N-dimethylformamide
(DMF) and the volume ratio of methanol is 9:In 1 mixed solution, DMF and methanol total volume are 30 mL, and 0.7 mL titanium is then added
Sour four butyl esters (TBOT), then pour the mixture into reaction kettle, and 150 C are heat-treated 24 h, then respectively with DMF and methanol from
The heart washs for several times, finally obtains yellow powder, as NH2-MIL-125。
A kind of preparation based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator of embodiment 7
Method：
（1）Glass-carbon electrode is polished with polishing powder, deionized water cleaning is reused, it is molten that electrode is placed in the 5 mmol/L potassium ferricyanides
It in liquid, and is scanned under -0.2 ~ 0.6 V current potential, makes the difference of spike potential less than 110 mV；
（2）By 8 μ L, 5 mg/mL AgNCs-Sem-AuNPs complex solutions are added dropwise on the electrode, dry at room temperature；
（3）By 8 μ L, 1 μ g/mL NT-Pro-BNP antibody is added dropwise on the electrode, after drying at room temperature, is cleaned, is removed with PBS
Excess antibody is removed, is dried at room temperature；
（4）By 3 μ L, mass fraction is that 1% BSA solution is added dropwise on electrode, to close nonspecific binding site, is dried
Extra BSA is washed away using PBS later, is dried at room temperature；
（5）By 8 μ L, 0.00001 ~ 50 ng mL-1A series of NT-Pro-BNP antigen standard solution of various concentrations is added dropwise
In on electrode, after drying at room temperature, is cleaned with PBS, remove extra antigen, dry at room temperature；
（6）By 8 μ L, 5 mg/mL are by NH2The secondary antibody of-MIL-125 label is added dropwise on electrode, after drying at room temperature, uses PBS
Cleaning, removes extra secondary antibody, dries at room temperature.
A kind of preparation based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator of embodiment 8
Method：
（1）Glass-carbon electrode is polished with polishing powder, deionized water cleaning is reused, it is molten that electrode is placed in the 5 mmol/L potassium ferricyanides
It in liquid, and is scanned under -0.2 ~ 0.6 V current potential, makes the difference of spike potential less than 110 mV；
（2）By 8 μ L, 6 mg/mL AgNCs-Sem-AuNPs complex solutions are added dropwise on the electrode, dry at room temperature；
（3）By 8 μ L, 1.5 μ g/mL NT-Pro-BNP antibody are added dropwise on the electrode, after drying at room temperature, are cleaned with PBS,
Excess antibody is removed, is dried at room temperature；
（4）By 3 μ L, mass fraction is that 1.5% BSA solution is added dropwise on electrode, to close nonspecific binding site, is done
Extra BSA is washed away using PBS after dry, is dried at room temperature；
（5）By 8 μ L, 0.00001 ~ 50 ng mL-1A series of NT-Pro-BNP antigen standard solution of various concentrations is added dropwise
In on electrode, after drying at room temperature, is cleaned with PBS, remove extra antigen, dry at room temperature；
（6）By 8 μ L, 6 mg/mL are by NH2The secondary antibody of-MIL-125 label is added dropwise on electrode, after drying at room temperature, uses PBS
Cleaning, removes extra secondary antibody, dries at room temperature.
A kind of preparation based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator of embodiment 9
Method：
（1）Glass-carbon electrode is polished with polishing powder, deionized water cleaning is reused, it is molten that electrode is placed in the 5 mmol/L potassium ferricyanides
It in liquid, and is scanned under -0.2 ~ 0.6 V current potential, makes the difference of spike potential less than 110 mV；
（2）By 8 μ L, 8 mg/mL AgNCs-Sem-AuNPs complex solutions are added dropwise on the electrode, dry at room temperature；
（3）By 8 μ L, 2 μ g/mL NT-Pro-BNP antibody are added dropwise on the electrode, after drying at room temperature, are cleaned, are removed with PBS
Excess antibody is removed, is dried at room temperature；
（4）By 3 μ L, mass fraction is that 2% BSA solution is added dropwise on electrode, to close nonspecific binding site, is dried
Extra BSA is washed away using PBS later, is dried at room temperature；
（5）By 8 μ L, 0.00001 ~ 50 ng mL-1A series of NT-Pro-BNP antigen standard solution of various concentrations is added dropwise
In on electrode, after drying at room temperature, is cleaned with PBS, remove extra antigen, dry at room temperature；
（6）By 8 μ L, 8 mg/mL are by NH2The secondary antibody of-MIL-125 label is added dropwise on electrode, after drying at room temperature, uses PBS
Cleaning, removes extra secondary antibody, dries at room temperature.
The inspection of 10 NT-Pro-BNP of embodiment, steps are as follows：
（1）It is tested using the three-electrode system of electrochemical workstation, Ag/AgCl electrode is as reference electrode, platinum electrode
For to electrode, prepared electrochemiluminescimmunosensor immunosensor is working electrode, electrochemical workstation and chemiluminescence are examined
Survey instrument, which links together, sets 700 V for the high pressure of photomultiplier tube, and cyclic voltammetry scan potential range is 0 ~ 1.3 V,
Sweep speed is 0.1 V/s；
（2）What it is in 10 mL, pH 6.0 ~ 8.5 is the PBS buffer solution of the mmol/L tripropyl amine (TPA) of 1 mmol/L ~ 12 containing concentration
In, by electrochemical luminescence method, detect the electrochemical luminescence signals generated to the NT-Pro-BNP standard solution of various concentration
Intensity draws working curve；
（3）NT-Pro-BNP standard solution is replaced to be measured NT-Pro-BNP sample solution to be measured；
（4）The range of linearity of NT-Pro-BNP detection is the ng/mL of 0.01 pg/mL ~ 100, and detection limit is 3.23 fg/mL.
Claims (3)
1. a kind of preparation method and application based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator,
It is characterized by comprising the following steps：
（1）Glass-carbon electrode is polished with polishing powder, deionized water cleaning is reused, it is molten that electrode is placed in the 5 mmol/L potassium ferricyanides
It in liquid, and is scanned under -0.2 ~ 0.6 V current potential, makes the difference of spike potential less than 110 mV；
（2）By 8 μ L, 5 ~ 8 mg/mL AgNCs-Sem-AuNPs complex solutions are added dropwise on the electrode, dry at room temperature；
（3）By 8 μ L, 1 ~ 2 μ g/mL NT-Pro-BNP antibody is added dropwise on the electrode, clear with PBS after drying at room temperature
It washes, removes Excess antibody, dry at room temperature；
（4）By 3 μ L, mass fraction is that 1 ~ 2% BSA solution is added dropwise on electrode, to close nonspecific binding site,
Extra BSA is washed away using PBS after drying, is dried at room temperature；
（5）By 8 μ L, 0.00001 ~ 100 ng mL-1A series of NT-Pro-BNP antigen standard solution of various concentrations is added dropwise
In on electrode, after drying at room temperature, is cleaned with PBS, remove extra antigen, dry at room temperature；
（6）By 8 μ L, 5 ~ 8 mg/mL are by NH2The secondary antibody of-MIL-125 label is added dropwise on electrode, after drying at room temperature, uses
PBS cleaning, removes extra secondary antibody, dries at room temperature；
A kind of preparation based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator as described in right 1
Method and application, described in AgNCs-Sem-AuNPs material preparation step it is as follows：
(1) preparation of AgNCs
0.15 ~ 0.18 g polyvinylpyrrolidone (PVP), 20 ~ 25 mL ethylene glycol (EG) are dissolved, then by 3 ~ 5
The AgNO of mL3The FeCl of (1 mM, be dissolved in EG) and 2 ~ 5 mL3(0.6 mM, be dissolved in EG) sequentially adds PVP solution
In, it then pours the mixture into the autoclave of 50 mL, 140 C react 1 h, and centrifuge washing for several times, is placed in 60 C vacuum
Dry 12 h, obtain AgNCs in drying box；
(2) preparation of AuNPs
Gold chloride (the HAuCl for being first 0.01 ~ 0.03% by 100 mL mass fractions4) 130 C oil bath heatings are to boiling, so
The sodium citrate that 2 ~ 3 mL mass fractions are 1 ~ 3% is added dropwise under agitation in above-mentioned solution afterwards, solution face
Color becomes rufous, as AuNPs from faint yellow, is kept in dark place after being cooled to room temperature in case using；
(3) preparation of AgNCs-Sem-AuNPs
200 ~ 300 μ L semicarbazides (Sem, 10 mM) solution is added to 300 ~ 500 μ L of above-mentioned synthesis under stiring
In AuNCs solution (1 mg/mL), 12 hours are stirred to obtain AgNCs-Sem, after being washed with deionized twice, collects precipitating
And disperse in deionized water, then, it is molten that AgNCs-Sem is added in the AuNPs solution (1 mg/mL) of 200 ~ 400 μ L preparation
It in liquid, stirs 12 hours, is then centrifuged for washing for several times, be placed in 60 C vacuum ovens dry 12 h, obtain AgNCs-
Sem-AuNPs。
2. a kind of system based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator as described in right 1
Preparation Method and application, described in NH2The preparation step of-MIL-125 material is as follows：
By 550 ~ 600 mg 2- amino terephthalic acid (TPA) (NH2- BDC) and the glacial acetic acid of 3.0 ~ 4.0 mL be dissolved in N, N- bis-
Methylformamide (DMF) and the volume ratio of methanol are 9:In 1 mixed solution, DMF and methanol total volume are 20 ~ 40 mL, with
0.5 ~ 0.7 mL butyl titanate (TBOT) is added afterwards, then pours the mixture into reaction kettle, 150 C are heat-treated 24 h,
It uses DMF and methanol centrifuge washing for several times respectively again, finally obtains yellow powder, as NH2-MIL-125。
3. a kind of system based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator as described in right 1
Preparation Method and application, which is characterized in that for the inspection of NT-Pro-BNP, detecting step is as follows：
（1）It is tested using the three-electrode system of electrochemical workstation, Ag/AgCl electrode is as reference electrode, platinum electrode
For to electrode, prepared electrochemiluminescimmunosensor immunosensor is working electrode, electrochemical workstation and chemiluminescence are examined
Survey instrument, which links together, sets 700 V for the high pressure of photomultiplier tube, and cyclic voltammetry scan potential range is 0 ~ 1.3 V,
Sweep speed is 0.1 V/s；
（2）What it is in 10 mL, pH 6.0 ~ 8.5 is the PBS buffer solution of the mmol/L tripropyl amine (TPA) of 1 mmol/L ~ 12 containing concentration
In, by electrochemical luminescence method, detect the electrochemical luminescence signals generated to the NT-Pro-BNP standard solution of various concentration
Intensity draws working curve；
（3）NT-Pro-BNP standard solution is replaced to be measured NT-Pro-BNP sample solution to be measured.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810856758.3A CN108918855B (en) | 2018-07-31 | 2018-07-31 | Preparation method and application of signal 'on-off' type electrochemiluminescence sensor based on AgNCs as luminophor |
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