CN107727714B - One kind being based on carbon nanohorn and TiO2The preparation method of the Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material - Google Patents
One kind being based on carbon nanohorn and TiO2The preparation method of the Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material Download PDFInfo
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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
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- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
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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
- G01N27/305—Electrodes, e.g. test electrodes; Half-cells optically transparent or photoresponsive electrodes
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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
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Abstract
The present invention discloses a kind of based on carbon nanohorn and TiO2The preparation method of the Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material, feature are based on carbon nanohorn (CNHs) and octahedra Detitanium-ore-type TiO2Mesomorphic (OAMs) nano material introduces perylene derivative (PTC-NH2) and two kinds of signal probes of luminol (luminol), a kind of Ratio-type electrochemical luminescence immunosensor of silicon phthalocyanine sensitization is prepared, and be used for the detection of prostate-specific antigen (PSA).The OAMs of CNHs and higher porosity with bigger serface can carry a large amount of biomolecule and signal probe as biosensor platform, simultaneously, the electron-withdrawing group of hexafluoro bisphenol-a-silicon phthalocyanine (HFSPC) can accelerate luminol to lose electronics, obtain the ECL signal of an enhancing.Based on above-mentioned advantage, obtained Ratio-type electrochemical luminescence immunosensor, have the advantages that high specificity, high sensitivity, stability are good, detection limit is low, can be used for the detection of prostate-specific antigen (PSA), there is more important application value and practical significance in terms of clinical application.
Description
Technical field
The invention belongs to new function materials and bio-sensing detection technique field, and in particular to one kind is based on carbon nanohorn
And TiO2The preparation side of Ratio-type prostate-specific antigen (PSA) electrochemical luminescence immunosensor of mesomorphic nano material
Method.
Background technique
Ratio method, a kind of new analysis method, quantization depend on the ratio of two signals rather than absolute value, gradually
The fields such as fluorescence, electrochemical luminescence, photoelectricity and electrochemistry are used in, other analytical technologies are compared, electrochemical luminescence has spirit
The advantages that sensitivity is high, simple, fast reaction, the development for being combined into biosensor with Ratio-type analytical technology provides more
Wide application prospect.Electrochemical luminescence immunosensor utilizes one kind biology of the specific binding between antigen and antibody
Sensor has high sensitivity, selectivity is good, easy to operate, is easy to miniaturization, continuously can fast and automatically change detection and analysis etc.
Advantage has a good application prospect.The present invention is prepared for a kind of based on carbon nanohorn and TiO2The ratio of mesomorphic nano material
Type electrochemical luminescence immunosensor, and realize the highly sensitive detection to prostate-specific antigen.
Carbon nanohorn is a kind of novel carbon nanomaterial, and since carbon nanohorn is in dahlia shape structure, surface has non-
Often more coordination site, shows the chemical characteristics such as good electron conduction, big specific surface area and high pore volume, in structure
It builds in good electrochemical sensor and gets the attention.TiO2Nano material because of its unique photocatalytic activity, nontoxicity,
Excellent chemically and physically stability, becomes the ideal material of photocatalysis and optical electro-chemistry sensor, performance generally by
The influence of crystal form, grain size, crystal face, crystallinity, specific surface area, micro-structure etc..TiO2Mesomorphic is that crystal subunit is orderly arranged
What column were constituted, compared to traditional TiO2Monocrystalline, TiO2Mesomorphic has more excellent performance.The present invention is based on carbon nanohorns
(CNHs) and octahedra Detitanium-ore-type TiO2Mesomorphic (OAMs) nano material introduces perylene derivative (PTC-NH2) and Rumi
Two kinds of signal probes of promise (luminol), prepare a kind of Ratio-type electrochemical luminescence immunosensor of silicon phthalocyanine sensitization, and are used for
The detection of prostate-specific antigen (PSA).The OAMs of CNHs and higher porosity with bigger serface are passed as biology
Sensor platform can carry a large amount of biomolecule and signal probe, meanwhile, the electrophilic of hexafluoro bisphenol-a-silicon phthalocyanine (HFSPC)
Group can accelerate luminol to lose electronics, obtain the ECL signal of an enhancing.Based on above-mentioned advantage, obtained Ratio-type electricity
Chemiluminescence immunoassay sensor has the advantages that high specificity, high sensitivity, stability are good, detection limit is low, can be used for prostate
The detection of specific antigen (PSA) has very important application value and practical significance in terms of clinical application.
Summary of the invention
An object of the present invention is based on carbon nanohorn and TiO2Mesomorphic nano material constructs a kind of unmarked, stabilization
Good, the electrochemical luminescence immunosensor of high sensitivity and preparation method thereof of property.
The second object of the present invention is to the electrochemical luminescence immunosensor is applied to the height of prostate-specific antigen
Sensitive Detection.
To realize goal of the invention, the present invention adopts the following technical scheme:
1. one kind is based on carbon nanohorn and TiO2Ratio-type prostate-specific antigen (PSA) electrification of mesomorphic nano material
Learn the preparation method of electrochemiluminescent immunoassay sensor, which comprises the following steps:
(1) glass-carbon electrode (GCE) mechanical grinding first on the chamois leather for be covered with alumina powder polishes, and is washed away with secondary water
Remained on surface powder, then move into ultrasonic water bath and clean, until cleaning up, ethyl alcohol is finally sequentially used, diluted acid and water are thoroughly washed
It washs；
(2) carbon nanohorn (CNHs) suspension that 3 μ L concentration are 3 mg/ml is added dropwise in clean glassy carbon electrode surface,
It dries, is cooled to room temperature under infrared lamp, CNHs modified glassy carbon electrode is made；
(3) 3 μ L 0.2mmol/L luminols (luminol), 2mg/mL carboxymethyl chitosan (CMCS) and 2mg/mL is added dropwise
Hexafluoro bisphenol-a-silicon phthalocyanine (HFSPC) mixed solution is dried under modified electrode surface, infrared lamp, is cooled to room temperature, and is made
Luminol@CMCS@HFSPC/CNHs modified glassy carbon electrode；
(4) luminol@CMCS@HFSPC/CNHs modified electrode is immersed into concentration than (1- (the 3- dimethylamino third for 1:2
Base) -3- ethyl-carbodiimide hydrochloride) (EDC) and 50min in the mixed liquor of n-hydroxysuccinimide (NHS), then immerse 30
μ L concentration is the prostate-specific antigen antibody (Ab of 2 mg/mL horseradish peroxidases (HRP) label1) in solution and at 4 °
It is incubated for 45min in C refrigerator, the Ab of physical absorption is washed with deionized water1, HRP-Ab is made1/luminol@CMCS@HFSPC/
CNHs modified glassy carbon electrode；
(5) it takes 60 μ L concentration to be added drop-wise in the refrigerator of step (4) 4 °C of modified electrode surface for the BSA of 1.0 wt.% and is incubated for 1
H is rinsed electrode surface with deionized water and is washed away physical absorption with nonspecific activity site on enclosed-electrode surface, and is saved
In 4 °C of refrigerators；
(6) modified electrode for obtaining step (5) immerses prostate-specific antigen (PSA) standard solution of various concentration
In and in 4 °C of refrigerators be incubated for 1 h, rinse electrode surface with the PBS buffer solution of pH7.5, PSA/HRP-Ab be made1/
Luminol@CMCS@HFSPC/CNHs modified glassy carbon electrode.
(7) modified electrode for obtaining step (6) immerses 50 μ L perylene derivative (PTC-NH2), octahedra rutile titania
Mine type TiO2Mesomorphic (OAMs) and prostate-specific antigen antibody (Ab2) (PTC-NH2@OAMs@Ab2) composition compound it is molten
50 min are incubated in liquid and in 4 °C of refrigerators, electrode surface is rinsed with the PBS buffer solution of pH7.5, PTC-NH is made2@
OAMs@ Ab2/PSA/HRP-Ab1/ luminol@CMCS@HFSPC/CNHs modified glassy carbon electrode, and be stored in 4 °C of refrigerators.
2. above-mentioned luminol (luminol), carboxymethyl chitosan (CMCS) and hexafluoro bisphenol-a-silicon phthalocyanine (HFSPC) are mixed
Close what solution was prepared by the following method: by the way that 2mmol/L luminol (luminol), 20mg/mL carboxymethyl chitosan is simply mixed
Sugared (CMCS) and 20mg/mL hexafluoro bisphenol-a-silicon phthalocyanine (HFSPC) solution are simply mixed and are suitably added the water of certain volume
It uniformly mixes and is prepared into again.
What 3. above-mentioned hexafluoro bisphenol-a-silicon phthalocyanine (HFSPC) was prepared by the following method: by 0.20 g SiPcCl2、0.24g
Hexafluoro bisphenol-a and 0.14 g anhydrous K2CO3It is placed in 30ml toluene solvant and flows back 48 hours at 110oC, it is then that reaction is mixed
Conjunction object is cooled to room temperature and the solvent was evaporated under reduced pressure to doing, and then solid product is placed in Soxhlet extractor pure with methylene chloride
Change 24 hours, is eluted after the material recrystallization being extracted with the volume ratio methanol for being 1:1 and the mixed liquor of deionized water, then weighed
Newly it is dissolved in methylene chloride.
4. above-mentioned perylene derivative (PTC-NH2), octahedra Detitanium-ore-type TiO2Mesomorphic (OAMs) and prostate-specific
Property antigen-antibody (Ab2) (PTC-NH2@OAMs@Ab2) complex solution is prepared by the following method: 1) volume ratio be 1:1
6mg/ml OAMs solution mixes 6 h of concussion with 1 wt % 3- aminopropyl triethoxysilane (APTES), and precipitating is collected by centrifugation
Then object is redispersed in deionized water, obtain NH2- OAMs solution；2) volume ratio is the 5 mg/ml PTC-NH of 1:42With 4
mg/ml NH2- OAMs solution mixing 1 h of concussion, is collected by centrifugation sediment, is then redispersed in deionized water, obtain PTC-NH2
@OAMs solution；3) by 100 μ L Ab2And 100 μ L PTC-NH2@OAMs solution and 60 μ l concentration are than (1- (the 3- bis- for 1:2
Methylaminopropyl) -3- ethyl-carbodiimide hydrochloride) (EDC) and n-hydroxysuccinimide (NHS) mixed liquor mixing and at 4 °
Place 12 h under C, sediment is collected by centrifugation and is dispersed in the PBS solution of pH 7.4, be then added 0.5 wt %BSA go unless
Specific adsorption site.
5. above-mentioned perylene derivative (PTC-NH2) be prepared by the following method: 20mg 3,4,9,10- tetracarboxylic acid
Acid dianhydride (PTCDA) stirring and dissolving in 2 mL acetone, then by 0.1ml anhydrous ethylenediamine (C2H8N4) slowly add under 4 °C
Enter into above-mentioned solution to be then centrifuged for, wash to pH to be 7.4, removes excessive ethylenediamine.Finally, products therefrom is uniformly divided
It is dispersed in 4ml deionized water, is stored in 4 °C of refrigerators.
6. above-mentioned octahedra Detitanium-ore-type TiO2Mesomorphic (OAMs) material is prepared by the following method: 1.2 grams of rutile titania
Mine type TiO2Powder is dissolved in the KOH solution that 60mL concentration is 18mol/ml, and stirring moves into one after twenty minutes, by suspension
In a 100ml polytetrafluoroethyllining lining stainless steel autoclave；Reactor is sealed, is reacted 72 hours under 170oC, is cooled to room
Temperature；Then, being washed with dilute acetic acid solution and being precipitated to precipitating pH value is 3.5, is obtained within dry 12 hours at 65oC after precipitating centrifugation
Product metatitanic acid nano wire；400mg presoma metatitanic acid nano wire is taken to be dispersed in 70ml spirit of vinegar, in 100 polytetrafluoroethylene (PTFE) high pressures
48h, obtained product distilled water and dehydrated alcohol centrifuge washing are reacted in reaction kettle under the conditions of 200oC, under the conditions of 60oC
It dries and is calcined 30 minutes under 12 h and 400oC, to remove remaining organic matter, OAMs is made.
7. the detecting step of prostate-specific antigen (PSA):
(1) it is measured using electrochemical workstation using three-electrode system, a kind of base prepared with above-mentioned preparation method
In carbon nanohorn and TiO2The Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material is working electrode, and Ag/AgCl is
Reference electrode, platinum electrode are to electrode, in 10 mmol/ml K2S2O8PH 8.0 PBS buffer solution in tested；
(2) prostate-specific antigen (PSA) standard solution of various concentration is detected using bipotential mode, just
- 0.8 V of beginning current potential, the pulse period 5 seconds, the burst length 40 seconds, 0.6 V of pulse potential and the pulse period 3 seconds, by electroluminescentization
It learns luminaire and acquires the ECL signal strength (Ic) of -0.8 V and the ECL signal strength (Ia) of 0.6 V, pass through its rate value
Relationship between (Ic/ Ia) and prostate-specific antigen (PSA) concentration of standard solution draws working curve；
(3) testing sample solution replaces prostate-specific antigen (PSA) solution to be detected, and the result of detection can pass through
Working curve checks in.
Remarkable advantage of the invention are as follows:
(1) ratio method, a kind of new analysis method, quantization depend on the ratio of two signals rather than absolute value,
Gradually it is used in the fields such as fluorescence, electrochemical luminescence, photoelectricity and electrochemistry.Compared to other analytical technologies, electrochemical luminescence tool
There are high sensitivity, simple, fast reaction, the development for being combined into biosensor with Ratio-type analytical technology provides
Broader practice prospect.
(2) CNHs with bigger serface and the OAMs of higher porosity can be carried greatly as biosensor platform
The biomolecule and signal probe of amount, meanwhile, the electron-withdrawing group of hexafluoro bisphenol-a-silicon phthalocyanine (HFSPC) can accelerate luminol to lose
De-electromation obtains the ECL signal of an enhancing.Immunosensor prepared by the present invention has preferable stability.
(3) present invention utilizes the immune response of antigen, antibody, improves the specificity of detection method.
Detailed description of the invention
Fig. 1 is octahedra Detitanium-ore-type TiO2Mesomorphic (OAMs) material, perylene derivative (PTC-NH2) and PTC-NH2
Infrared spectroscopy (IR) figure of@OAMs complex solution.
Fig. 2 is the electrochemical luminescence response signal and prostate-specific antigen (PSA) standard solution of immune sensing electrode
The linear relationship chart of concentration.
Specific embodiment
The present invention is further illustrated by the following examples, but protection scope of the present invention is not limited to following reality
Apply example.
Embodiment 1
One kind being based on carbon nanohorn and TiO2Ratio-type prostate-specific antigen (PSA) electrochemistry of mesomorphic nano material
The preparation of electrochemiluminescent immunoassay sensor:
(1) glass-carbon electrode (GCE) mechanical grinding first on the chamois leather for be covered with alumina powder polishes, and is washed away with secondary water
Remained on surface powder, then move into ultrasonic water bath and clean, until cleaning up, ethyl alcohol is finally sequentially used, diluted acid and water are thoroughly washed
It washs；
(2) carbon nanohorn (CNHs) suspension that 3 μ L concentration are 3 mg/ml is added dropwise in clean glassy carbon electrode surface,
It dries, is cooled to room temperature under infrared lamp, CNHs modified glassy carbon electrode is made；Carbon nanohorn uses persons skilled in the art energy
It is prepared by the method for realization.
(3) 3 μ L 0.2mmol/L luminols (luminol), 2mg/mL carboxymethyl chitosan (CMCS) and 2mg/mL is added dropwise
Hexafluoro bisphenol-a-silicon phthalocyanine (HFSPC) mixed solution is dried under modified electrode surface, infrared lamp, is cooled to room temperature, and is made
Luminol@CMCS@HFSPC/CNHs modified glassy carbon electrode；
(4) luminol@CMCS@HFSPC/CNHs modified electrode is immersed into concentration than (1- (the 3- dimethylamino third for 1:2
Base) -3- ethyl-carbodiimide hydrochloride) (EDC) and 50min in the mixed liquor of n-hydroxysuccinimide (NHS), then immerse 30
μ L concentration is the prostate-specific antigen antibody (Ab of 2 mg/mL horseradish peroxidases (HRP) label1) in solution and at 4 °
It is incubated for 45min in C refrigerator, the Ab of physical absorption is washed with deionized water1, HRP-Ab is made1/luminol@CMCS@HFSPC/
CNHs modified glassy carbon electrode；Prostate-specific antigen antibody can be produced using Beijing Bo Ao Bioisystech Co., Ltd.
(5) it takes 60 μ L concentration to be added drop-wise in the refrigerator of step (4) 4 °C of modified electrode surface for the BSA of 1.0 wt.% and is incubated for 1
H is rinsed electrode surface with deionized water and is washed away physical absorption with nonspecific activity site on enclosed-electrode surface, and is saved
In 4 °C of refrigerators；
(6) modified electrode for obtaining step (5) immerses prostate-specific antigen (PSA) standard solution of various concentration
In and in 4 °C of refrigerators be incubated for 1 h, rinse electrode surface with the PBS buffer solution of pH7.5, PSA/HRP-Ab be made1/
Luminol@CMCS@HFSPC/CNHs modified glassy carbon electrode.
(7) modified electrode for obtaining step (6) immerses 50 μ L perylene derivative (PTC-NH2), octahedra rutile titania
Mine type TiO2Mesomorphic (OAMs) and prostate-specific antigen antibody (Ab2) (PTC-NH2@OAMs@Ab2) composition compound it is molten
50 min are incubated in liquid and in 4 °C of refrigerators, electrode surface is rinsed with the PBS buffer solution of pH7.5, PTC-NH is made2@
OAMs@ Ab2/PSA/HRP-Ab1/ luminol@CMCS@HFSPC/CNHs modified glassy carbon electrode, and be stored in 4 °C of refrigerators.
Luminol (luminol) that above-mentioned concentration is 0.2mmol/L, concentration be 2mg/mL carboxymethyl chitosan (CMCS) and
Hexafluoro bisphenol-a-silicon phthalocyanine (HFSPC) mixed solution that concentration is 2mg/mL is prepared by the following method: being 2mmol/L by concentration
Luminol (luminol), concentration is 20mg/mL carboxymethyl chitosan (CMCS) and concentration is 20mg/mL hexafluoro bisphenol-a-silicon
The solution of phthalocyanine (HFSPC) is simply mixed and is suitably added the water of certain volume and uniformly mixes and be prepared into again.
What above-mentioned hexafluoro bisphenol-a-silicon phthalocyanine (HFSPC) was prepared by the following method: by 0.20 g SiPcCl2, 0.24g six
Fluorine bisphenol-A and 0.14 g anhydrous K2CO3It is placed in 30ml toluene solvant and flows back 48 hours at 110oC, then mix reaction
Object is cooled to room temperature and the solvent was evaporated under reduced pressure to doing, and then solid product is placed in Soxhlet extractor and is purified with methylene chloride
It 24 hours, is eluted with the volume ratio methanol for being 1:1 and the mixed liquor of deionized water after the material recrystallization being extracted, then again
It is dissolved in methylene chloride.
Embodiment 2
Octahedra Detitanium-ore-type TiO in embodiment 12The preparation of mesomorphic (OAMs) material: 1.2 grams of Detitanium-ore-type TiO2
Powder is dissolved in the KOH solution that 60mL concentration is 18mol/ml, and after twenty minutes, one 100ml of suspension immigration is gathered for stirring
In tetrafluoroethene inner liner stainless steel autoclave；Reactor is sealed, is reacted 72 hours under 170oC, is cooled to room temperature；Then, it uses
It is 3.5 that dilute acetic acid solution washing, which is precipitated to precipitating pH value, obtains product metatitanic acid and receives within dry 12 hours at 65oC after precipitating centrifugation
Rice noodles；400mg presoma metatitanic acid nano wire is taken to be dispersed in 70ml spirit of vinegar, in 100 polytetrafluoroethylene (PTFE) autoclaves
48h, obtained product distilled water and dehydrated alcohol centrifuge washing are reacted under the conditions of 200oC, dry 12 h under the conditions of 60oC
And calcined 30 minutes under 400oC, to remove remaining organic matter, OAMs is made.
Embodiment 3
Perylene derivative (PTC-NH in embodiment 12) preparation: 20mg 3,4,9,10- tetracarboxylic dianhydride
(PTCDA) stirring and dissolving in 2 mL acetone, then by 0.1ml anhydrous ethylenediamine (C2H8N4) be slowly added under 4 °C
It states in solution to be then centrifuged for, wash to pH to be 7.4, removes excessive ethylenediamine.Finally, products therefrom is dispersed in 4ml
In deionized water, it is stored in 4 °C of refrigerators.
Embodiment 4
Perylene derivative (PTC-NH in embodiment 12), octahedra Detitanium-ore-type TiO2Mesomorphic (OAMs) (PTC-
NH2@OAMs) complex solution preparation: 1) volume ratio be 1:1 three second of 6mg/ml OAMs solution and 1 wt % 3- aminopropyl
Oxysilane (APTES) mixing 6 h of concussion, are collected by centrifugation sediment, are then redispersed in deionized water, obtain NH2- OAMs
Solution；2) volume ratio is the 5 mg/ml PTC-NH of 1:42With 4 mg/ml NH2- OAMs solution mixing 1 h of concussion, centrifugation are received
Collect sediment, is then redispersed in deionized water, obtains PTC-NH2@OAMs solution.Fig. 1 is octahedra Detitanium-ore-type TiO2It is situated between
Brilliant (OAMs) material, perylene derivative (PTC-NH2) and PTC-NH2The infrared spectroscopy (IR) of@OAMs complex solution
Figure.Compared to OAMs, PTC-NH2@OAMs is in 1599 cm-1There is the characteristic peak of the condensation aromatic ring of perylene skeleton at place, this
Outside, in 1771 and 1301 cm-1Locate C=O and C-N stretching vibration characteristic peak, and in 1637 cm-1The bending vibration for locating N-H is special
Peak is levied, illustrates PTC-NH2Successfully it is fixed on OAMs.
Embodiment 5
The detecting step of prostate-specific antigen (PSA):
(1) it is measured using electrochemical workstation using three-electrode system, is received with one kind prepared by embodiment 1 based on carbon
Rice angle and TiO2The Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material is working electrode, and Ag/AgCl is reference electricity
Pole, platinum electrode are to electrode, in 10 mmol/ml K2S2O8PH 8.0 PBS buffer solution in tested；
(2) prostate-specific antigen (PSA) standard solution of various concentration is detected using bipotential mode, just
- 0.8 V of beginning current potential, the pulse period 5 seconds, the burst length 40 seconds, 0.6 V of pulse potential and the pulse period 3 seconds, by electroluminescentization
It learns luminaire and acquires the ECL signal strength (Ic) of -0.8 V and the ECL signal strength (Ia) of 0.6 V, pass through its rate value
Relationship between (Ic/ Ia) and prostate-specific antigen (PSA) concentration of standard solution draws working curve, and Fig. 2 is immune
The electrochemical luminescence response signal of sensing electrode and the linear relationship chart of prostaglandin E1 concentration of standard solution；
(3) testing sample solution replaces prostate-specific antigen (PSA) solution to be detected, and the result of detection can pass through
Working curve checks in.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (8)
1. one kind is based on carbon nanohorn and TiO2The preparation side of the Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material
Method, which comprises the following steps:
(1) glass-carbon electrode (GCE) mechanical grinding first on the chamois leather for be covered with alumina powder polishes, and washes away surface with secondary water
Residual powder, then move into ultrasonic water bath and clean, until cleaning up, ethyl alcohol is finally sequentially used, diluted acid and water thoroughly wash；
(2) it is carbon nanohorn (CNHs) suspension of 3 mg/ml in clean glassy carbon electrode surface that 3 μ L concentration, which are added dropwise, infrared
It dries, is cooled to room temperature under lamp, CNHs modified glassy carbon electrode is made；
(3) it is 2mg/mL carboxymethyl chitosan that luminol (luminol), concentration that 3 μ L concentration are 0.2mmol/L, which is added dropwise,
(CMCS) and concentration is hexafluoro bisphenol-a-silicon phthalocyanine (HFSPC) mixed solution of 2mg/mL under modified electrode surface, infrared lamp
Drying, is cooled to room temperature, and luminol@CMCS@HFSPC/CNHs modified glassy carbon electrode is made；
(4) luminol@CMCS@HFSPC/CNHs modified electrode is immersed into concentration than the 1- (3- dimethylamino-propyl)-for 1:2
50min in the mixed liquor of 3- ethyl-carbodiimide hydrochloride (EDC) and n-hydroxysuccinimide (NHS), then to immerse 30 μ L dense
Degree is the prostate-specific antigen antibody (Ab of 2 mg/mL horseradish peroxidases (HRP) label1) in solution and in 4 °C of ice
It is incubated for 45min in case, the Ab of physical absorption is washed with deionized water1, HRP-Ab is made1/luminol@CMCS@HFSPC/CNHs
Modified glassy carbon electrode；
(5) it takes 60 μ L concentration to be added drop-wise in 4 °C of the modified electrode surface refrigerator of step (4) for the BSA of 1.0 wt.% and is incubated for 1
H is rinsed electrode surface with deionized water and is washed away physical absorption with nonspecific activity site on enclosed-electrode surface, and is saved
In 4 °C of refrigerators；
(6) modified electrode for obtaining step (5) immerses in prostate-specific antigen (PSA) standard solution of various concentration simultaneously
It is incubated for 1 h in 4 °C of refrigerators, electrode surface is rinsed with the PBS buffer solution of pH7.5, PSA/HRP-Ab is made1/luminol@
CMCS@HFSPC/CNHs modified glassy carbon electrode；
(7) modified electrode for obtaining step (6) immerses 50 μ L perylene derivative (PTC-NH2), octahedra Detitanium-ore-type
TiO2Mesomorphic (OAMs) and prostate-specific antigen antibody (Ab2) composition compound PTC-NH2@OAMs@Ab2In solution simultaneously
50 min are incubated in 4 °C of refrigerators, electrode surface is rinsed with the PBS buffer solution of pH7.5, PTC-NH is made2@OAMs@ Ab2/
PSA/HRP-Ab1/ luminol@CMCS@HFSPC/CNHs modified glassy carbon electrode, and be stored in 4 °C of refrigerators.
2. the method according to claim 1, wherein the concentration is the luminol of 0.2mmol/L
(luminol), hexafluoro bisphenol-a-silicon phthalocyanine that concentration is 2mg/mL carboxymethyl chitosan (CMCS) and concentration is 2mg/mL
(HFSPC) mixed solution is prepared by the following method: being 2mmol/L luminol (luminol), concentration 20mg/ by concentration
ML carboxymethyl chitosan (CMCS) and concentration are that 20mg/mL hexafluoro bisphenol-a-silicon phthalocyanine (HFSPC) solution is simply mixed and fits
When the water that certain volume is added uniformly mixes again and it is prepared into.
3. method according to claim 1 or 2, which is characterized in that the hexafluoro bisphenol-a-silicon phthalocyanine (HFSPC) is under
State method preparation: by 0.20 g SiPcCl2, 0.24g hexafluoro bisphenol-a and 0.14 g anhydrous K2CO3It is placed in 30ml toluene solvant
In flow back 48 hours at 110oC, then reaction mixture is cooled to room temperature and the solvent was evaporated under reduced pressure to dry, then will
Solid product is placed in Soxhlet extractor is purified 24 hours with methylene chloride, and it is 1 that volume ratio is used after the material recrystallization being extracted:
1 methanol and the mixed liquor elution of deionized water, are then redissolved in methylene chloride.
4. the method according to claim 1, wherein the perylene derivative (PTC-NH2), it is octahedra
Detitanium-ore-type TiO2Mesomorphic (OAMs) and prostate-specific antigen antibody (Ab2) compound PTC-NH2@OAMs@ Ab2Solution
It is prepared by the following method: 1) the 6mg/ml OAMs solution and 1wt% 3- aminopropyl triethoxysilane that volume ratio is 1:1
(APTES) 6 h of mixing concussion, are collected by centrifugation sediment, are then redispersed in deionized water, obtain NH2- OAMs solution；2)
Volume ratio is the 5 mg/ml PTC-NH of 1:42With 4 mg/ml NH2- OAMs solution mixing 1 h of concussion, is collected by centrifugation precipitating
Then object is redispersed in deionized water, obtain PTC-NH2@OAMs solution；3) by 100 μ L Ab2And 100 μ L PTC-NH2 @
OAMs solution and 60 μ l concentration are than (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride) (EDC) and N- for 1:2
HOSu NHS (NHS) mixes into mixed liquor and places 12 h under 4 °C, and sediment is collected by centrifugation and is dispersed in pH
In 7.4 PBS solution, 0.5wt%BSA is then added and removes non-specific adsorption sites.
5. method according to claim 1 or 4, which is characterized in that the perylene derivative (PTC-NH2) under
Method preparation: 3,4,9,10- tetracarboxylic dianhydride (PTCDA) of 20mg stirring and dissolving in 2 mL acetone is stated, then by 0.1ml
Anhydrous ethylenediamine (C2H8N2) be slowly added under 4 °C in above-mentioned solution be then centrifuged for, wash to pH be 7.4, removed
The ethylenediamine of amount is stored in 4 °C of refrigerators finally, products therefrom is dispersed in 4ml deionized water.
6. method according to claim 1 or 4, which is characterized in that the octahedra Detitanium-ore-type TiO2Mesomorphic (OAMs)
What material was prepared by the following method: 1.2 grams of Detitanium-ore-type TiO2It is molten that powder is dissolved in the KOH that 60mL concentration is 18mol/ml
In liquid, after twenty minutes, suspension is moved into a 100ml polytetrafluoroethyllining lining stainless steel autoclave for stirring；Sealing reaction
Device reacts 72 hours under 170oC, is cooled to room temperature；Then, being washed with dilute acetic acid solution and being precipitated to precipitating pH value is 3.5, is sunk
It forms sediment dry 12 hours at 65oC after being centrifuged and obtains product metatitanic acid nano wire；400mg presoma metatitanic acid nano wire is taken to be dispersed in
In 70ml spirit of vinegar, 48h is reacted under the conditions of 200oC in 100 polytetrafluoroethylene (PTFE) autoclaves, obtained product steams
Distilled water and dehydrated alcohol centrifuge washing are dried under the conditions of 60oC and calcines 30 minutes under 12 h and 400oC, to remove having for remnants
OAMs is made in machine object.
7. one kind of any method preparation of claim 1-6 is based on carbon nanohorn and TiO2The ratio of mesomorphic nano material
Type prostate-specific antigen (PSA) electrochemical luminescence immunosensor.
8. as claimed in claim 7 be based on carbon nanohorn and TiO2The Ratio-type electrochemical luminescence immune sensing of mesomorphic nano material
Device, which is characterized in that be used for prostate-specific antigen (PSA), detecting step is as follows:
(1) it is measured using electrochemical workstation using three-electrode system, with as claimed in claim 7 a kind of based on carbon nanometer
Angle and TiO2The Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material is working electrode, and Ag/AgCl is reference electrode,
Platinum electrode is to electrode, in 10 mmol/ml K2S2O8PH 8.0 PBS buffer solution in tested；
(2) prostate-specific antigen (PSA) standard solution of various concentration is detected using bipotential mode, initial electricity
- 0.8 V of position, the pulse period 5 seconds, the burst length 40 seconds, 0.6 V of pulse potential and the pulse period 3 seconds passed through electroluminescent chemistry hair
Light device acquires the ECL signal strength (Ic) of -0.8 V and the ECL signal strength (Ia) of 0.6 V, passes through its rate value (Ic/
Ia) the relationship between prostate-specific antigen (PSA) concentration of standard solution draws working curve；
(3) testing sample solution replaces prostate-specific antigen (PSA) solution to be detected, and the result of detection can pass through work
Curve checks in.
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| CN108535345B (en) * | 2018-04-15 | 2019-06-21 | 福建师范大学 | A kind of nontoxic optical electro-chemistry competitive immunoassay method of the zearalenone based on peptide sensor |
| CN108827946B (en) * | 2018-04-30 | 2021-03-02 | 福建师范大学 | Vomitoxin ratio type electrochemiluminescence immunosensor sharing co-reaction reagent type and detection method thereof |
| CN109490282B (en) * | 2018-11-18 | 2021-05-11 | 福建师范大学 | Based on NiFe2O4Nano-tube catalysis enhanced ovarian cancer marker ratio type electrochemiluminescence sensing platform |
| CN109884319B (en) * | 2019-03-21 | 2022-05-10 | 福建师范大学 | Dual-mode immunoassay method for ovarian cancer tumor marker |
| CN110031527B (en) * | 2019-04-30 | 2021-07-02 | 福建师范大学 | Double-reading biosensor for human thyroglobulin |
| CN110361533A (en) * | 2019-08-02 | 2019-10-22 | 福建师范大学 | A kind of interlayer type prostate-specific antigen optical electro-chemistry immunoassay method based on signals in situ amplification |
| CN110501487A (en) * | 2019-09-08 | 2019-11-26 | 浙江理工大学 | A kind of construction method of the electrochemical immunosensor based on Au@Zn-MOFs composite material |
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| CN104777157A (en) * | 2015-04-02 | 2015-07-15 | 西南大学 | Non-enzyme electrogenerated chemiluminescence (ECL) glucose sensor |
| CN105954346A (en) * | 2016-07-27 | 2016-09-21 | 济南大学 | Preparation method of a three-dimensional paper-based electrochemical ratio meter |
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