CN107727714A - One kind is based on carbon nanohorn and TiO2The preparation method of the Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material - Google Patents
One kind is 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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Abstract
The present invention discloses one kind and is based on carbon nanohorn and TiO2The preparation method of the Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material, feature are to be based on carbon nanohorn (CNHs) and octahedra Detitanium-ore-type TiO2Mesomorphic（OAMs）Nano material, introduce perylene derivative（PTC‑NH2）And luminol（luminol）Two kinds of signal probes, a kind of Ratio-type electrochemical luminescence immunosensor of silicon phthalocyanine sensitization is prepared, and be used for PSA（PSA）Detection.The OAMs of CNHs and higher porosity with bigger serface can carry substantial amounts of biomolecule and signal probe as biosensor platform, meanwhile, hexafluoro bisphenol-a silicon phthalocyanine（HFSPC）Electron withdraw group luminol can be accelerated to lose electronics, obtain one enhancing ECL signals.Based on above-mentioned advantage, obtained Ratio-type electrochemical luminescence immunosensor, there is high specificity, high sensitivity, the advantage such as stability is good, test limit is low, available for PSA（PSA）Detection, there is more important application value and practical significance in terms of clinical practice.
Description
Technical field
The invention belongs to new function material and bio-sensing detection technique field, and in particular to one kind is based on carbon nanohorn
And TiO2The Ratio-type PSA of mesomorphic nano material（PSA）The preparation side of electrochemical luminescence immunosensor
Method.
Background technology
Ratio method, a kind of new analysis method, it quantifies the ratio rather than absolute value depending on two signals, progressively
The fields such as fluorescence, electrochemical luminescence, photoelectricity and electrochemistry are used in, compared to other analytical technologies, electrochemical luminescence has spirit
The advantages that sensitivity is high, simple, fast reaction, the development for being combined into biology sensor of itself and Ratio-type analytical technology provide more
Wide application prospect.Electrochemical luminescence immunosensor, utilize one kind biology of the specific binding between antigen and antibody
Sensor, there is high sensitivity, selectivity is good, easy to operate, is easy to miniaturization, continuously can fast and automatically change detection and analysis etc.
Advantage, have a good application prospect.The present invention is prepared for one kind and is based on carbon nanohorn and TiO2The ratio of mesomorphic nano material
Type electrochemical luminescence immunosensor, and realize the highly sensitive detection to PSA.
Carbon nanohorn is a kind of new carbon nanomaterial, and because 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
Build in good electrochemical sensor and get 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, its performance typically by
The influence of crystal formation, grain size, crystal face, crystallinity, specific surface area, micro-structural etc..TiO2Mesomorphic is that crystal subunit is arranged in order
What row were formed, compared to traditional TiO2Monocrystalline, TiO2Mesomorphic has more excellent performance.The present invention is based on carbon nanohorn
And octahedra Detitanium-ore-type TiO (CNHs)2Mesomorphic（OAMs）Nano material, introduce perylene derivative（PTC-NH2）And Rumi
Promise（luminol）Two kinds of signal probes, a kind of Ratio-type electrochemical luminescence immunosensor of silicon phthalocyanine sensitization is prepared, and be used for
PSA（PSA）Detection.The OAMs of CNHs and higher porosity with bigger serface pass as biology
Sensor platform can carry substantial amounts of biomolecule and signal probe, meanwhile, hexafluoro bisphenol-a-silicon phthalocyanine（HFSPC）Electrophilic
Group can accelerate luminol to lose electronics, obtain the ECL signals of an enhancing.Based on above-mentioned advantage, obtained Ratio-type electricity
Chemiluminescence immunoassay sensor, there is high specificity, high sensitivity, the advantage such as stability is good, test limit is low, available for prostate
Specific antigen（PSA）Detection, there is very important application value and practical significance in terms of clinical practice.
The content of the invention
An object of the present invention is to be based on carbon nanohorn and TiO2Mesomorphic nano material, structure is a kind of unmarked, stable
Good, the electrochemical luminescence immunosensor of high sensitivity and preparation method thereof of property.
The second object of the present invention is that the electrochemical luminescence immunosensor is applied to the height of PSA
Sensitive Detection.
To realize goal of the invention, the present invention adopts the following technical scheme that：
1. one kind is based on carbon nanohorn and TiO2The Ratio-type PSA of mesomorphic nano material（PSA）Electrochemistry is sent out
The preparation method of light immunosensor, it is characterised in that comprise the following steps：
（1）Glass-carbon electrode（GCE）Mechanical grinding is polished first on the chamois leather for be covered with alumina powder, and surface is washed away with secondary water
Residual powder, then move into ultrasonic water bath and clean, until cleaning up, ethanol is finally sequentially used, diluted acid and water thoroughly wash；
（2）The carbon nanohorn that 3 μ L concentration are 3 mg/ml is added dropwise（CNHs）Suspension is infrared in clean glassy carbon electrode surface
Dried under lamp, be cooled to room temperature, CNHs modified glassy carbon electrodes are made；
（3）3 μ L 0.2mmol/L luminols are added dropwise（luminol）, 2mg/mL carboxymethyl chitosans（CMCS）And 2mg/mL hexafluoros
Bisphenol-A-silicon phthalocyanine（HFSPC）Mixed solution is dried under modified electrode surface, infrared lamp, is cooled to room temperature, is made
Luminol@CMCS@HFSPC/CNHs modified glassy carbon electrodes；
（4）It is 1 that luminol@CMCS@HFSPC/CNHs modified electrodes are immersed into concentration ratio:2（1- (3- dimethylamino-propyls)-
3- ethyl-carbodiimide hydrochlorides）（EDC）With n-hydroxysuccinimide（NHS）Mixed liquor in 50min, then immerse 30 μ L
Concentration is 2 mg/mL horseradish peroxidases（HRP）The PSA antibody of mark（Ab1）In solution and at 4 °C
45min is incubated in refrigerator, the Ab of physical absorption is washed with deionized water1, HRP-Ab is made1/luminol@CMCS@HFSPC/
CNHs modified glassy carbon electrodes；
（5）The BSA for taking 60 μ L concentration to be 1.0 wt.% is added drop-wise to step（4）1 h is incubated in the refrigerator of 4 °C of modified electrode surface,
With nonspecific activity site on enclosed-electrode surface, physical absorption is washed away with deionized water rinsing electrode surface, and be stored in
In 4 °C of refrigerators；
（6）By step（5）The modified electrode of acquisition immerses the PSA of various concentrations（PSA）In standard liquid simultaneously
1 h is incubated in 4 °C of refrigerators, electrode surface is rinsed with pH7.5 PBS cushioning liquid, PSA/HRP-Ab is made1/luminol@
CMCS@HFSPC/CNHs modified glassy carbon electrodes.
（7）By step（6）The modified electrode of acquisition immerses 50 μ L perylene derivatives（PTC-NH2）, octahedra rutile titania
Ore deposit type TiO2Mesomorphic（OAMs）With PSA antibody（Ab2）（PTC-NH2@OAMs@ Ab2）The compound of composition is molten
50 min are incubated in liquid and in 4 °C of refrigerators, electrode surface is rinsed with pH7.5 PBS cushioning liquid, PTC-NH is made2@
OAMs@ Ab2/PSA/HRP-Ab1/ luminol@CMCS@HFSPC/CNHs modified glassy carbon electrodes, and be stored in 4 °C of refrigerators.
2. above-mentioned luminol（luminol）, carboxymethyl chitosan（CMCS）And hexafluoro bisphenol-a-silicon phthalocyanine（HFSPC）It is mixed
Close what solution was prepared by following methods：By the way that 2mmol/L luminols are simply mixed（luminol）, 20mg/mL carboxymethyl chitosans
Sugar（CMCS）And 20mg/mL hexafluoro bisphenol-as-silicon phthalocyanine（HFSPC）Solution be simply mixed and be suitably added the water of certain volume
Uniformly mix and be prepared into again.
3. above-mentioned hexafluoro bisphenol-a-silicon phthalocyanine（HFSPC）Prepared by following methods：By 0.20 g SiPcCl2、0.24g
Hexafluoro bisphenol-a and 0.14 g anhydrous Ks2CO3It is placed in 30ml toluene solvants and is flowed back 48 hours under 110oC, it is then that reaction is mixed
Compound is cooled to room temperature and the solvent was evaporated under reduced pressure to doing, and then solid product is placed in apparatus,Soxhlet's pure with dichloromethane
Change 24 hours, it is 1 that volume ratio is used after the material recrystallization being extracted：1 methanol and the mixed liquor elution of deionized water, Ran Houchong
Newly it is dissolved in dichloromethane.
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 following methods：1）Volume ratio is 1:1
6mg/ml OAMs solution and 1 wt % 3- aminopropyl triethoxysilanes（APTES）Mixing 6 h of concussion, are collected by centrifugation precipitation
Thing, deionized water is then redispersed in, obtains NH2- OAMs solution；2) volume ratio is 1:45 mg/ml PTC-NH2With 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 ratios are 1:2（1- (3- bis-
Methylaminopropyl) -3- ethyl-carbodiimide hydrochlorides）（EDC）And n-hydroxysuccinimide（NHS）Mixed liquor mixes and at 4 °
12 h are placed under C, sediment is collected by centrifugation and is dispersed in pH 7.4 PBS solution, then add 0.5 wt %BSA go unless
Specific adsorption site.
5. above-mentioned perylene derivative（PTC-NH2）Prepared by following methods：20mg 3,4,9,10- tetracarboxylic acids
Acid dianhydride（PTCDA）The stirring and dissolving in 2 mL acetone, then by 0.1ml anhydrous ethylenediamines（C2H8N4）Slowly add under 4 °C
Enter into above-mentioned solution and be then centrifuged for, wash to pH to be 7.4, remove excessive ethylenediamine.Finally, products therefrom is uniformly divided
It is dispersed in 4ml deionized waters, is stored in 4 °C of refrigerators.
6. above-mentioned octahedra Detitanium-ore-type TiO2Mesomorphic（OAMs）Material is prepared by following methods：1.2 grams of rutile titania
Ore deposit type TiO2Powder is dissolved in the KOH solution that 60mL concentration is 18mol/ml, and after stirring 20 minutes, suspension is moved into one
In individual 100ml polytetrafluoroethyllining linings stainless steel autoclave；Reactor is sealed, is reacted 72 hours under 170oC, is cooled to room
Temperature；Then, it is 3.5 to be washed with dilute acetic acid solution and be precipitated to precipitation pH value, dries 12 hours and obtains under 65oC after pelleting centrifugation
Product metatitanic acid nano wire；400mg presoma metatitanic acid nano wires are taken to be dispersed in 70ml spirit of vinegars, in 100 polytetrafluoroethylene (PTFE) high pressures
React 48h in reactor under the conditions of 200oC, resulting product distilled water and absolute ethyl alcohol centrifuge washing, under the conditions of 60oC
Dry and calcined 30 minutes under 12 h and 400oC, to remove remaining organic matter, OAMs is made.
7. PSA（PSA）Detecting step：
（1）It is measured using electrochemical workstation using three-electrode system, carbon is based on one kind prepared by above-mentioned preparation method
Nanometer angle 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 cushioning liquid in tested；
（2）PSA using bipotential pattern to various concentrations（PSA）Standard liquid is detected, initial electricity
- 0.8 V of position, 5 seconds pulse periods, 40 seconds burst lengths, the V of pulse potential 0.6 and 3 seconds pulse periods, sent out by electroluminescent chemistry
Light device gathers -0.8 V ECL signal intensities (Ic) and 0.6 V ECL signal intensities (Ia), passes through its rate value (Ic/
) and PSA Ia（PSA）Relation between concentration of standard solution, drawing curve；
（3）Testing sample solution replaces PSA（PSA）Solution is detected, and the result of detection can pass through work
Curve checks in.
The present invention remarkable advantage be：
(1) ratio method, a kind of new analysis method, it quantifies the ratio rather than absolute value depending on two signals, progressively
It is used in the fields such as fluorescence, electrochemical luminescence, photoelectricity and electrochemistry.Compared to other analytical technologies, electrochemical luminescence has spirit
The advantages that sensitivity is high, simple, fast reaction, the development for being combined into biology sensor of itself and Ratio-type analytical technology provide more
Wide application 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, hexafluoro bisphenol-a-silicon phthalocyanine（HFSPC）Electron withdraw group luminol can be accelerated to lose
De-electromation, obtain the ECL signals 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.
Brief description of the drawings
Fig. 1 is octahedra Detitanium-ore-type TiO2Mesomorphic（OAMs）Material, perylene derivative（PTC-NH2）And PTC-NH2
The infrared spectrum of@OAMs complex solutions（IR）Figure.
Fig. 2 is the electrochemical luminescence response signal and PSA of immune sensing electrode（PSA）Standard liquid
The linear relationship chart of concentration.
Embodiment
The present invention further illustrates the present invention with the following example, but protection scope of the present invention is not limited to following reality
Apply example.
Embodiment 1
One kind is based on carbon nanohorn and TiO2The Ratio-type PSA of mesomorphic nano material（PSA）Electrochemical luminescence
The preparation of immunosensor：
（1）Glass-carbon electrode（GCE）Mechanical grinding is polished first on the chamois leather for be covered with alumina powder, and table is washed away with secondary water
Face residual powder, then move into ultrasonic water bath and clean, until cleaning up, ethanol is finally sequentially used, diluted acid and water thoroughly wash；
（2）The carbon nanohorn that 3 μ L concentration are 3 mg/ml is added dropwise（CNHs）Suspension is infrared in clean glassy carbon electrode surface
Dried under lamp, be cooled to room temperature, CNHs modified glassy carbon electrodes are made；Carbon nanohorn can be realized using persons skilled in the art
Method prepare.
（3）3 μ L 0.2mmol/L luminols are added dropwise（luminol）, 2mg/mL carboxymethyl chitosans（CMCS）And 2mg/
ML hexafluoro bisphenol-as-silicon phthalocyanine（HFSPC）Mixed solution is dried under modified electrode surface, infrared lamp, is cooled to room temperature, is made
Luminol@CMCS@HFSPC/CNHs modified glassy carbon electrodes；
（4）It is 1 that luminol@CMCS@HFSPC/CNHs modified electrodes are immersed into concentration ratio:2（1- (3- dimethylaminos third
Base) -3- ethyl-carbodiimide hydrochlorides）（EDC）With n-hydroxysuccinimide（NHS）Mixed liquor in 50min, then immerse 30
μ L concentration is 2 mg/mL horseradish peroxidases（HRP）The PSA antibody of mark（Ab1）In solution and at 4 °
45min is incubated in C refrigerators, the Ab of physical absorption is washed with deionized water1, HRP-Ab is made1/luminol@CMCS@HFSPC/
CNHs modified glassy carbon electrodes；PSA antibody can use Beijing Bo Ao Bioisystech Co., Ltd to produce.
（5）The BSA for taking 60 μ L concentration to be 1.0 wt.% is added drop-wise to step（4）It is incubated in the refrigerator of 4 °C of modified electrode surface
1 h, with nonspecific activity site on enclosed-electrode surface, physical absorption is washed away with deionized water rinsing electrode surface, and protect
In the presence of in 4 °C of refrigerators；
（6）By step（5）The modified electrode of acquisition immerses the PSA of various concentrations（PSA）In standard liquid
And 1 h is incubated in 4 °C of refrigerators, electrode surface is rinsed with pH7.5 PBS cushioning liquid, PSA/HRP-Ab is made1/
Luminol@CMCS@HFSPC/CNHs modified glassy carbon electrodes.
（7）By step（6）The modified electrode of acquisition immerses 50 μ L perylene derivatives（PTC-NH2）, octahedra rutile titania
Ore deposit type TiO2Mesomorphic（OAMs）With PSA antibody（Ab2）（PTC-NH2@OAMs@ Ab2）The compound of composition is molten
50 min are incubated in liquid and in 4 °C of refrigerators, electrode surface is rinsed with pH7.5 PBS cushioning liquid, PTC-NH is made2@
OAMs@ Ab2/PSA/HRP-Ab1/ luminol@CMCS@HFSPC/CNHs modified glassy carbon electrodes, and be stored in 4 °C of refrigerators.
Above-mentioned concentration is 0.2mmol/L luminol（luminol）, concentration be 2mg/mL carboxymethyl chitosans（CMCS）And
Concentration is 2mg/mL hexafluoro bisphenol-a-silicon phthalocyanine（HFSPC）Mixed solution is prepared by following methods：It is 2mmol/L by concentration
Luminol（luminol）, concentration is 20mg/mL carboxymethyl chitosans（CMCS）And concentration is 20mg/mL hexafluoro bisphenol-as-silicon
Phthalocyanine（HFSPC）Solution be simply mixed and be suitably added the water of certain volume and uniformly mix and be prepared into again.
Above-mentioned hexafluoro bisphenol-a-silicon phthalocyanine（HFSPC）Prepared by following methods：By 0.20 g SiPcCl2, 0.24g six
Fluorine bisphenol-A and 0.14 g anhydrous Ks2CO3It is placed in 30ml toluene solvants and is flowed back 48 hours under 110oC, then mixes reaction
Thing is cooled to room temperature and the solvent was evaporated under reduced pressure to doing, and then solid product is placed in apparatus,Soxhlet's and purified with dichloromethane
24 hours, with volume ratio be 1 after the material recrystallization being extracted：1 methanol and the mixed liquor elution of deionized water, then again
It is dissolved in dichloromethane.
Embodiment 2
Octahedra Detitanium-ore-type TiO in embodiment 12Mesomorphic（OAMs）The preparation of material：1.2 grams of Detitanium-ore-type TiO2Powder
It is dissolved in the KOH solution that 60mL concentration is 18mol/ml, after stirring 20 minutes, suspension is moved into a 100ml polytetrafluoro
In ethene inner liner stainless steel autoclave；Reactor is sealed, is reacted 72 hours under 170oC, is cooled to room temperature；Then, with dilute vinegar
It is 3.5 that acid solution wash, which is precipitated to precipitation pH value, is dried 12 hours under 65oC after pelleting centrifugation and obtains product metatitanic acid nanometer
Line；400mg presoma metatitanic acid nano wires are taken to be dispersed in 70ml spirit of vinegars, the 200o in 100 polytetrafluoroethylene (PTFE) autoclaves
React 48h under the conditions of C, resulting product distilled water and absolute ethyl alcohol centrifuge washing, dry 12 h simultaneously under the conditions of 60oC
Calcined 30 minutes under 400oC, to remove remaining organic matter, OAMs is made.
Embodiment 3
Perylene derivative in embodiment 1（PTC-NH2）Preparation：20mg 3,4,9,10- tetracarboxylic dianhydrides
（PTCDA）The stirring and dissolving in 2 mL acetone, then by 0.1ml anhydrous ethylenediamines（C2H8N4）It is slowly added under 4 °C
State in solution and be then centrifuged for, wash to pH to be 7.4, remove excessive ethylenediamine.Finally, products therefrom is dispersed in 4ml
In deionized water, it is stored in 4 °C of refrigerators.
Embodiment 4
Perylene derivative in embodiment 1（PTC-NH2）, octahedra Detitanium-ore-type TiO2Mesomorphic（OAMs）（PTC-NH2@
OAMs）The preparation of complex solution：1）Volume ratio is 1:1 6mg/ml OAMs solution and 1 wt % 3- aminopropyl-triethoxies
Silane（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 1:45 mg/ml PTC-NH2With 4 mg/ml NH2- OAMs solution mixing 1 h of concussion, it is heavy to be collected by centrifugation
Starch, deionized water is then redispersed in, obtains PTC-NH2@OAMs solution.Fig. 1 is octahedra Detitanium-ore-type TiO2Mesomorphic
（OAMs）Material, perylene derivative（PTC-NH2）And PTC-NH2The infrared spectrum of@OAMs complex solutions（IR）Figure.
Compared to OAMs, PTC-NH2@OAMs are in 1599 cm-1There is the characteristic peak of the condensation aromatic ring of perylene skeleton at place, in addition,
1771 and 1301 cm-1Locate C=O and C-N stretching vibration characteristic peaks, and in 1637 cm-1Locate N-H flexural vibrations characteristic peak,
Illustrate PTC-NH2Successfully it is fixed on OAMs.
Embodiment 5
PSA（PSA）Detecting step：
（1）It is measured using electrochemical workstation using three-electrode system, carbon nanohorn is based on one kind prepared by embodiment 1
And TiO2The Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material is working electrode, and Ag/AgCl is reference electrode, platinum
Silk electrode is to electrode, in 10 mmol/ml K2S2O8PH 8.0 PBS cushioning liquid in tested；
（2）PSA using bipotential pattern to various concentrations（PSA）Standard liquid is detected, initial electricity
- 0.8 V of position, 5 seconds pulse periods, 40 seconds burst lengths, the V of pulse potential 0.6 and 3 seconds pulse periods, sent out by electroluminescent chemistry
Light device gathers -0.8 V ECL signal intensities (Ic) and 0.6 V ECL signal intensities (Ia), passes through its rate value (Ic/
) and PSA Ia（PSA）Relation between concentration of standard solution, drawing curve, Fig. 2 are immune sensing electricity
The electrochemical luminescence response signal of pole and the linear relationship chart of prostaglandin E1 concentration of standard solution；
（3）Testing sample solution replaces PSA（PSA）Solution is detected, and the result of detection can pass through work
Curve checks in.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, it should all belong to the covering scope of 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, it is characterised in that comprise the following steps：
（1）Glass-carbon electrode（GCE）Mechanical grinding is polished first on the chamois leather for be covered with alumina powder, and table is washed away with secondary water
Face residual powder, then move into ultrasonic water bath and clean, until cleaning up, ethanol is finally sequentially used, diluted acid and water thoroughly wash；
（2）The carbon nanohorn that 3 μ L concentration are 3 mg/ml is added dropwise（CNHs）Suspension is infrared in clean glassy carbon electrode surface
Dried under lamp, be cooled to room temperature, CNHs modified glassy carbon electrodes are made；
（3）The luminol that 3 μ L concentration are 0.2mmol/L is added dropwise（luminol）, concentration be 2mg/mL carboxymethyl chitosans
（CMCS）And hexafluoro bisphenol-a-silicon phthalocyanine that concentration is 2mg/mL（HFSPC）Mixed solution is under modified electrode surface, infrared lamp
Drying, is cooled to room temperature, and luminol@CMCS@HFSPC/CNHs modified glassy carbon electrodes are made；
（4）It is 1 that luminol@CMCS@HFSPC/CNHs modified electrodes are immersed into concentration ratio:2（1- (3- dimethylaminos third
Base) -3- ethyl-carbodiimide hydrochlorides）（EDC）With n-hydroxysuccinimide（NHS）Mixed liquor in 50min, then immerse 30
μ L concentration is 2 mg/mL horseradish peroxidases（HRP）The PSA antibody of mark（Ab1）In solution and at 4 °
45min is incubated in C refrigerators, the Ab of physical absorption is washed with deionized water1, HRP-Ab is made1/luminol@CMCS@HFSPC/
CNHs modified glassy carbon electrodes；
（5）The BSA for taking 60 μ L concentration to be 1.0 wt.% is added drop-wise to step（4）4 °C of modified electrode surface refrigerator in be incubated 1
H, with nonspecific activity site on enclosed-electrode surface, physical absorption is washed away with deionized water rinsing electrode surface, and preserve
In 4 °C of refrigerators；
（6）By step（5）The modified electrode of acquisition immerses the PSA of various concentrations（PSA）In standard liquid
And 1 h is incubated in 4 °C of refrigerators, electrode surface is rinsed with pH7.5 PBS cushioning liquid, PSA/HRP-Ab is made1/
Luminol@CMCS@HFSPC/CNHs modified glassy carbon electrodes.
（7）By step（6）The modified electrode of acquisition immerses 50 μ L perylene derivatives（PTC-NH2）, octahedra Detitanium-ore-type
TiO2Mesomorphic（OAMs）With PSA antibody（Ab2）（PTC-NH2@OAMs@ Ab2）In the complex solution of composition
And 50 min are incubated in 4 °C of refrigerators, electrode surface is rinsed with pH7.5 PBS cushioning liquid, PTC-NH is made2@OAMs@
Ab2/PSA/HRP-Ab1/ luminol@CMCS@HFSPC/CNHs modified glassy carbon electrodes, and be stored in 4 °C of refrigerators.
2. according to the method for claim 1, it is characterised in that described concentration is 0.2mmol/L luminol
（luminol）, concentration be 2mg/mL carboxymethyl chitosans（CMCS）And hexafluoro bisphenol-a-silicon phthalocyanine that concentration is 2mg/mL
（HFSPC）Mixed solution is prepared by following methods：It is 2mmol/L luminols by concentration（luminol）, concentration 20mg/
ML carboxymethyl chitosans（CMCS）And concentration is 20mg/mL hexafluoro bisphenol-as-silicon phthalocyanine（HFSPC）Solution be simply mixed and fit
When the water for adding certain volume uniformly mixes again and it is prepared into.
3. method according to claim 1 or 2, it is characterised in that described hexafluoro bisphenol-a-silicon phthalocyanine（HFSPC）Under
State method preparation：By 0.20 g SiPcCl2, 0.24g hexafluoro bisphenol-as and 0.14 g anhydrous Ks2CO3It is placed in 30ml toluene solvants
In flowed back 48 hours under 110oC, reactant mixture is then cooled to room temperature and the solvent was evaporated under reduced pressure to dry, then will
Solid product is placed in apparatus,Soxhlet's is purified 24 hours with dichloromethane, 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 dichloromethane.
4. according to the method for claim 1, it is characterised in that described perylene derivative（PTC-NH2）, it is octahedra
Detitanium-ore-type TiO2Mesomorphic（OAMs）With PSA antibody（Ab2）（PTC-NH2@OAMs@ Ab2）Complex solution
Prepared by following methods：1）Volume ratio is 1:1 6mg/ml OAMs solution and 1wt% 3- aminopropyl triethoxysilanes
（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 1:45 mg/ml PTC-NH2With 4 mg/ml NH2- OAMs solution mixing 1 h of concussion, is collected by centrifugation precipitation
Thing, deionized water is then redispersed in, obtains PTC-NH2@OAMs solution；3）By 100 μ L Ab2And 100 μ L PTC-NH2 @
OAMs solution and 60 μ l concentration ratios are 1:2（1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides）（EDC）And N-
HOSu NHS（NHS）Mix into mixed liquor and 12 h are placed under 4 °C, sediment is collected by centrifugation and is dispersed in pH
In 7.4 PBS solution, then add 0.5wt%BSA and remove non-specific adsorption sites.
5. the method according to claim 1 or 4, it is characterised in that described perylene derivative（PTC-NH2）Under
State method preparation：20mg 3,4,9,10- tetracarboxylic dianhydrides（PTCDA）The stirring and dissolving in 2 mL acetone, then by 0.1ml
Anhydrous ethylenediamine（C2H8N4）In above-mentioned solution is slowly added under 4 °C and is then centrifuged for, washs to pH to be 7.4, was removed
The ethylenediamine of amount.Finally, products therefrom is dispersed in 4ml deionized waters, be stored in 4 °C of refrigerators.
6. the method according to claim 1 or 4, it is characterised in that described octahedra Detitanium-ore-type TiO2Mesomorphic（OAMs）
Material is prepared by following methods：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 stirring 20 minutes, suspension is moved into a 100ml polytetrafluoroethyllining lining stainless steel autoclave；Sealing reaction
Device, react 72 hours under 170oC, be cooled to room temperature；Then, it is 3.5 to be washed with dilute acetic acid solution and be precipitated to precipitation pH value, is sunk
Form sediment to dry 12 hours under 65oC after centrifuging and obtain product metatitanic acid nano wire；400mg presoma metatitanic acid nano wires are taken to be dispersed in
In 70ml spirit of vinegars, 48h is reacted under the conditions of 200oC in 100 polytetrafluoroethylene (PTFE) autoclaves, resulting product steams
Distilled water and absolute ethyl alcohol centrifuge washing, dry under the conditions of 60oC and calcine 30 minutes under 12 h and 400oC, to remove having for remnants
Machine thing, OAMs is made.
7. one kind prepared by any described methods of claim 1-6 is based on carbon nanohorn and TiO2The ratio of mesomorphic nano material
Type PSA（PSA）Electrochemical luminescence immunosensor.
8. described in claim 7 based on carbon nanohorn and TiO2The Ratio-type electrochemical luminescence immune sensing of mesomorphic nano material
Device, it is characterised in that for PSA（PSA）, detecting step is as follows：
（1）It is measured using electrochemical workstation using three-electrode system, carbon nanometer is based on one kind described in claim 7
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 cushioning liquid in tested；
（2）PSA using bipotential pattern to various concentrations（PSA）Standard liquid is detected, initial electricity
- 0.8 V of position, 5 seconds pulse periods, 40 seconds burst lengths, the V of pulse potential 0.6 and 3 seconds pulse periods, sent out by electroluminescent chemistry
Light device gathers -0.8 V ECL signal intensities (Ic) and 0.6 V ECL signal intensities (Ia), passes through its rate value (Ic/
) and PSA Ia（PSA）Relation between concentration of standard solution, drawing curve；
（3）Testing sample solution replaces PSA（PSA）Solution is detected, and the result of detection can pass through work
Curve checks in.
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