CN104133069B - Preparation method for double-functional mark photo-electrochemical sensor and application - Google Patents
Preparation method for double-functional mark photo-electrochemical sensor and application Download PDFInfo
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Abstract
The invention provides a preparation method for a double-functional mark photo-electrochemical sensor and application and belongs to the technical fields of nano-functional materials, clinical analysis, bio-sensing and electrochemistry. A TiO2-CdSe semiconductor compound is prepared based on a ligand principle between TiO2 and carboxyl; a remarkably-enhanced photoelectric conversion effect of the prepared TiO2-CdSe semiconductor compound is displayed on a visible region; a photocurrent value is 10 times as much as that of a single TiO2 photocurrent value; the TiO2-CdSe semiconductor compound has large specific surface area and good biocompatibility and is used for marking a second antibody (Ab2) of CA125 so that a TiO2-CdSe-Ab2 hatched matter is prepared. The difference between the double-functional mark photo-electrochemical sensor and other electrochemical or photo-induced electrochemical sensors is as follows: the TiO2-CdSe semiconductor compound can be used for not only carrying out photoelectric conversion and but also generating Cd2<+>, so that an immunosensor taking the TiO2-CdSe semiconductor compound as a second antibody marker can adopt an electrochemical analysis technology and a photo-induced electrochemical analysis technology to rapidly, sensitively and accurately detect the CA125. The method has certain guiding significance and application value on early diagnosis of cancers.
Description
Technical field
The invention belongs to functionalized nano material, immunoassay and biosensor technique field, be specifically related to based on the difunctional TiO of one
2the preparations and applicatio of the sandwich type immunosensor that/CdSe semiconductor label builds.
Background technology
Semiconductor material can be with because of it exists energy level difference between valence band, makes it can produce the transition of electronics under the exciting of light homenergic, thus presents the semiconductive state between conductor and insulator.
TiO
2it is a kind of semiconductor material through aspect application such as photocatalysis of being everlasting, solar cell and fuel cells.TiO
2there is the feature of easily batch synthesis and environmentally safe.Compared to block TiO
2, the TiO of nano shape
2have that specific surface area is large, bioaffinity good and be convenient to the advantages such as functionalization.But unfortunately, TiO
2bandwidth wider (3.2 eV), thus make it effectively can only be excited at ultraviolet region, the photoelectric transformation efficiency in visible region is extremely low.In order to the light transfer characteristic that can show in visible-range, to TiO
2sensitization is the Hot Contents of researcher research always.
CdSe quantum dots (CdSe QDs) is a kind of narrow semiconductor nano material that can be with, and its bandwidth is about 1.7 eV, almost close to TiO
250% size of bandwidth.Narrow energy gap makes CdSe QDs just can show good photocatalysis and opto-electronic conversion character within the scope of visible region.In addition, show according to correlative study, when wide can be with semiconductor and narrow can be with the mutual compound of semiconductor after, the compound obtained can show and be better than both photoelectric conversion result itself.Therefore say, CdSe is except inherently a kind of good opto-electronic conversion original paper, and it also can be used as TiO
2sensitizer in order to improve TiO
2in the photoelectric transformation efficiency of visible region.
Photic electrochemical immunosensor is a kind of using luminous energy as excitation energy, and electric signal is as the sensor of detection signal.In sensors with auxiliary electrode, photo-electric conversion element-semiconductor material plays irreplaceable effect.Different just because of detection signal and excitation energy mode, make photic electrochemical sensor have high sensitivity.Immuno analytical method is combined with photoelectric detecting technology, greatly can improves the sensitivity that measured object detects, and can detectability be reduced.By the impact of measured matter on the photosignal that semiconductor produces, realize the quantitative detection to measured matter.Semiconductor nano material has high specific surface area and good bioaffinity, therefore, it is possible to biomolecule such as the more antigen of load, antibody, thus improves the sensitivity detected further.
Different from photic galvanochemistry, electrochemical analysis is that a kind of electric signal is by different way as exciting the analysis and detection technology with detection signal.Electrochemical sensor a kind of electrochemical analysis is combined produced a kind of senser element with sensing technology.Its Cleaning Principle is the electrochemical signals affecting electrode system based on measured matter, thus realizes the quantitative-qualitative analysis to measured matter.Electrochemical sensor is also sensitive analysis detection technique emerging in recent years, and obtains research and apply widely in fields such as life science, bio-science and Surface Sciences.Nano material is often used in the building process of electrochemical sensor because of its excellent physics, chemical property.
Tumor markers refers to that tumour cell produces a kind of material of secretion in growth course, and existing of its content and tumour cell is closely related.By detecting tumor markers, the monitoring to tumour can be realized clinically, realizing, to the early diagnosis of tumor disease and prevention etc., having important directive significance to clinical detection.CA125 is a kind of Specific marker of oophoroma.In human ovarian cancer patients's serum, CA125 level can apparently higher than normal value, and chemotherapy and operation responder CA125 level decline very soon, if when having recurrence, CA125 raises can prior to before clinical symptoms.Therefore realize the rapid and accurate determination of CA125, Timeliness coverage also understands the state of an illness, has important directive significance to clinical detection.
The present invention is based on TiO
2and the coordination between carboxyl, has prepared TiO by simply shaking method
2-CdSe semiconductor compound.Prepared TiO
2-CdSe semiconductor compound shows 10 times to TiO in visible region
2photo-current intensity.First by prepared TiO
2-CdSe semiconductor compound, as the two anti-labels of tumor markers CA125, successfully constructs the sandwich type immunosensor for Sensitive Detection CA125.
With TiO
2-CdSe is two anti-labels, this sandwich type immunosensor can be used up send a telegraph the quantitative detection that chemistry and galvanochemistry two kinds of analysis and detection technologies realize CA125.Testing result shows, no matter be electrochemical method or photic electrochemical method, this sensor all has high sensitivity, low detection limit and the wide range of linearity to the detection of CA125.With prepared TiO
2the sandwich type immunosensor of-CdSe semiconductor compound constructed by the anti-label of CA125 bis-, the analysis that can expand to other tumor markerses such as CA153, CA199 detects.
Summary of the invention
An object of the present invention is to provide the TiO that preparation has high-photoelectric transformation efficiency
2the short-cut method of-CdSe heterojunction composite semiconductor.
Two of object of the present invention is by prepared TiO
2-CdSe semiconductor compound as the anti-label of CA125 bis-for building sandwich type immunosensor.
Three of object of the present invention uses galvanochemistry and photic galvanochemistry two kinds of methods to realize detecting the rapid sensitive of CA125.
Technical scheme of the present invention, comprises the following steps.
1. the preparation method of a difunctional mark Optical Electro-Chemistry sensor
(1) pre-service of ITO electrode: ito glass is cut to 3 cm × 0.5 cm sizes, use liquid detergent, acetone, ethanol and ultrapure water ultrasonic cleaning 30 min successively, finally dry up glass with nitrogen, reserve the area of 0.5 cm × 0.5 cm size in order to modified biological molecule with insulating tape in one end of ITO electrode;
(2) be in the methanol solution of 3-aminopropyl triethoxysilane APTES of 1% ~ 3% by the ITO electrode after process being immersed in volume fraction, lucifuge soaks 10 ~ 14 h, rinses electrode, to remove unconjugated APTES molecule with water;
(3) the 10 μ L gold nano colloidal sols getting fresh preparation are added drop-wise to electrode surface, in conjunction with CA125 primary antibodie molecule;
(4) 6 μ L, 8 ~ 12 μ g/mL CA125 primary antibodie solution are added drop-wise on electrode, are placed in 4 DEG C of constant temperature refrigerators and hatch 2 h, rinse electrode surface to remove unconjugated primary antibodie molecule;
(5) drip painting 2 μ L, massfraction be the BSA solution of 0.5% ~ 1.5% on electrode, be placed in 4 DEG C of Refrigerator stores, dry;
(6) clean with ultrapure water, dry in the air to moisture state, the CA125 antigenic solution of 6 μ L, 0.05 pg/mL ~ 100 ng/mL variable concentrations is dripped and is applied to electrode surface, make, between itself and CA125 primary antibodie, specific immune response occurs and continue hatching 2 h, with ultrapure water to remove unconjugated CA125, dry;
(7) 6 μ LTiO are got
2-CdSe-Ab
2two anti-compound solution of incubating drip and are applied to electrode surface, hatch 2 h post-flush electrode surfaces, dry, obtained difunctional mark Optical Electro-Chemistry sensor.
2. TiO
2-CdSe-Ab
2two anti-preparation methods of incubating compound solution
(1) TiO
2the preparation of nano particle
Getting 6 ~ 8 mL butyl titanates is dissolved in 30 mL ethanol, stir, get 10 mL ultrapure waters under agitation slowly to add in solution, room temperature magnetic agitation reacts 120 min, is transferred in autoclave by this mixed liquor, 200 DEG C of hydro-thermal reaction 10 ~ 12 h, obtained white solid, centrifuging, and clean 5 times respectively with intermediate water and ethanol, vacuum drying 10 ~ 14 h at 80 DEG C, the obtained TiO of grinding
2nano particle.
(2) preparation of carboxyl-functional CdSe quantum dot
By 1.0 ~ 1.3 g CdCl
22.5H
2o is dissolved in 40 mL water, adds the mixed liquor of mercaptoacetic acid, regulates pH to 9 ~ 11, add the NaSeSO of 12 mL, 0.2 mol/L under stirring with the NaOH of 1 mol/L
3solution, reactant oil bath is heated to boiling, reflux 4 h, finally obtains carboxylated CdSe quantum dot solution;
The mixed liquor of described mercaptoacetic acid be by 50 ~ 60 mL high purity waters and 0.84 mL mercaptoacetic acid blended obtained; The NaSeSO of described 0.2 mol/L
3solution, gets 10 mmol Se and 12 mmol Na
2sO
3add in 50 mL water, react 3 h, filter obtained.
(3) TiO
2-CdSe-Ab
2two anti-preparations of incubating compound solution
Take 3 ~ 5 mg TiO
2be scattered in the carboxylated CdSe quantum dot solution of 4 mL, ultrasonic and stirring reaction 10 ~ 14 h, after question response completes, with ultrapure water centrifuge washing 3 times, obtained TiO
2-CdSe semiconductor compound; By TiO
2-CdSe semiconductor compound is scattered in 1 mL water, with the carboxyl on 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides EDC/N-N-Hydroxysuccinimide NHS crosslinking chemical activated camplex surface, by the anti-Ab of CA125 bis-of 1 mL, 8 ~ 12 μ g/mL
2add mixing, and shake 12 h in 4 DEG C of isothermal vibration devices, remove unconjugated Ab with ultrapure water centrifuge washing
2molecule, obtained TiO
2-CdSe-Ab
2two anti-incubate compound solution.
3. galvanochemistry and photic electrochemical method realize the quantitative detection to human body CA125
(1) difunctional mark Optical Electro-Chemistry sensor is placed in 0.3 ~ 0.5 mL, the salpeter solution of 0.1 mol/L soaks 4 h, makes CdSe quantum dot be decomposed into Cd
2+, joined 3.6 mL, 30 ~ 50 mg/mL Hg
2+pH 4.6 NaAc_HAc buffer solution in, use three-electrode system, carry out Electrochemical Detection;
Described three electrode saturated calomel electrodes are contrast electrode, and platinum electrode is auxiliary electrode, and glass-carbon electrode is working electrode;
(2) scanning voltage scope is from-0.9 ~-0.6 V, and the electromotive force rank that jump are 4 mV, frequency 25 Hz, amplitude 25 mV;
(3) Cd
2+there is redox reaction and produce signal near-0.7 V, according to the linear relationship between gained strength of current and analysis thing, drawing curve;
(4) Photoelectric Detection adopts i-t means of testing, and bias voltage is set to 0 V; Carry out switch lamp every 20 s, before and after record switch lamp, the changing value of electric current, produces the photocurrent values of different size, drawing curve according to the antigen of variable concentrations;
(5) testing sample solution is replaced the standard solution of CA125, detect according to the method for drafting of the working curve of described CA125.
useful achievement of the present invention
(1) photoelectricity successful is prepared higher than TiO based on coordination principle
2tiO
2-CdSe semiconductor composite nano material.Prepared TiO
2-CdSe semiconductor compound shows 10 times to TiO in visible region
2photo-current intensity, thus significantly improve sensing system detect sensitivity, system detectability is reduced.
(2) TiO is prepared
2the CA125 bis-of-CdSe compound mark resists, build sandwich type immunosensor realize to CA125 sensitive, accurately detect.
(3) with other galvanochemistry or photic electrochemical sensor unlike, due to TiO
2-CdSe semiconductor compound can carry out opto-electronic conversion, can produce Cd again
2+.Therefore, TiO
2-CdSe semiconductor compound as the immunosensor of two anti-labels can adopt galvanochemistry and photic galvanochemistry two kinds of analytical technologies realize to CA125 quick, sensitive, accurately detect, and can the accuracy of the result mutually.
(4) adopt electrochemical method to be 0.1 pg/mL ~ 20 ng/mL to the sensing range of CA125, detect and be limited to 0.02 pg/mL; Adopt photic electrochemical method to be 0.05 pg/mL ~ 100 ng/mL to the sensing range of CA125, detect and be limited to 0.01 pg/mL.
Embodiment
embodiment 1the preparation method of difunctional mark Optical Electro-Chemistry sensor
(1) pre-service of ITO electrode: ito glass is cut to 3 cm × 0.5 cm sizes, use liquid detergent, acetone, ethanol and ultrapure water ultrasonic cleaning 30 min successively, finally dry up glass with nitrogen, reserve the area of 0.5 cm × 0.5 cm size in order to modified biological molecule with insulating tape in one end of ITO electrode;
(2) be in the methanol solution of 3-aminopropyl triethoxysilane APTES of 1% by the ITO electrode after process being immersed in volume fraction, lucifuge soaks 10h, rinses electrode, to remove unconjugated APTES molecule with water;
(3) the 10 μ L gold nano colloidal sols getting fresh preparation are added drop-wise to electrode surface, in conjunction with CA125 primary antibodie molecule;
(4) 6 μ L, 8 μ g/mL CA125 primary antibodie solution are added drop-wise on electrode, are placed in 4 DEG C of constant temperature refrigerators and hatch 2 h, rinse electrode surface to remove unconjugated primary antibodie molecule;
(5) drip painting 2 μ L, massfraction be the BSA solution of 0.5% on electrode, be placed in 4 DEG C of Refrigerator stores, dry;
(6) clean with ultrapure water, dry in the air to moisture state, the CA125 antigenic solution of 6 μ L, 0.05 pg/mL ~ 100 ng/mL variable concentrations is dripped and is applied to electrode surface, make, between itself and CA125 primary antibodie, specific immune response occurs and continue hatching 2 h, with ultrapure water to remove unconjugated CA125, dry;
(7) 6 μ LTiO are got
2-CdSe-Ab
2two anti-compound solution of incubating drip and are applied to electrode surface, hatch 2 h post-flush electrode surfaces, dry, obtained difunctional mark Optical Electro-Chemistry sensor.
embodiment 2the preparation method of difunctional mark Optical Electro-Chemistry sensor
(1) pre-service of ITO electrode: ito glass is cut to 3 cm × 0.5 cm sizes, use liquid detergent, acetone, ethanol and ultrapure water ultrasonic cleaning 30 min successively, finally dry up glass with nitrogen, reserve the area of 0.5 cm × 0.5 cm size in order to modified biological molecule with insulating tape in one end of ITO electrode;
(2) be that in the methanol solution of 3-aminopropyl triethoxysilane APTES of 2%, lucifuge soaks 12 h, rinses electrode with water by the ITO electrode after process being immersed in volume fraction, to remove unconjugated APTES molecule;
(3) the 10 μ L gold nano colloidal sols getting fresh preparation are added drop-wise to electrode surface, in conjunction with CA125 primary antibodie molecule;
(4) 6 μ L, 10 μ g/mL CA125 primary antibodie solution are added drop-wise on electrode, are placed in 4 DEG C of constant temperature refrigerators and hatch 2 h, rinse electrode surface to remove unconjugated primary antibodie molecule;
(5) drip painting 2 μ L, massfraction be the BSA solution of 1.0% on electrode, be placed in 4 DEG C of Refrigerator stores, dry;
(6) clean with ultrapure water, dry in the air to moisture state, the CA125 antigenic solution of 6 μ L, 0.05 pg/mL ~ 100 ng/mL variable concentrations is dripped and is applied to electrode surface, make, between itself and CA125 primary antibodie, specific immune response occurs and continue hatching 2 h, with ultrapure water to remove unconjugated CA125, dry;
(7) 6 μ LTiO are got
2-CdSe-Ab
2two anti-compound solution of incubating drip and are applied to electrode surface, hatch 2 h post-flush electrode surfaces, dry, obtained difunctional mark Optical Electro-Chemistry sensor.
embodiment 3the preparation method of difunctional mark Optical Electro-Chemistry sensor
(1) pre-service of ITO electrode: ito glass is cut to 3 cm × 0.5 cm sizes, use liquid detergent, acetone, ethanol and ultrapure water ultrasonic cleaning 30 min successively, finally dry up glass with nitrogen, reserve the area of 0.5 cm × 0.5 cm size in order to modified biological molecule with insulating tape in one end of ITO electrode;
(2) be that in the methanol solution of 3-aminopropyl triethoxysilane APTES of 3%, lucifuge soaks 14 h, rinses electrode with water by the ITO electrode after process being immersed in volume fraction, to remove unconjugated APTES molecule;
(3) the 10 μ L gold nano colloidal sols getting fresh preparation are added drop-wise to electrode surface, in conjunction with CA125 primary antibodie molecule;
(4) 6 μ L, 12 μ g/mL CA125 primary antibodie solution are added drop-wise on electrode, are placed in 4 DEG C of constant temperature refrigerators and hatch 2 h, rinse electrode surface to remove unconjugated primary antibodie molecule;
(5) drip painting 2 μ L, massfraction be the BSA solution of 1.5% on electrode, be placed in 4 DEG C of Refrigerator stores, dry;
(6) clean with ultrapure water, dry in the air to moisture state, the CA125 antigenic solution of 6 μ L, 0.05 pg/mL ~ 100 ng/mL variable concentrations is dripped and is applied to electrode surface, make, between itself and CA125 primary antibodie, specific immune response occurs and continue hatching 2 h, with ultrapure water to remove unconjugated CA125, dry;
(7) 6 μ LTiO are got
2-CdSe-Ab
2two anti-compound solution of incubating drip and are applied to electrode surface, hatch 2 h post-flush electrode surfaces, dry, obtained difunctional mark Optical Electro-Chemistry sensor.
embodiment 4tiO
2the preparation of nano particle
Getting 6 mL butyl titanates is dissolved in 30 mL ethanol, stir, get 10 mL ultrapure waters under agitation slowly to add in solution, room temperature magnetic agitation reacts 120 min, is transferred in autoclave by this mixed liquor, 200 DEG C of hydro-thermal reaction 10 h, obtained white solid, centrifuging, and clean 5 times respectively with intermediate water and ethanol, vacuum drying 10 h at 80 DEG C, the obtained TiO of grinding
2nano particle.
embodiment 5tiO
2the preparation of nano particle
Getting 7 mL butyl titanates is dissolved in 30 mL ethanol, stir, get 10 mL ultrapure waters under agitation slowly to add in solution, room temperature magnetic agitation reacts 120 min, is transferred in autoclave by this mixed liquor, 200 DEG C of hydro-thermal reaction 11 h, obtained white solid, centrifuging, and clean 5 times respectively with intermediate water and ethanol, vacuum drying 12 h at 80 DEG C, the obtained TiO of grinding
2nano particle.
embodiment 6tiO
2the preparation of nano particle
Getting 8 mL butyl titanates is dissolved in 30 mL ethanol, stir, get 10 mL ultrapure waters under agitation slowly to add in solution, room temperature magnetic agitation reacts 120 min, is transferred in autoclave by this mixed liquor, 200 DEG C of hydro-thermal reaction 12 h, obtained white solid, centrifuging, and clean 5 times respectively with intermediate water and ethanol, vacuum drying 14 h at 80 DEG C, the obtained TiO of grinding
2nano particle.
embodiment 7the preparation of carboxyl-functional CdSe quantum dot
By 1.0 gCdCl
22.5H
2o is dissolved in 40mL water, adds the mixed liquor of mercaptoacetic acid, regulates pH to 9, add the NaSeSO of 12mL, 0.2mol/L under stirring with the NaOH of 1mol/L
3solution, is heated to boiling by reactant oil bath, backflow 4h, finally obtains carboxylated CdSe quantum dot solution;
The mixed liquor of described mercaptoacetic acid be by 50 mL high purity waters and 0.84mL mercaptoacetic acid blended obtained; The NaSeSO of described 0.2mol/L
3solution, gets 10mmolSe and 12mmolNa
2sO
3add in 50mL water, reaction 3h, filter obtained.
embodiment 8the preparation of carboxyl-functional CdSe quantum dot
By 1.2 gCdCl
22.5H
2o is dissolved in 40mL water, adds the mixed liquor of mercaptoacetic acid, regulates pH to 10, add the NaSeSO of 12mL, 0.2mol/L under stirring with the NaOH of 1mol/L
3solution, is heated to boiling by reactant oil bath, backflow 4h, finally obtains carboxylated CdSe quantum dot solution;
The mixed liquor of described mercaptoacetic acid be by 55 mL high purity waters and 0.84mL mercaptoacetic acid blended obtained; The NaSeSO of described 0.2mol/L
3solution, gets 10mmolSe and 12mmolNa
2sO
3add in 50mL water, reaction 3h, filter obtained.
embodiment 9the preparation of carboxyl-functional CdSe quantum dot
By 1.3 gCdCl
22.5H
2o is dissolved in 40mL water, adds the mixed liquor of mercaptoacetic acid, regulates pH to 11, add the NaSeSO of 12mL, 0.2mol/L under stirring with the NaOH of 1mol/L
3solution, is heated to boiling by reactant oil bath, backflow 4h, finally obtains carboxylated CdSe quantum dot solution;
The mixed liquor of described mercaptoacetic acid be by 60 mL high purity waters and 0.84mL mercaptoacetic acid blended obtained; The NaSeSO of described 0.2mol/L
3solution, gets 10mmolSe and 12mmolNa
2sO
3add in 50mL water, reaction 3h, filter obtained.
embodiment 10tiO
2-CdSe-Ab
2the preparation of two anti-hatching things
Take 3 mg TiO
2be scattered in the carboxylated CdSe quantum dot solution of 4 mL, ultrasonic and stirring reaction 10 ~ 14 h, after question response completes, with ultrapure water centrifuge washing 3 times, obtained TiO
2-CdSe semiconductor compound; By TiO
2-CdSe semiconductor compound is scattered in 1 mL water, with the carboxyl on 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides EDC/N-N-Hydroxysuccinimide NHS crosslinking chemical activated camplex surface, by the anti-Ab of CA125 bis-of 1 mL, 8 μ g/mL
2add mixing, and shake 12 h in 4 DEG C of isothermal vibration devices, remove unconjugated Ab with ultrapure water centrifuge washing
2molecule;
Described TiO
2, be the TiO of embodiment 4, embodiment 5 or embodiment 6 preparation
2nano particle.
Described carboxylated CdSe quantum dot solution is the carboxylated CdSe quantum dot solution of embodiment 7, embodiment 8 or embodiment 9 preparation.
embodiment 11tiO
2-CdSe-Ab
2the preparation of two anti-hatching things
Take 4 mg TiO
2be scattered in the carboxylated CdSe quantum dot solution of 4 mL, ultrasonic and stirring reaction 10 ~ 14 h, after question response completes, with ultrapure water centrifuge washing 3 times, obtained TiO
2-CdSe semiconductor compound; By TiO
2-CdSe semiconductor compound is scattered in 1 mL water, with the carboxyl on 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides EDC/N-N-Hydroxysuccinimide NHS crosslinking chemical activated camplex surface, by the anti-Ab of CA125 bis-of 1 mL, 10 μ g/mL
2add mixing, and shake 12 h in 4 DEG C of isothermal vibration devices, remove unconjugated Ab with ultrapure water centrifuge washing
2molecule;
Described TiO
2for the TiO of embodiment 5, embodiment 6 or embodiment 7 preparation
2nano particle.
Described carboxylated CdSe quantum dot solution is the carboxylated CdSe quantum dot solution of embodiment 7, embodiment 8 or embodiment 9 preparation.
embodiment 12tiO
2-CdSe-Ab
2the preparation of two anti-hatching things
Take 5 mg TiO
2be scattered in the carboxylated CdSe quantum dot solution of 4 mL, ultrasonic and stirring reaction 10 ~ 14 h, after question response completes, with ultrapure water centrifuge washing 3 times, obtained TiO
2-CdSe semiconductor compound; By TiO
2-CdSe semiconductor compound is scattered in 1 mL water, with the carboxyl on 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides EDC/N-N-Hydroxysuccinimide NHS crosslinking chemical activated camplex surface, by the anti-Ab of CA125 bis-of 1 mL, 12 μ g/mL
2add mixing, and shake 12 h in 4 DEG C of isothermal vibration devices, remove unconjugated Ab with ultrapure water centrifuge washing
2molecule;
Described TiO
2for the TiO of embodiment 5, embodiment 6 or embodiment 7 preparation
2nano particle.
Described carboxylated CdSe quantum dot solution is the carboxylated CdSe quantum dot solution of embodiment 7, embodiment 8 or embodiment 9 preparation.
embodiment 13galvanochemistry and photic galvanochemistry two kinds of methods detect CA125
(1) difunctional mark Optical Electro-Chemistry sensor is placed in 0.3 mL, the salpeter solution of 0.1 mol/L soaks 4 h, makes CdSe quantum dot be decomposed into Cd
2+, joined 3.6 mL, 30 mg/mL Hg
2+pH 4.6 NaAc_HAc buffer solution in, use three-electrode system, carry out Electrochemical Detection;
Described three electrode saturated calomel electrodes are contrast electrode, and platinum electrode is auxiliary electrode, and glass-carbon electrode is working electrode;
(2) scanning voltage scope is from-0.9 ~-0.6 V, and the electromotive force rank that jump are 4 mV, frequency 25 Hz, amplitude 25 mV;
(3) Cd
2+there is redox reaction and produce signal near-0.7 V, according to the linear relationship between gained strength of current and analysis thing, drawing curve;
(4) Photoelectric Detection adopts i-t means of testing, and bias voltage is set to 0 V; Carry out switch lamp every 20 s, before and after record switch lamp, the changing value of electric current, produces the photocurrent values of different size, drawing curve according to the antigen of variable concentrations;
(5) testing sample solution is replaced the standard solution of CA125, detect according to the method for drafting of the working curve of described CA125;
(6) for the detection of CA125 in 1# blood serum sample, adopt using standard samples recovery, often kind of sample parallel detects 7 times, and its relative standard deviation is lower than 3.2%, and the recovery is 90.9% ~ 105%, and the preci-sion and accuracy of detection method of the present invention is all satisfactory.
embodiment 14galvanochemistry and photic galvanochemistry two kinds of methods detect CA125
(1) difunctional mark Optical Electro-Chemistry sensor is placed in 0.4 mL, the salpeter solution of 0.1 mol/L soaks 4 h, makes CdSe quantum dot be decomposed into Cd
2+, joined 3.6 mL, 40 mg/mL Hg
2+pH 4.6 NaAc_HAc buffer solution in, use three-electrode system, carry out Electrochemical Detection;
Described three electrode saturated calomel electrodes are contrast electrode, and platinum electrode is auxiliary electrode, and glass-carbon electrode is working electrode;
(2) scanning voltage scope is from-0.9 ~-0.6 V, and the electromotive force rank that jump are 4 mV, frequency 25 Hz, amplitude 25 mV;
(3) Cd
2+there is redox reaction and produce signal near-0.7 V, according to the linear relationship between gained strength of current and analysis thing, drawing curve;
(4) Photoelectric Detection adopts i-t means of testing, and bias voltage is set to 0 V; Carry out switch lamp every 20 s, before and after record switch lamp, the changing value of electric current, produces the photocurrent values of different size, drawing curve according to the antigen of variable concentrations;
(5) adopt electrochemical method to be 0.1 pg/mL ~ 20 ng/mL to the sensing range of CA125, detect and be limited to 0.02 pg/mL;
(6) adopt PhotoelectrochemicalMethod Method to be 0.05 pg/mL ~ 100 ng/mL to the sensing range of CA125, detect and be limited to 0.01 pg/mL;
(7) testing sample solution is replaced the standard solution of CA125, detect according to the method for drafting of the working curve of described CA125;
(8) for the detection of CA125 in 2# blood serum sample, adopt using standard samples recovery, often kind of sample parallel detects 7 times, and its relative standard deviation is lower than 1.9%, and the recovery is 95.6% ~ 97.6%, and the preci-sion and accuracy of detection method of the present invention is all satisfactory.
embodiment 15galvanochemistry and photic galvanochemistry two kinds of methods detect CA125
(1) difunctional mark Optical Electro-Chemistry sensor is placed in 0.5 mL, the salpeter solution of 0.1 mol/L soaks 4 h, makes CdSe quantum dot be decomposed into Cd
2+, joined 3.6 mL, 50 mg/mL Hg
2+pH 4.6 NaAc_HAc buffer solution in, use three-electrode system, carry out Electrochemical Detection;
Described three electrode saturated calomel electrodes are contrast electrode, and platinum electrode is auxiliary electrode, and glass-carbon electrode is working electrode;
(2) scanning voltage scope is from-0.9 ~-0.6 V, and the electromotive force rank that jump are 4 mV, frequency 25 Hz, amplitude 25 mV;
(3) Cd
2+there is redox reaction and produce signal near-0.7 V, according to the linear relationship between gained strength of current and analysis thing, drawing curve;
(4) Photoelectric Detection adopts i-t means of testing, and bias voltage is set to 0 V; Carry out switch lamp every 20 s, before and after record switch lamp, the changing value of electric current, produces the photocurrent values of different size, drawing curve according to the antigen of variable concentrations;
(5) for the detection of CA125 in 3# blood serum sample, adopt using standard samples recovery, often kind of sample parallel detects 7 times, and its relative standard deviation is lower than 1.8%, and the recovery is 95.8% ~ 98.0%, and the preci-sion and accuracy of detection method of the present invention is all satisfactory.
Claims (2)
1. a preparation method for difunctional mark Optical Electro-Chemistry sensor, is characterized in that, comprise the following steps:
(1) TiO
2the preparation of nano particle
Getting 6 ~ 8 mL butyl titanates is dissolved in 30 mL ethanol, stir, get 10 mL ultrapure waters under agitation slowly to add in solution, room temperature magnetic agitation reacts 120 min, is transferred in autoclave by this mixed liquor, 200 DEG C of hydro-thermal reaction 10 ~ 12 h, obtained white solid, centrifuging, and clean 5 times respectively with intermediate water and ethanol, vacuum drying 10 ~ 14 h at 80 DEG C, the obtained TiO of grinding
2nano particle;
(2) preparation of carboxyl-functional CdSe quantum dot
By 1.0 ~ 1.3 g CdCl
22.5H
2o is dissolved in 40 mL water, adds the mixed liquor of mercaptoacetic acid, regulates pH to 9 ~ 11, add the NaSeSO of 12 mL, 0.2 mol/L under stirring with the NaOH of 1 mol/L
3solution, reactant oil bath is heated to boiling, reflux 4 h, finally obtains carboxylated CdSe quantum dot solution;
The mixed liquor of described mercaptoacetic acid be by 50 ~ 60 mL high purity waters and 0.84 mL mercaptoacetic acid blended obtained; The NaSeSO of described 0.2 mol/L
3solution, gets 10 mmol Se and 12 mmol Na
2sO
3add in 50 mL water, react 3 h, filter obtained;
(3) TiO
2-CdSe-Ab
2two anti-preparations of incubating compound solution
Take 3 ~ 5 mg TiO
2be scattered in the carboxylated CdSe quantum dot solution of 4 mL, ultrasonic and stirring reaction 10 ~ 14 h, after question response completes, with ultrapure water centrifuge washing 3 times, obtained TiO
2-CdSe semiconductor compound; By TiO
2-CdSe semiconductor compound is scattered in 1 mL water, with the carboxyl on 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides EDC/N-N-Hydroxysuccinimide NHS crosslinking chemical activated camplex surface, by the anti-Ab of CA125 bis-of 1 mL, 8 ~ 12 μ g/mL
2add mixing, and shake 12 h in 4 DEG C of isothermal vibration devices, remove unconjugated Ab with ultrapure water centrifuge washing
2molecule, obtained TiO
2-CdSe-Ab
2two anti-incubate compound solution;
(4) preparation of difunctional mark Optical Electro-Chemistry sensor
1) pre-service of ITO electrode: ito glass is cut to 3 cm × 0.5 cm sizes, use liquid detergent, acetone, ethanol and ultrapure water ultrasonic cleaning 30 min successively, finally dry up glass with nitrogen, reserve the area of 0.5 cm × 0.5 cm size in order to modified biological molecule with insulating tape in one end of ITO electrode;
2) be in the methanol solution of 3-aminopropyl triethoxysilane APTES of 1% ~ 3% by the ITO electrode after process being immersed in volume fraction, lucifuge soaks 10 ~ 14 h, rinses electrode, to remove unconjugated APTES molecule with water;
3) the 10 μ L gold nano colloidal sols getting fresh preparation are added drop-wise to electrode surface, in conjunction with CA125 primary antibodie molecule;
4) 6 μ L, 8 ~ 12 μ g/mL CA125 primary antibodie solution are added drop-wise on electrode, are placed in 4 DEG C of constant temperature refrigerators and hatch 2 h, rinse electrode surface to remove unconjugated primary antibodie molecule;
5) drip painting 2 μ L, massfraction be the BSA solution of 0.5% ~ 1.5% on electrode, be placed in 4 DEG C of Refrigerator stores, dry;
6) clean with ultrapure water, dry in the air to moisture state, the CA125 antigenic solution of 6 μ L, 0.05 pg/mL ~ 100 ng/mL variable concentrations is dripped and is applied to electrode surface, make, between itself and CA125 primary antibodie, specific immune response occurs and continue hatching 2 h, with ultrapure water to remove unconjugated CA125, dry;
7) 6 μ L TiO are got
2-CdSe-Ab
2two anti-compound solution of incubating drip and are applied to electrode surface, hatch 2 h post-flush electrode surfaces, dry, obtained difunctional mark Optical Electro-Chemistry sensor.
2. the difunctional mark Optical Electro-Chemistry sensor prepared of preparation method as claimed in claim 1, is characterized in that, use the quantitative detection that galvanochemistry and photic electrochemical method realize human body CA125, step is as follows:
(1) difunctional mark Optical Electro-Chemistry sensor is placed in 0.3 ~ 0.5 mL, the salpeter solution of 0.1 mol/L soaks 4 h, makes CdSe quantum dot be decomposed into Cd
2+, joined 3.6 mL, 30 ~ 50 mg/mL Hg
2+pH 4.6 NaAc_HAc buffer solution in, use three-electrode system, carry out Electrochemical Detection;
Described three electrode saturated calomel electrodes are contrast electrode, and platinum electrode is auxiliary electrode, and glass-carbon electrode is working electrode;
(2) scanning voltage scope is from-0.9 ~-0.6 V, and the electromotive force rank that jump are 4 mV, frequency 25 Hz, amplitude 25 mV;
(3) Cd
2+there is redox reaction and produce signal near-0.7 V, according to the linear relationship between gained strength of current and analysis thing, drawing curve;
(4) Photoelectric Detection adopts i-t means of testing, and bias voltage is set to 0 V; Carry out switch lamp every 20 s, before and after record switch lamp, the changing value of electric current, produces the photocurrent values of different size, drawing curve according to the antigen of variable concentrations;
(5) testing sample solution is replaced the standard solution of CA125, detect according to the method for drafting of the working curve of described CA125.
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