CN104297479A - Preparation method and application of electrochemiluminescence immunoassay sensor for detecting tumor marker - Google Patents

Preparation method and application of electrochemiluminescence immunoassay sensor for detecting tumor marker Download PDF

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CN104297479A
CN104297479A CN201410492036.6A CN201410492036A CN104297479A CN 104297479 A CN104297479 A CN 104297479A CN 201410492036 A CN201410492036 A CN 201410492036A CN 104297479 A CN104297479 A CN 104297479A
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silver
polyaniline
preparation
solution
metal oxide
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CN104297479B (en
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颜梅
王秀
于京华
申蕾
邓文平
葛慎光
张彦
刘海云
杨春蕾
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University of Jinan
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/66—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light electrically excited, e.g. electroluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA

Abstract

The invention discloses an electrochemiluminescence immunoassay sensor for detecting a tumor marker. A preparation method of the sensor comprises the following steps: synthesizing polyaniline on conducting glass by an electropolymerization method, preparing g-C3N4, a silver-gold core shell nanosphere or copper oxide or manganese dioxide by the existing method, connecting the g-C3N4 on the polyaniline by 1-(3-dimethylamino propyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide mixed solution used as coupling agent, modifying the primary antibody on the prepared conducting glass, coating the surface of zinc oxide by the silver-gold core shell nanosphere, modifying second antibody on the silver-gold core shell nanosphere coating the surface of the zinc oxide, and detecting by electrochemiluminescence. The sensor has strong specificity, high sensitivity, simplicity in operation, wide linearity range and low detection limit.

Description

Detect the preparations and applicatio of tumor markers electrochemiluminescimmunosensor immunosensor
Technical field
The present invention relates to immune marker detection technique field, is a kind of preparation of electrochemiluminescimmunosensor immunosensor in particular, and the Electrochemiluminescsensor sensor that the invention still further relates to described in employing detects the method for tumor markers.
Background technology
All the time, malignant tumour i.e. cancer are serious threats of human health and life security.Cancer cell, refers to that cell that some have division potential there occurs vicious transformation and Clonal hyperplasia and a kind of neoformation of producing under the effect of carcinogenic factor.And cancer cell, except self growth is out of control, also can invades normal surrounding tissue even can transfer to health other parts via the circulation system in body or lymphatic system, thus cause health pathology even dead.So the diagnosis that cancer is early stage and treatment early have great importance.But early stage lower being difficult to of antigenic content of cancer is found, and the mensuration of the antigen of low content becomes the important pursuit of analyst.
In recent years, immunoassay is the clinical means of an important tumor disease in clinical examination.Immunoassay utilizes antibody and antigentic specificity association reaction to detect various material, as the high selectivity biochemical method of medicine, hormone, protein, microorganism etc.Biological immune analytical technology has that sensitivity is high, simple to operate, cost is low, specificity is high, it is little to disturb, the feature such as safety non-toxic and favorable reproducibility.And conventional analysis can be carried out to a large amount of sample, for the qualitative screening of sample, quantitative measurement, determine the content of component to be measured in sample.Current immunoassay detection method has almost been used on all common drugs, major part applies to the detection of drug residue, along with being gradually improved of analytical technology, monoclonal antibody, recombinant single chain antibody, and the development of immunosensor and chip technology, this technology will play an increasingly important role at food security, express-analysis detection field.
Immunoassay comprises non-marking immunoassay and markup immunoassay.Non-marked immunity comprises Immune proliferation and immunoelectrophoresis, is that the antigen of solubility contacts in solution or gel with corresponding antibody, thus forms the technology that a kind of insoluble antigen-antibody complex carries out being separated.Markup immuno analytical method is that the probe materials such as employing photoelectric material, isotope or enzyme carry out labelled antibody or antigen carries out antigen-antibody reaction, by the mensuration to the label signal in immune complex, thus reaches the object of monitoring immune response.Markup immuno analytical method comprises radiommunoassay, enzyme linked immunosorbent assay, capillary electrophoresis chemiluminescence immunoassay, high performance liquid chromatography chemiluminescence immune assay, fluidic chip chemiluminescence immunoassay, electrochemical immunoanalytical, electroluminescence immunoassay, immunofluorence technic, colloid gold immune technology and ferritin immunological technique etc.But these immune analysis methods are generally consuming time longer and have the operating process such as loaded down with trivial details application of sample, incubation, washing, and operating personnel are more with contacting of immune substance, may work the mischief to the healthy of personnel of operation.And the detection means that some finding speed is fast, highly sensitive, detectability is low needs expensive equipment, can not popularize in developing country.
Based on above technology, we need to look for a kind of cheap shirtsleeve operation method, realize high-sensitivity detection.Electro-conductive glass is a kind of relatively cheap material, sensor is based upon on electro-conductive glass by we, a kind of portable immunoassay glass-chip can be become, then in conjunction with the advantage of composite nano materials, can realize detecting the high sensitivity low content of tumor markers.
Summary of the invention
The technical problem to be solved in the present invention there is provided a kind of highly sensitive, detection speed is fast, reagent dosage is few, detects the Electrochemiluminescsensor sensor of tumor markers.
In order to solve the problems of the technologies described above, the present invention is realized by following measures: a kind of preparation method detecting the Electrochemiluminescsensor sensor of tumor markers, and it comprises the following steps:
(1) electro-conductive glass is cut into required size, and carry out pre-service;
(2) utilize existing method in ITO electro-conductive glass square region, be polymerized polyaniline, and synthesize g-C 3n 4, silver-colored golden core-shell nanospheres and metal oxide, oxide comprises zinc paste, cupric oxide, manganese dioxide;
(3) carboxylated g-C 3n 4, be fixed on polyaniline, for fixing primary antibodie with coupling agent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxysuccinimide mixed liquor;
(4) golden for silver core-shell nanospheres is modified on an metal oxide, then resist two and be modified on silver-colored golden core-shell nanospheres;
(5) Electrochemiluminescsensor sensor made is detected tumor markers in conjunction with electrogenerated chemiluminescence instrument.
Polyaniline/g-C of the present invention 3n 4and the preparation of metal oxide/silver-colored billon comprises the following steps:
(1) electro-conductive glass is first cut into the rectangular of 4 cm × 1 cm, 15 min ultrasonic washings are carried out successively with ethanol, acetone and intermediate water, made the mould being used for vaseline printing stencil by Adobe Illustrator, vaseline is coated in the specific region on ITO electro-conductive glass;
(2) preparation of polyaniline: the preparation of polyaniline is prepared by electropolymerization, make working electrode by electro-conductive glass, platinum electrode is done electrode, Ag/AgCl electrode is done to carry out in the three-electrode system of contrast electrode composition, electrolytic solution is made up of the nitric acid of 4.00 mL 3 M and 0.40 mL aniline, and voltage applied on the working electrode (s is with 0.1 Vs -1sweep velocity circulate between-0.2-1.2 V, obtained polyaniline thus;
(3) g-C 3n 4preparation: take 10 g melamines in crucible, calcination 4 h in 550 DEG C of high temperature furnaces;
(4) by g-C that (3) obtain 3n 4be placed in the there-necked flask filling salpeter solution, condensing reflux 24 h, then centrifuge washing 4 times, be finally dissolved in 10 mL intermediate waters for subsequent use;
(5) the polyaniline surface of preparing in (2) drips coupling agent solution, then gets 10 μ L(4) g-C for preparing 3n 4be added drop-wise to surface, wash unreacted g-C with water 3n 4, dry for subsequent use under room temperature;
(6) preparation of silver nanoparticle ball: a solution comprises 3mL ethylene glycol, 90 mM silver nitrates, b solution comprises 3 mL ethylene glycol, 147 mM polyvinylpyrrolidones, 0.22 mM sodium chloride; Get a there-necked flask and add 5mL ethylene glycol, be heated to 153 DEG C, then dropwise join the while of a, b two solution slowly in there-necked flask, become yellow through a series of reaction final color, with acetone and water centrifuge washing respectively, collecting precipitation;
(7) the silver nanoparticle ball that (6) prepare being joined 3 mL contains in the water of polyvinylpyrrolidone, be heated to 100 DEG C and keep 10 min, then the chlorauric acid solution of 100 μ L 1 % is added, heat 10 min to colour stable, then with the silver chloride of saturated sodium-chloride cleaning the inside, finally use water centrifuge washing polyvinylpyrrolidone and sodium chloride, the silver obtained gold core-shell nanospheres is distributed in intermediate water;
(8) metal oxide involved in the present invention comprises zinc paste, cupric oxide, manganese dioxide, prepares this three kinds of metal oxides under different conditions;
(9) metal oxide that (8) obtain is joined in the suspending liquid of (7), stirring reaction 24 h, intermediate water centrifuge washing, be distributed in 1 mL intermediate water, obtain the suspending liquid of metal oxide/silver-colored golden core-shell nanospheres thus;
(10) two anti-joining in the suspending liquid (9) prepared are reacted 30 min, finally close avtive spot with BSA, obtain metal oxide/silver-colored golden core-shell nanospheres thus to modify two anti-composite particles, the solution prepared is kept in the refrigerator of 4 DEG C stand-by;
(11) electrode prepared in (5) is modified successively upper primary antibodie, two anti-composite particles that antigen, (10) obtain, carry out the detection of electrogenerated chemiluminescence in containing the phosphate buffered solution of nitric acid.
The wash solution used in the present invention is pH 7.4 phosphate buffered solution.
Tumor markers of the present invention is carcinomebryonic antigen, CA 125, prostatein antigen, human chorionic gonadotrophin, cancer embryo ferritin, pancreatic oncofetal antigen, cytokeratin, alpha-fetoprotein, acid phosphatase, Catecholamine matter.
beneficial effect of the present invention:
(1) the present invention utilizes the method for electrogenerated chemiluminescence to carry out measurement operation fast simply, and reaction and result complete and record automatically by instrument, avoid the impact of subjective factor, and ensure good repeatability, be convenient to Site Detection;
(2) utilize electropolymerization synthesized polyaniline, play and connect electro-conductive glass and g-C 3n 4effect, and synthesized polyaniline/g-C first 3n 4can electric conductivity be strengthened, reduce impedance, be beneficial to the transmission of photosignal;
(3) g-C is utilized 3n 4as luminescent substance, there is good biocompatibility, reduce the impact of luminescent substance on bioanalysis;
(4) utilize metal oxide/silver-colored golden core-shell nanospheres to modify two to resist, two anti-content can be improved, play the effect of amplifying signal, strengthen sensitivity.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
The preparation of [Fig. 1] electrode and the modification of sensor.
Embodiment
Detect a preparation method for the Electrochemiluminescsensor sensor of immune marker, it comprises the following steps:
(1) electro-conductive glass is cut into required size, and carry out pre-service;
(2) utilize existing method in ITO square region, be polymerized polyaniline, and synthesize g-C 3n 4, silver-colored golden core-shell nanospheres and metal oxide, metal oxide comprises zinc paste, cupric oxide, manganese dioxide;
(3) carboxylated g-C 3n 4, be fixed on polyaniline with coupling agent, for fixing primary antibodie;
(4) golden for silver core-shell nanospheres is modified on an metal oxide, then resist two and be modified on silver-colored golden core-shell nanospheres;
(5) Electrochemiluminescsensor sensor made is detected tumor markers in conjunction with electrogenerated chemiluminescence instrument.
Polyaniline/g-C of the present invention 3n 4, metal oxide/Yin Jin and section's Nano microsphere and sensor preparation comprise the following steps:
(1) electro-conductive glass is first cut into the rectangular of 4 cm × 1 cm, 15 min ultrasonic washings are carried out successively with ethanol, acetone and intermediate water, made the mould being used for vaseline printing stencil by Adobe Illustrator, vaseline is coated in the specific region on ITO electro-conductive glass;
(2) preparation of polyaniline: the preparation of polyaniline is prepared by electropolymerization, make working electrode by electro-conductive glass, platinum electrode is done electrode, Ag/AgCl electrode is done to carry out in the three-electrode system of contrast electrode composition, electrolytic solution is made up of the nitric acid of 4.00 mL 3 M and 0.40 mL aniline, and voltage applied on the working electrode (s is with 0.1 Vs -1sweep velocity circulate between-0.2-1.2 V, obtained polyaniline thus;
(3) g-C 3n 4preparation: take 10 g melamines in crucible, calcination 4 h in 550 DEG C of high temperature furnaces;
(4) by g-C that (3) obtain 3n 4be placed in the there-necked flask filling salpeter solution, condensing reflux 24 h, then centrifuge washing 4 times, be finally dissolved in 10 mL intermediate waters for subsequent use;
(5) the polyaniline surface of preparing in (2) drips coupling agent solution, then gets 10 μ L(4) g-C for preparing 3n 4be added drop-wise to surface, wash unreacted g-C with water 3n 4, dry for subsequent use under room temperature;
(6) preparation of silver nanoparticle ball: a solution comprises 3mL ethylene glycol, 90 mM silver nitrates, b solution comprises 3 mL ethylene glycol, 147 mM polyvinylpyrrolidones, 0.22 mM sodium chloride; Get a there-necked flask and add 5mL ethylene glycol, be heated to 153 DEG C, then dropwise join the while of a, b two solution slowly in there-necked flask, become yellow through a series of reaction final color, with acetone and water centrifuge washing respectively, collecting precipitation;
(7) the silver nanoparticle ball that (6) prepare being joined 3 mL contains in the water of polyvinylpyrrolidone, be heated to 100 DEG C and keep 10 min, then the chlorauric acid solution of 100 μ L 1 % is added, heat 10 min to colour stable, then with the silver chloride of saturated sodium-chloride cleaning the inside, finally use water centrifuge washing polyvinylpyrrolidone and sodium chloride, the silver obtained gold core-shell nanospheres is distributed in intermediate water;
(8) metal oxide involved in the present invention comprises zinc paste, cupric oxide, manganese dioxide, prepares this three kinds of metal oxides under different conditions;
(9) metal oxide that (8) obtain is joined in the suspending liquid of (7), stirring reaction 24 h, intermediate water centrifuge washing, be distributed in 1 mL intermediate water, obtain the suspending liquid of metal oxide/silver-colored golden core-shell nanospheres thus;
(10) two anti-joining in the suspending liquid (9) prepared are reacted 30 min, finally close avtive spot with BSA, obtain metal oxide/silver-colored golden core-shell nanospheres thus to modify two anti-composite particles, the solution prepared is kept in the refrigerator of 4 DEG C stand-by;
(11) electrode prepared in (5) is modified successively upper primary antibodie, two anti-composite particles that antigen, (10) obtain, carry out the detection of electrogenerated chemiluminescence in containing the phosphate buffered solution of nitric acid.
The wash solution used in the present invention is pH 7.4 phosphate buffered solution.
embodiment 1(embryonal antigen class, as alpha-fetoprotein, AFP)
(1) the higher AFP of clinical onset is selected to measure;
(2) electro-conductive glass is first cut into the rectangular of 4 cm × 1 cm, 15 min ultrasonic washings are carried out successively with ethanol, acetone and intermediate water, made the mould being used for vaseline printing stencil by Adobe Illustrator, vaseline is coated in the specific region on ITO electro-conductive glass;
(3) preparation of polyaniline: the preparation of polyaniline is prepared by electropolymerization, make working electrode by electro-conductive glass, platinum electrode is done electrode, Ag/AgCl electrode is done to carry out in the three-electrode system of contrast electrode composition, electrolytic solution is made up of the nitric acid of 4.00 mL 3 M and 0.40 mL aniline, and voltage applied on the working electrode (s is with 0.1 Vs -1sweep velocity circulate between-0.2-1.2 V, obtained polyaniline thus;
(4) g-C 3n 4preparation: take 10 g melamines in crucible, calcination 4 h in 550 DEG C of high temperature furnaces;
(5) by g-C that (3) obtain 3n 4be placed in the there-necked flask filling salpeter solution, condensing reflux 24 h, then centrifuge washing 4 times, be finally dissolved in 10 mL intermediate waters for subsequent use;
(6) the polyaniline surface of preparing in (3) drips coupling agent solution, then gets 10 μ L(5) g-C for preparing 3n 4be added drop-wise to surface, wash unreacted g-C with water 3n 4, dry for subsequent use under room temperature;
(7) preparation of silver nanoparticle ball: a solution comprises 3 mL ethylene glycol, 90 mM silver nitrates, b solution comprises 3 mL ethylene glycol, 147 mM polyvinylpyrrolidones, 0.22 mM sodium chloride; Get a there-necked flask and add 5 mL ethylene glycol, be heated to 153 DEG C, then dropwise join the while of a, b two solution slowly in there-necked flask, become yellow through a series of reaction final color, with acetone and water centrifuge washing respectively, collecting precipitation;
(8) the silver nanoparticle ball that (7) prepare being joined 3 mL contains in the water of polyvinylpyrrolidone, be heated to 100 DEG C and keep 10 min, then the chlorauric acid solution of 100 μ L 1 % is added, heat 10 min to colour stable, then with the silver chloride of saturated sodium-chloride cleaning the inside, finally use water centrifuge washing polyvinylpyrrolidone and sodium chloride, the silver obtained gold core-shell nanospheres is distributed in intermediate water;
(9) preparation of zinc paste: take zinc acetate as raw material, Triammonium citrate is modifier, prepares certain density zinc acetate solution, after stirring, be placed in thermostatted water wish, then under the condition of agitating heating, add absolute ethyl alcohol uniformly, after 2 h, zinc acetate dissolves completely, generates hydrogen metal oxide deposits, then appropriate peptizator ammoniacal liquor is added, hydrogen metal oxide deposits disappears and forms colloidal sol, leaves standstill and becomes hydrogen metal oxide wet gel, and in muffle furnace, calcining obtains metal oxide powder;
(10) zinc paste that (9) obtain is joined in the suspending liquid of (8), stirring reaction 24 h, intermediate water centrifuge washing, be distributed in 1 mL intermediate water, obtain the suspending liquid of metal oxide/silver-colored golden core-shell nanospheres thus;
(11) two of AFP anti-joining in the suspending liquid (9) prepared are reacted 30 min, finally close avtive spot with BSA, obtain metal oxide/silver-colored golden core-shell nanospheres thus to modify two anti-composite particles, the solution prepared is kept in the refrigerator of 4 DEG C stand-by;
(12) electrode prepared in (6) is modified successively the primary antibodie of upper AFP, two anti-composite particles that antigen, (11) obtain, carry out the detection of electrogenerated chemiluminescence in containing the phosphate buffered solution of nitric acid.
AFP Electrochemiluminescsensor sensor detects the AFP in human body, animal blood serum sample, the results are shown in Table 1.Utilize existing method, preparation AFP electrochemical immunosensor, connects electrochemical workstation, carries out actual detection, the results are shown in Table 1 to the AFP in human body, animal blood serum sample extracting solution.
 
Table 1 AFP electrochemiluminescimmunosensor immunosensor of the present invention and AFP electrochemical immunosensor Detection results contrast
From table 1, result can find out that AFP electrochemiluminescimmunosensor immunosensor has the wider range of linearity, higher sensitivity and lower detectability than AFP electrochemical immunosensor.
 
embodiment 2(carbohydrate mark, as CA 125)
(1) the higher CA125 of clinical onset is selected to measure;
(2) electro-conductive glass is first cut into the rectangular of 4 cm × 1 cm, 15 min ultrasonic washings are carried out successively with ethanol, acetone and intermediate water, made the mould being used for vaseline printing stencil by Adobe Illustrator, vaseline is coated in the specific region on ITO electro-conductive glass;
(3) preparation of polyaniline: the preparation of polyaniline is prepared by electropolymerization, make working electrode by electro-conductive glass, platinum electrode is done electrode, Ag/AgCl electrode is done to carry out in the three-electrode system of contrast electrode composition, electrolytic solution is made up of the nitric acid of 4.00 mL 3 M and 0.40 mL aniline, and voltage applied on the working electrode (s is with 0.1 Vs -1sweep velocity circulate between-0.2-1.2 V, obtained polyaniline thus;
(4) g-C 3n 4preparation: take 10 g melamines in crucible, calcination 4 h in 550 DEG C of high temperature furnaces;
(5) by g-C that (3) obtain 3n 4be placed in the there-necked flask filling salpeter solution, condensing reflux 24 h, then centrifuge washing 4 times, be finally dissolved in 10 mL intermediate waters for subsequent use;
(6) the polyaniline surface of preparing in (3) drips coupling agent solution, then gets 10 μ L(5) g-C for preparing 3n 4be added drop-wise to surface, wash unreacted g-C with water 3n 4, dry for subsequent use under room temperature;
(7) preparation of silver nanoparticle ball: a solution comprises 3mL ethylene glycol, 90 mM silver nitrates, b solution comprises 3 mL ethylene glycol, 147 mM polyvinylpyrrolidones, 0.22 mM sodium chloride; Get a there-necked flask and add 5mL ethylene glycol, be heated to 153 DEG C, then dropwise join the while of a, b two solution slowly in there-necked flask, become yellow through a series of reaction final color, with acetone and water centrifuge washing respectively, collecting precipitation;
(8) the silver nanoparticle ball that (7) prepare being joined 3 mL contains in the water of polyvinylpyrrolidone, be heated to 100 DEG C and keep 10 min, then the chlorauric acid solution of 100 μ L 1 % is added, heat 10 min to colour stable, then with the silver chloride of saturated sodium-chloride cleaning the inside, finally use water centrifuge washing polyvinylpyrrolidone and sodium chloride, the silver obtained gold core-shell nanospheres is distributed in intermediate water;
(9) preparation of cupric oxide: 1 mM sodium dodecylsulphonate and 1mM APP to join in 25mL intermediate water vigorous stirring under room temperature, then add 1 mM CuCl 22H 2o, adds the NaOH of 15 mL 4 M, stirs 15 min, react 12 h in autoclave at 120 DEG C under stirring, by hot water and absolute ethyl alcohol centrifuge washing precipitation, at 60 DEG C, finally dry 10 h obtain solid oxidation copper nano flower;
(10) cupric oxide that (9) obtain is joined in the suspending liquid of (8), stirring reaction 24 h, intermediate water centrifuge washing, be distributed in 1mL intermediate water, obtain the suspending liquid of metal oxide/silver-colored golden core-shell nanospheres thus;
(11) two of CA 125 anti-joining in the suspending liquid (9) prepared are reacted 30 min, finally close avtive spot with BSA, obtain metal oxide/silver-colored golden core-shell nanospheres thus to modify two anti-composite particles, the solution prepared is kept in the refrigerator of 4 DEG C stand-by;
(12) electrode prepared in (6) is modified successively the primary antibodie of upper CA 125, two anti-composite particles that antigen, (11) obtain, carry out the detection of electrogenerated chemiluminescence in containing the phosphate buffered solution of nitric acid.
CA 125 Flow-injection/Chemiluminescence Sensor detects the CA 125 in human body, animal blood serum sample, the results are shown in Table 2.Utilize existing method, preparation CA 125 electrochemical immunosensor, connects electrochemical workstation, carries out actual detection, the results are shown in Table 2 to the CA 125 in human body, animal blood serum sample extracting solution.
 
Table 2 CA 125 of the present invention electrochemiluminescimmunosensor immunosensor and CA 125 electrochemical immunosensor Detection results ratio
From table 2, result can find out that CA 125 electrochemiluminescimmunosensor immunosensor has the wider range of linearity, higher sensitivity and lower detectability than CA 125 electrochemical immunosensor.
 
embodiment 3(steroids, as HCG)
(1) the higher HCG of clinical onset is selected to measure;
(2) electro-conductive glass is first cut into the rectangular of 4 cm × 1 cm, 15 min ultrasonic washings are carried out successively with ethanol, acetone and intermediate water, made the mould being used for vaseline printing stencil by Adobe Illustrator, vaseline is coated in the specific region on ITO electro-conductive glass;
(3) preparation of polyaniline: the preparation of polyaniline is prepared by electropolymerization, make working electrode by electro-conductive glass, platinum electrode is done electrode, Ag/AgCl electrode is done to carry out in the three-electrode system of contrast electrode composition, electrolytic solution is made up of the nitric acid of 4.00 mL 3 M and 0.40 mL aniline, and voltage applied on the working electrode (s is with 0.1 Vs -1sweep velocity circulate between-0.2-1.2 V, obtained polyaniline thus;
(4) g-C 3n 4preparation: take 10 g melamines in crucible, calcination 4 h in 550 DEG C of high temperature furnaces;
(5) by g-C that (3) obtain 3n 4be placed in the there-necked flask filling salpeter solution, condensing reflux 24 h, then centrifuge washing 4 times, be finally dissolved in 10 mL intermediate waters for subsequent use;
(6) the polyaniline surface of preparing in (3) drips coupling agent solution, then gets 10 μ L(5) g-C for preparing 3n 4be added drop-wise to surface, wash unreacted g-C with water 3n 4, dry for subsequent use under room temperature;
(7) preparation of silver nanoparticle ball: a solution comprises 3 mL ethylene glycol, 90 mM silver nitrates, b solution comprises 3 mL ethylene glycol, 147 mM polyvinylpyrrolidones, 0.22 mM sodium chloride; Get a there-necked flask and add 5 mL ethylene glycol, be heated to 153 DEG C, then dropwise join the while of a, b two solution slowly in there-necked flask, become yellow through a series of reaction final color, with acetone and water centrifuge washing respectively, collecting precipitation;
(8) the silver nanoparticle ball that (7) prepare being joined 3 mL contains in the water of polyvinylpyrrolidone, be heated to 100 DEG C and keep 10 min, then the chlorauric acid solution of 100 μ L 1 % is added, heat 10 min to colour stable, then with the silver chloride of saturated sodium-chloride cleaning the inside, finally use water centrifuge washing polyvinylpyrrolidone and sodium chloride, the silver obtained gold core-shell nanospheres is distributed in intermediate water;
(9) preparation of manganese dioxide: 0.2500 g potassium permanganate is dissolved in 125 mL distilled water, 2.5 mL oleic acid are added after stir about 0.5 h, oleic acid reduction potassium permanganate defines stable emulsion, emulsion is put 24 h in ambient temperatare, the product of the brownish black generated with intermediate water and ethanol centrifuge washing is to remove residual reactants, finally, in 60 ° of C vacuum drying chambers, drying obtains flower-shaped manganese dioxide nano ball;
(10) manganese dioxide that (9) obtain is joined in the suspending liquid of (8), stirring reaction 24h, intermediate water centrifuge washing, be distributed in 1mL intermediate water, obtain the suspending liquid of metal oxide/silver-colored golden core-shell nanospheres thus;
(11) two of HCG anti-joining in the suspending liquid (9) prepared are reacted 30 min, finally close avtive spot with BSA, obtain metal oxide/silver-colored golden core-shell nanospheres thus to modify two anti-composite particles, the solution prepared is kept in the refrigerator of 4 DEG C stand-by;
(12) electrode prepared in (6) is modified successively the primary antibodie of upper HCG, two anti-composite particles that antigen, (11) obtain, carry out the detection of electrogenerated chemiluminescence in containing the phosphate buffered solution of nitric acid.
HCG Flow-injection/Chemiluminescence Sensor detects the HCG in human body, animal blood serum sample, the results are shown in Table 3.Utilize existing method, preparation HCG electrochemical immunosensor, connects electrochemical workstation, carries out actual detection, the results are shown in Table 3 to the HCG in human body, animal blood serum sample extracting solution.
 
Table 3 HCG electrochemiluminescimmunosensor immunosensor of the present invention and HCG electrochemical immunosensor Detection results contrast
Result as can be seen from table 3: HCG electrochemiluminescimmunosensor immunosensor has the wider range of linearity, higher sensitivity and lower detectability than HCG electrochemical immunosensor.

Claims (2)

1. detect a preparation method for the Electrochemiluminescsensor sensor of tumor markers, it comprises the following steps:
(1) electro-conductive glass is cut into required size, and carry out pre-service;
(2) utilize existing method in electro-conductive glass square region, be polymerized polyaniline, and synthesize g-C 3n 4, silver-colored golden core-shell nanospheres and metal oxide;
(3) carboxylated g-C 3n 4, be fixed on polyaniline, for fixing primary antibodie with coupling agent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxysuccinimide mixed liquor;
(4) golden for silver core-shell nanospheres is modified on an metal oxide, then resist two and be modified on silver-colored golden core-shell nanospheres;
(5) Electrochemiluminescsensor sensor made is detected tumor markers in conjunction with electrogenerated chemiluminescence instrument.
2. polyaniline/g-C of the present invention 3n 4, metal oxide/silver-colored golden core-shell nanospheres and sensor preparation comprise the following steps:
(1) electro-conductive glass is first cut into the rectangular of 4 cm × 1 cm, 15 min ultrasonic washings are carried out successively with ethanol, acetone and intermediate water, made the mould being used for vaseline printing stencil by Adobe Illustrator, vaseline is coated in the specific region on ITO electro-conductive glass;
(2) preparation of polyaniline: the preparation of polyaniline is prepared by electropolymerization, make working electrode by electro-conductive glass, platinum electrode is done electrode, Ag/AgCl electrode is done to carry out in the three-electrode system of contrast electrode composition, electrolytic solution is made up of the nitric acid of 4.00 mL 3 M and 0.40 mL aniline, and voltage applied on the working electrode (s is with 0.1 Vs -1sweep velocity circulate between-0.2-1.2 V, obtained polyaniline thus;
(3) g-C 3n 4preparation: take 10 g melamines in crucible, calcination 4 h in 550 DEG C of high temperature furnaces;
(4) by g-C that (3) obtain 3n 4be placed in the there-necked flask filling salpeter solution, condensing reflux 24 h, then centrifuge washing 4 times, be finally dissolved in 10 mL intermediate waters for subsequent use;
(5) the polyaniline surface of preparing in (2) drips coupling agent solution, then gets 10 μ L(4) g-C for preparing 3n 4be added drop-wise to surface, wash unreacted g-C with water 3n 4, dry for subsequent use under room temperature;
(6) preparation of silver nanoparticle ball: a solution comprises 3mL ethylene glycol, 90 mM silver nitrates, b solution comprises 3 mL ethylene glycol, 147 mM polyvinylpyrrolidones, 0.22 mM sodium chloride; Get a there-necked flask and add 5mL ethylene glycol, be heated to 153 DEG C, then dropwise join the while of a, b two solution slowly in there-necked flask, become yellow through a series of reaction final color, with acetone and water centrifuge washing respectively, collecting precipitation;
(7) the silver nanoparticle ball that (6) prepare being joined 3 mL contains in the water of polyvinylpyrrolidone, be heated to 100 DEG C and keep 10 min, then the chlorauric acid solution of 100 μ L 1 % is added, heat 10 min to colour stable, then with the silver chloride of saturated sodium-chloride cleaning the inside, finally use water centrifuge washing polyvinylpyrrolidone and sodium chloride, the silver obtained gold core-shell nanospheres is distributed in intermediate water;
(8) metal oxide involved in the present invention comprises zinc paste, cupric oxide, manganese dioxide, prepares this three kinds of metal oxides under different conditions;
(9) metal oxide that (8) obtain is joined in the suspending liquid of (7), stirring reaction 24 h, intermediate water centrifuge washing, be distributed in 1 mL intermediate water, obtain the suspending liquid of metal oxide/silver-colored golden core-shell nanospheres thus;
(10) two anti-joining in the suspending liquid (9) prepared are reacted 30 min, finally close avtive spot with bovine serum albumin(BSA), obtain metal oxide/silver-colored golden core-shell nanospheres thus to modify two anti-composite particles, the solution prepared is kept in the refrigerator of 4 DEG C stand-by;
(11) electrode prepared in (5) is modified successively upper primary antibodie, two anti-composite particles that antigen, (10) obtain, carry out the detection of electrogenerated chemiluminescence in containing the phosphate buffered solution of nitric acid.
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