CN105842460A - Preparation method of electro-chemiluminescence immunosensor based on silver-hybridized bismuth sulfide - Google Patents
Preparation method of electro-chemiluminescence immunosensor based on silver-hybridized bismuth sulfide Download PDFInfo
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Abstract
The invention relates to a preparation method of an electro-chemiluminescence immunosensor based on silver-hybridized bismuth sulfide and belongs to the field of electro-chemiluminescence sensors. With cerium dioxide as an electro-chemiluminescence signal source, supporting quantity of an antibody can be increased by means of excellent biocompatibility and large specific surface area of gold-hybridized aminated graphene. With the silver-hybridized bismuth sulfide as a second antibody marker, detection on a prostate specific antigen is achieved according to different electro-chemiluminescence signal intensity of to-be-detected substance in different concentrations.
Description
Technical field
The present invention relates to the preparation method of a kind of electrochemiluminescimmunosensor immunosensor based on argentaization bismuth sulfide.It is specifically related to a kind of auraization amination Graphene/ceria as base material, the bio-compatibility of golden nanometer particle and the big specific surface area of amination Graphene is utilized to come immobilized ceria and detection antibody, it is prepared for a kind of electrochemiluminescimmunosensor immunosensor detecting prostate specific antigen, belongs to new function material and bio-sensing detection technique field.
Background technology
Prostate specific antigen (Prostate Specific Antigen, PSA) it is a kind of single chain glycoprotein of epithelial cells by prostatic acini and conduit, functionally belong to a kind of serine protease of class kallikrein, participate in the liquefaction process of seminal fluid, be routine clinical optimum with malignant disease Diagnosis and differential diaggnosis and the important indicator of patients with prostate cancer Follow-up After for prostate.
The clinical testing procedure of current existing prostate specific antigen is a lot, such as enzyme immunoassay, radioimmunoassay, RIA, chemiluminescence immune assay etc..The shortcomings such as but said method has radioactive pollution, sensitivity is low, and the detection cycle is long, complex steps.Immunosensor is a kind of biosensor combined with analytical chemistry method by immunological method, by the functionality combination between antigen and antibody so that it has high sensitivity, high selectivity, analyzes the advantages such as quick and easy and simple to handle.
Electrochemiluminescimmunosensor immunosensor have highly sensitive, selectivity good, simple in construction, easy and simple to handle, be prone to miniaturization, can the advantage such as detection analysis continuous, rapid automatized, therefore the present invention is prepared for electrochemiluminescimmunosensor immunosensor based on argentaization bismuth sulfide, it is achieved that the detection to prostate specific antigen.
Amination Graphene is combined by the present invention with ceria, and amination Graphene has bigger specific surface area, the immobilized more luminescent substance of energy, improves the electrogenerated chemiluminescence performance of sensor.By golden nanometer particle in-situ reducing to amination Graphene/ceria surface, improve the electric conductivity of sensing platform.And use the bismuth sulfide of argentaization as two anti-labels, realize the cancellation to ceria electrochemiluminescence signal by the effect of resonance energy transfer, effectively enhance the sensitivity of electrochemiluminescimmunosensor immunosensor.The method has the advantages such as low cost, highly sensitive, specificity good, detection is quick, and preparation process is relatively simple, provides new way for the most effectively detection prostate specific antigen.
Summary of the invention
An object of the present invention is that synthesis has the auraization amination Graphene/ceria material of large specific surface area, its can immobilized substantial amounts of luminescent substance, improve the electric conductivity of base material, strengthen the sensitivity of sensor.
The two of the purpose of the present invention are to obtain argentaization bismuth sulfide by the method for reduction, by the resonance energy transfer effect of bismuth sulfide Yu ceria, it is achieved the cancellation of electrochemiluminescence signal, improve the stability of electrochemiluminescence signal.
The three of the purpose of the present invention are that to utilize auraization amination Graphene/ceria be mark layer as base material, argentaization bismuth sulfide, build a kind of without enzyme, quick and overdelicate sandwich type electrochemiluminescimmunosensor immunosensor.
The four of the purpose of the present invention are quick, the Sensitive Detection that this sandwich type electrochemiluminescimmunosensor immunosensor is applied to prostate specific antigen.
Technical scheme is as follows:
1. the preparation method of an electrochemical luminous sensor based on argentaization bismuth sulfide, it is characterised in that comprise the following steps:
(1) glass-carbon electrode of a diameter of 4 mm is taken turns doing polishing with 1.0 μm, 0.3 μm, 0.05 μm aluminum oxide polishing powder successively, clean with ultrapure water；
(2) 6 L, 1 ~ 3 mg/mL are taken
Auraization amination Graphene/ceria solution is added drop-wise under electrode surface, room temperature dry, and uses ultrapure water electrode surface, dries；
(3) drip 6 L, the antibody of 5 ~ 10 g/mL, use ultrapure water electrode surface after drying, 4 DEG C of refrigerators dry；
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L, mass fraction are 1%, in 4 DEG C of refrigerators, hatch 3 h, clean up；
(5) 6 μ L prostate specific antigen droppings are used for capturing antibody, incubated at room temperature 1 h on electrode, clean up；
(6) it is 1 ~ 3 by 6 μ L concentration
The antibody-solutions of the argentaization bismuth sulfide labelling of mg/mL drop on electrode with antigenic specificity identification, incubated at room temperature 1 h, clean up, store for future use in 4 DEG C of refrigerators.
2. the preparation of base material auraization amination Graphene/ceria solution
(1) preparation of amination Graphene
75 ~ 125 mg graphene oxides are joined in 40 mL ethylene glycol solutions, the most ultrasonic 1 h, it is subsequently adding 1.0 mL ammonia, acutely after vibration 1 min, solution is transferred in 50 mL reactors, at 180 DEG C, heats 6 h, centrifuge washing 20 min under 8000 r/min, 35 DEG C of vacuum drying, i.e. obtain amination Graphene；
(2) preparation of auraization amination Graphene/ceria
By in 40 ~ 60 mg amination graphene dispersion to 50 mL ultra-pure waters, ultrasonic 2 h, it is subsequently adding 1.1 g six nitric hydrate ceriums, stirring 30 min, add ammonia regulation pH to 11, adding 2.5 mL mass fractions is the chlorauric acid solution of 1%, solution stirs 6 h at 90 DEG C, centrifuge washing 20 min under 8000 r/min, 35 DEG C of vacuum drying, i.e. obtain auraization amination Graphene/ceria.
3. the preparation of the antibody-solutions of the prostate specific antigen of argentaization bismuth sulfide labelling
(1) preparation of bismuth sulfide
1.6 ~ 2.0 g five nitric hydrate bismuths are dissolved in 25 mL ethylene glycol, with nitrogen bubble 15 minutes, obtain solution A；Simultaneously by 1.2 ~ 1.5
G sodium sulfide is dissolved in the mixed solution of 10 mL ethylene glycol and 20 mL ultra-pure waters, stirs 15 min, obtains solution B；Under agitation, solution B is added dropwise in solution A, by 1.8 ~ 2.0
G urea joins in above-mentioned solution, adds 20 mL ultra-pure waters, stirs 30 minutes, is transferred in reactor by mixed solution, heats 24 h at 180 DEG C, centrifuge washing 20 min under 8000 r/min, and 35 DEG C of vacuum drying obtain bismuth sulfide；
(2) preparation of argentaization bismuth sulfide label
230 ~ 250 mg bismuth sulfides are distributed in 100 mL ultra-pure waters, ultrasonic 10 h, are subsequently adding 110 ~ 130
Mg silver nitrate and 10 ~ 20 mg surfactants (PVP), stir 6 h, add the sodium citrate solution of 6 mL 5mmol/L, stirring 24 h, centrifuge washing 20 min under 8000 r/min, in vacuum drying oven, 35 DEG C are dried, obtain argentaization bismuth sulfide label；
(3) preparation of the antibody-solutions of argentaization bismuth sulfide labelling
It is 5 ~ 15 by argentaization bismuth sulfide label solution and 300 μ L concentration that 3 mL concentration are 1 ~ 3 mg/mL
The antibody-solutions mixing of μ g/mL prostate specific antigen, vibrate 24 h, centrifuge washing 15 min under 5000 r/min, obtains the antibody hatching thing of argentaization bismuth sulfide labelling, it is scattered in the phosphate buffered solution that 3 mL, pH are 7.4, is prepared the antibody-solutions of argentaization bismuth sulfide labelling.
4. the detection method of prostate specific antigen
(1) three-electrode system of electrochemical workstation is used to test, Ag/AgCl electrode is as reference electrode, platinum electrode is to electrode, prepared electrochemical luminous sensor is working electrode, electrochemical workstation and chemiluminescence detector are linked together the high pressure of photomultiplier tube is set to 750 V, cyclic voltammetry scan current potential is-2.0 V, and sweep speed is 0.1 V/s；
(2) 10 mL, pH 5.5 ~
In the phosphate buffered solution containing 100 ~ 150 mmol/L potassium peroxydisulfates of 8.0, by electrochemical luminescence system, detect the electrochemical luminescence signals intensity that the determinand antigen to variable concentrations produces, drawing curve；
(3) testing sample solution replace prostate specific antigen solution detect.
The useful achievement of the present invention
(1) amination Graphene is combined by the present invention with ceria, and amination Graphene has bigger specific surface area, the immobilized more luminescent substance of energy, improves the electrogenerated chemiluminescence performance of sensor.By golden nanometer particle in-situ reducing to amination Graphene/ceria surface, improve the electric conductivity of sensing platform.
(2) and use the bismuth sulfide of argentaization as two anti-labels, realize the cancellation to ceria electrochemiluminescence signal by the effect of resonance energy transfer, effectively enhance the sensitivity of electrochemiluminescimmunosensor immunosensor.
(3) the sandwich electrochemiluminescimmunosensor immunosensor of preparation is used for the detection of prostate specific antigen by the present invention, and detection limit is low, range of linearity width, it is possible to achieve simple, quick, sensitive and specific detection.The present invention is 0.001 ~ 10 to the prostate specific antigen detection range of linearity
Ng/mL, detection limit reaches 0.3 pg/mL.
Detailed description of the invention
Embodiment 1 A kind of preparation method of prostate specific antigen Electrochemiluminescsensor sensor based on auraization amination Graphene/ceria
(1) glass-carbon electrode of a diameter of 4 mm is taken turns doing polishing with 1.0 μm, 0.3 μm, 0.05 μm aluminum oxide polishing powder successively, clean with ultrapure water；
(2) by 6 μ L, concentration be the base material auraization amination Graphene/ceria solution of 1 mg/mL in electrode surface, 4 DEG C of preservations；
(3) drip 6 L, the antibody of 5 g/mL, use ultrapure water electrode surface after drying, 4 DEG C of refrigerators dry；
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L, mass fraction are 1%, in 4 DEG C of refrigerators, hatch 3 h, clean up；
(5) 6 μ L prostate specific antigen droppings are used for capturing antibody, incubated at room temperature 1 h on electrode, clean up；
(6) antibody-solutions of the argentaization bismuth sulfide labelling that 6 μ L concentration are 1 mg/mL is dropped on electrode with antigenic specificity identification, incubated at room temperature 1 h, clean up, store for future use in 4 DEG C of refrigerators.
Embodiment 2 A kind of preparation method of prostate specific antigen Electrochemiluminescsensor sensor based on auraization amination Graphene/ceria
(1) glass-carbon electrode of a diameter of 4 mm is taken turns doing polishing with 1.0 μm, 0.3 μm, 0.05 μm aluminum oxide polishing powder successively, clean with ultrapure water；
(2) by 6 μ L, concentration be the base material auraization amination Graphene/ceria solution of 2 mg/mL in electrode surface, 4 DEG C of preservations；
(3) drip 6 L, the antibody of 8 g/mL, use ultrapure water electrode surface after drying, 4 DEG C of refrigerators dry；
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L, mass fraction are 1%, in 4 DEG C of refrigerators, hatch 3 h, clean up；
(5) specificity that 6 μ L prostate specific antigen droppings are used for capturing on electrode antibody is known, and incubated at room temperature 1 h cleans up；
The antibody-solutions of the argentaization bismuth sulfide labelling that 6 μ L concentration are 2 mg/mL is dropped on electrode with antigenic specificity identification, incubated at room temperature 1 h, clean up, store for future use in 4 DEG C of refrigerators.
Embodiment 3 A kind of preparation method of prostate specific antigen Electrochemiluminescsensor sensor based on auraization amination Graphene/ceria
(1) glass-carbon electrode of a diameter of 4 mm is taken turns doing polishing with 1.0 μm, 0.3 μm, 0.05 μm aluminum oxide polishing powder successively, clean with ultrapure water；
(2) by 6 μ L, concentration be the base material auraization amination Graphene/ceria solution of 3 mg/mL in electrode surface, 4 DEG C of preservations；
(3) drip 6 L, the antibody of 10 g/mL, use ultrapure water electrode surface after drying, 4 DEG C of refrigerators dry；
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L, mass fraction are 1%, in 4 DEG C of refrigerators, hatch 3 h, clean up；
(5) 6 μ L prostate specific antigen droppings are used for capturing antibody, incubated at room temperature 1 h on electrode, clean up；
(6) antibody-solutions of the argentaization bismuth sulfide labelling that 6 μ L concentration are 3 mg/mL is dropped on electrode with antigenic specificity identification, incubated at room temperature 1 h, clean up, store for future use in 4 DEG C of refrigerators.
Embodiment 4 The preparation of base material auraization amination Graphene/ceria solution
(1) preparation of amination Graphene
75 mg graphene oxides are joined in 40 mL ethylene glycol solutions, the most ultrasonic 1 h, it is subsequently adding 1.0 mL ammonia, acutely after vibration 1 min, solution is transferred in 50 mL reactors, at 180 DEG C, heats 6 h, centrifuge washing 20 min under 8000 r/min, 35 DEG C of vacuum drying, i.e. obtain amination Graphene；
(2) preparation of auraization amination Graphene/ceria
By in 40 mg amination graphene dispersion to 50 mL ultra-pure waters, ultrasonic 2 h, it is subsequently adding 1.1 g six nitric hydrate ceriums, stirring 30 min, add ammonia regulation pH to 11, adding 2.5 mL mass fractions is the chlorauric acid solution of 1%, solution stirs 6 h at 90 DEG C, centrifuge washing 20 min under 8000 r/min, 35 DEG C of vacuum drying, i.e. obtain auraization amination Graphene/ceria.
Embodiment 5 The preparation of base material auraization amination Graphene/ceria solution
(1) preparation of amination Graphene
100 mg graphene oxides are joined in 40 mL ethylene glycol solutions, the most ultrasonic 1 h, it is subsequently adding 1.0 mL ammonia, acutely after vibration 1 min, solution is transferred in 50 mL reactors, at 180 DEG C, heats 6 h, centrifuge washing 20 min under 8000 r/min, 35 DEG C of vacuum drying, i.e. obtain amination Graphene；
(2) preparation of auraization amination Graphene/ceria
By in 50 mg amination graphene dispersion to 50 mL ultra-pure waters, ultrasonic 2 h, it is subsequently adding 1.1 g six nitric hydrate ceriums, stirring 30 min, add ammonia regulation pH to 11, adding 2.5 mL mass fractions is the chlorauric acid solution of 1%, solution stirs 6 h at 90 DEG C, centrifuge washing 20 min under 8000 r/min, 35 DEG C of vacuum drying, i.e. obtain auraization amination Graphene/ceria.
Embodiment 6 The preparation of base material auraization amination Graphene/ceria solution
(1) preparation of amination Graphene
125 mg graphene oxides are joined in 40 mL ethylene glycol solutions, the most ultrasonic 1 h, it is subsequently adding 1.0 mL ammonia, acutely after vibration 1 min, solution is transferred in 50 mL reactors, at 180 DEG C, heats 6 h, centrifuge washing 20 min under 8000 r/min, 35 DEG C of vacuum drying, i.e. obtain amination Graphene；
(2) preparation of auraization amination Graphene/ceria
By in 60 mg amination graphene dispersion to 50 mL ultra-pure waters, ultrasonic 2 h, it is subsequently adding 1.1 g six nitric hydrate ceriums, stirring 30 min, add ammonia regulation pH to 11, adding 2.5 mL mass fractions is the chlorauric acid solution of 1%, solution stirs 6 h at 90 DEG C, centrifuge washing 20 min under 8000 r/min, 35 DEG C of vacuum drying, i.e. obtain auraization amination Graphene/ceria.
Embodiment 7 The preparation of the antibody-solutions of the prostate specific antigen of argentaization bismuth sulfide labelling
(1) preparation of bismuth sulfide
1.6 g five nitric hydrate bismuths are dissolved in 25 mL ethylene glycol, with nitrogen bubble 15 minutes, obtain solution A；1.2 g sodium sulfide are dissolved in the mixed solution of 10 mL ethylene glycol and 20 mL ultra-pure waters simultaneously, stir 15 min, obtain solution B；Under agitation, solution B is added dropwise in solution A, 1.8 g ureas are joined in above-mentioned solution, add 20 mL ultra-pure waters, stir 30 minutes, mixed solution is transferred in reactor, 24 h are heated at 180 DEG C, centrifuge washing 20 min under 8000 r/min, 35 DEG C of vacuum drying, obtain bismuth sulfide；
(2) preparation of argentaization bismuth sulfide label
230 mg bismuth sulfides are distributed in 100 mL ultra-pure waters, ultrasonic 10 h, are subsequently adding 110 mg silver nitrate and 10 mg surfactants (PVP), stir 6 h, add 6 mL
The sodium citrate solution of 5mmol/L, stirs 24 h, centrifuge washing 20 min under 8000 r/min, and in vacuum drying oven, 35 DEG C are dried, obtain argentaization bismuth sulfide label；
(3) preparation of the antibody-solutions of argentaization bismuth sulfide labelling
Argentaization bismuth sulfide label solution and the antibody-solutions that 300 μ L concentration are 5 μ g/mL prostate specific antigen that 3 mL concentration are 1 mg/mL are mixed, vibrate 24 h, centrifuge washing 15 min under 5000 r/min, obtain the antibody hatching thing of argentaization bismuth sulfide labelling, it is scattered in the phosphate buffered solution that 3 mL, pH are 7.4, is prepared the antibody-solutions of argentaization bismuth sulfide labelling.
Embodiment 8 The preparation of the antibody-solutions of the prostate specific antigen of argentaization bismuth sulfide labelling
(1) preparation of bismuth sulfide
1.8 g five nitric hydrate bismuths are dissolved in 25 mL ethylene glycol, with nitrogen bubble 15 minutes, obtain solution A；1.4 g sodium sulfide are dissolved in the mixed solution of 10 mL ethylene glycol and 20 mL ultra-pure waters simultaneously, stir 15 min, obtain solution B；Under agitation, solution B is added dropwise in solution A, 1.9 g ureas are joined in above-mentioned solution, add 20 mL ultra-pure waters, stir 30 minutes, mixed solution is transferred in reactor, 24 h are heated at 180 DEG C, centrifuge washing 20 min under 8000 r/min, 35 DEG C of vacuum drying, obtain bismuth sulfide；
(2) preparation of argentaization bismuth sulfide label
240 mg bismuth sulfides are distributed in 100 mL ultra-pure waters, ultrasonic 10 h, are subsequently adding 120 mg silver nitrate and 15 mg surfactants (PVP), stir 6 h, add 6 mL
The sodium citrate solution of 5mmol/L, stirs 24 h, centrifuge washing 20 min under 8000 r/min, and in vacuum drying oven, 35 DEG C are dried, obtain argentaization bismuth sulfide label；
(3) preparation of the antibody-solutions of argentaization bismuth sulfide labelling
Argentaization bismuth sulfide label solution and the antibody-solutions that 300 μ L concentration are 10 μ g/mL prostate specific antigen that 3 mL concentration are 2 mg/mL are mixed, vibrate 24 h, centrifuge washing 15 min under 5000 r/min, obtain the antibody hatching thing of argentaization bismuth sulfide labelling, it is scattered in the phosphate buffered solution that 3 mL, pH are 7.4, is prepared the antibody-solutions of argentaization bismuth sulfide labelling.
Embodiment 9 The preparation of the antibody-solutions of the prostate specific antigen of argentaization bismuth sulfide labelling
(1) preparation of bismuth sulfide
2.0 g five nitric hydrate bismuths are dissolved in 25 mL ethylene glycol, with nitrogen bubble 15 minutes, obtain solution A；1.5 g sodium sulfide are dissolved in the mixed solution of 10 mL ethylene glycol and 20 mL ultra-pure waters simultaneously, stir 15 min, obtain solution B；Under agitation, solution B is added dropwise in solution A, 2.0 g ureas are joined in above-mentioned solution, add 20 mL ultra-pure waters, stir 30 minutes, mixed solution is transferred in reactor, 24 h are heated at 180 DEG C, centrifuge washing 20 min under 8000 r/min, 35 DEG C of vacuum drying, obtain bismuth sulfide；
(2) preparation of argentaization bismuth sulfide label
250 mg bismuth sulfides are distributed in 100 mL ultra-pure waters, ultrasonic 10 h, are subsequently adding 130 mg silver nitrate and 20 mg surfactants (PVP), stir 6 h, add 6 mL
The sodium citrate solution of 5mmol/L, stirs 24 h, centrifuge washing 20 min under 8000 r/min, and in vacuum drying oven, 35 DEG C are dried, obtain argentaization bismuth sulfide label；
(3) preparation of the antibody-solutions of argentaization bismuth sulfide labelling
Argentaization bismuth sulfide label solution and the antibody-solutions that 300 μ L concentration are 15 μ g/mL prostate specific antigen that 3 mL concentration are 3 mg/mL are mixed, vibrate 24 h, centrifuge washing 15 min under 5000 r/min, obtain the antibody hatching thing of argentaization bismuth sulfide labelling, it is scattered in the phosphate buffered solution that 3 mL, pH are 7.4, is prepared the antibody-solutions of argentaization bismuth sulfide labelling.
Embodiment 10 The detection method of prostate specific antigen
(1) three-electrode system of electrochemical workstation is used to test, Ag/AgCl electrode is as reference electrode, platinum electrode is to electrode, prepared electrochemiluminescimmunosensor immunosensor is working electrode, electrochemical workstation and chemiluminescence detector are linked together the high pressure of photomultiplier tube is set to 750 V, cyclic voltammetry scan current potential is-2.0 V, and sweep speed is 0.1 V/s；
(2) in the phosphate buffered solution containing 100 mmol/L potassium peroxydisulfates of 10 mL, pH 5.5, by electrochemical luminescence system, the electrochemical luminescence signals intensity that the determinand antigen to variable concentrations produces, drawing curve are detected；
(3) testing sample solution replace prostate specific antigen solution detect.
Embodiment 11 The detection method of prostate specific antigen
(1) three-electrode system of electrochemical workstation is used to test, Ag/AgCl electrode is as reference electrode, platinum electrode is to electrode, prepared electrochemiluminescimmunosensor immunosensor is working electrode, electrochemical workstation and chemiluminescence detector are linked together the high pressure of photomultiplier tube is set to 750 V, cyclic voltammetry scan current potential is-2.0 V, and sweep speed is 0.1 V/s；
(2) in the phosphate buffered solution containing 125 mmol/L potassium peroxydisulfates of 10 mL, pH 6.5, by electrochemical luminescence system, the electrochemical luminescence signals intensity that the determinand antigen to variable concentrations produces, drawing curve are detected；
(3) testing sample solution replace prostate specific antigen solution detect.
Embodiment 12 The detection method of prostate specific antigen
(1) three-electrode system of electrochemical workstation is used to test, Ag/AgCl electrode is as reference electrode, platinum electrode is to electrode, prepared electrochemiluminescimmunosensor immunosensor is working electrode, electrochemical workstation and chemiluminescence detector are linked together the high pressure of photomultiplier tube is set to 750 V, cyclic voltammetry scan current potential is-2.0 V, and sweep speed is 0.1 V/s；
(2) in the phosphate buffered solution containing 150 mmol/L potassium peroxydisulfates of 10 mL, pH 8.0, by electrochemical luminescence system, the electrochemical luminescence signals intensity that the determinand antigen to variable concentrations produces, drawing curve are detected；
(3) testing sample solution replace prostate specific antigen solution detect.
Claims (4)
1. the preparation method of an electrochemiluminescimmunosensor immunosensor based on argentaization bismuth sulfide, it is characterised in that comprise the following steps:
(1) glass-carbon electrode of a diameter of 4 mm is taken turns doing polishing with 1.0 μm, 0.3 μm, 0.05 μm aluminum oxide polishing powder successively, clean with ultrapure water；
(2) take 6 L, 1 ~ 3 mg/mL auraization amination Graphene/ceria solution is added drop-wise under electrode surface, room temperature dry, and uses ultrapure water electrode surface, dries；
(3) drip 6 L, the antibody of 5 ~ 10 g/mL, use ultrapure water electrode surface after drying, 4 DEG C of refrigerators dry；
(4) close nonspecific activity site with the bovine serum albumin solution that 3 μ L, mass fraction are 1%, in 4 DEG C of refrigerators, hatch 3 h, clean up；
(5) 6 μ L prostate specific antigen droppings are used for capturing antibody, incubated at room temperature 1 h on electrode, clean up；
(6) antibody-solutions of the argentaization bismuth sulfide labelling that 6 μ L concentration are 1 ~ 3 mg/mL is dropped on electrode with antigenic specificity identification, incubated at room temperature 1 h, clean up, store for future use in 4 DEG C of refrigerators.
A kind of preparation method of electrochemiluminescimmunosensor immunosensor based on argentaization bismuth sulfide, the preparation process of described auraization amination Graphene/ceria solution is as follows:
(1) preparation of amination Graphene
75 ~ 125 mg graphene oxides are joined in 40 mL ethylene glycol solutions, the most ultrasonic 1 h, it is subsequently adding 1.0 mL ammonia, acutely after vibration 1 min, solution is transferred in 50 mL reactors, at 180 DEG C, heats 6 h, centrifuge washing 20 min under 8000 r/min, 35 DEG C of vacuum drying, i.e. obtain amination Graphene；
(2) preparation of auraization amination Graphene/ceria
By in 40 ~ 60 mg amination graphene dispersion to 50 mL ultra-pure waters, ultrasonic 2 h, it is subsequently adding 1.1 g six nitric hydrate ceriums, stirring 30 min, add ammonia regulation pH to 11, adding 2.5 mL mass fractions is the chlorauric acid solution of 1%, solution stirs 6 h at 90 DEG C, centrifuge washing 20 min under 8000 r/min, 35 DEG C of vacuum drying, i.e. obtain auraization amination Graphene/ceria.
A kind of preparation method of electrochemiluminescimmunosensor immunosensor based on argentaization bismuth sulfide, the preparation process of the antibody-solutions of described argentaization bismuth sulfide labelling is as follows:
(1) preparation of bismuth sulfide
1.6 ~ 2.0 g five nitric hydrate bismuths are dissolved in 25 mL ethylene glycol, with nitrogen bubble 15 minutes, obtain solution A；1.2 ~ 1.5 g sodium sulfide are dissolved in the mixed solution of 10 mL ethylene glycol and 20 mL ultra-pure waters simultaneously, stir 15 min, obtain solution B；Under agitation, solution B is added dropwise in solution A, 1.8 ~ 2.0 g ureas are joined in above-mentioned solution, add 20 mL ultra-pure waters, stir 30 minutes, mixed solution is transferred in reactor, 24 h are heated at 180 DEG C, centrifuge washing 20 min under 8000 r/min, 35 DEG C of vacuum drying, obtain bismuth sulfide；
(2) preparation of argentaization bismuth sulfide label
230 ~ 250 mg bismuth sulfides are distributed in 100 mL ultra-pure waters, ultrasonic 10 h, it is subsequently adding 110 ~ 130 mg silver nitrate and 10 ~ 20 mg surfactants (PVP), stir 6 h, add the sodium citrate solution of 6 mL 5mmol/L, stir 24 h, centrifuge washing 20 min under 8000 r/min, in vacuum drying oven, 35 DEG C are dried, obtain argentaization bismuth sulfide label；
(3) preparation of the antibody-solutions of argentaization bismuth sulfide labelling
Argentaization bismuth sulfide label solution and the antibody-solutions that 300 μ L concentration are 5 ~ 15 μ g/mL prostate specific antigen that 3 mL concentration are 1 ~ 3 mg/mL are mixed, vibrate 24 h, centrifuge washing 15 min under 5000 r/min, obtain the antibody hatching thing of argentaization bismuth sulfide labelling, it is scattered in the phosphate buffered solution that 3 mL, pH are 7.4, is prepared the antibody-solutions of argentaization bismuth sulfide labelling.
4. the detection method of electrochemiluminescimmunosensor immunosensor prepared by preparation method as claimed in claim 1, it is characterised in that step is as follows:
(1) three-electrode system of electrochemical workstation is used to test, Ag/AgCl electrode is as reference electrode, platinum electrode is to electrode, prepared electrochemiluminescimmunosensor immunosensor is working electrode, electrochemical workstation and chemiluminescence detector being linked together and the high pressure of photomultiplier tube is set to 750 V, cyclic voltammetry scan current potential is-2.0
V, sweep speed is 0.1 V/s；
(2) in the phosphate buffered solution containing 100 ~ 150 mmol/L potassium peroxydisulfates of 10 mL, pH 5.5 ~ 8.0, by electrochemical luminescence system, the electrochemical luminescence signals intensity that the determinand antigen to variable concentrations produces, drawing curve are detected；
(3) testing sample solution replace prostate specific antigen solution detect.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106501239A (en) * | 2016-10-17 | 2017-03-15 | 济南大学 | A kind of preparation method and application of the Electrochemiluminescsensor sensor based on poly-dopamine Nano microsphere |
| CN108918856A (en) * | 2018-07-31 | 2018-11-30 | 济南大学 | A kind of preparation method and application of double MOFs material quenching type electrochemiluminescimmunosensor immunosensors |
| CN108918855A (en) * | 2018-07-31 | 2018-11-30 | 济南大学 | A kind of preparation method and application based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator |
| CN113588752A (en) * | 2021-09-01 | 2021-11-02 | 集美大学 | Preparation method and application of electrochemiluminescence aptamer sensor |
| CN115557525A (en) * | 2022-03-23 | 2023-01-03 | 黑龙江大学 | Method for preparing lanthanide series metal oxide array material and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104391117A (en) * | 2014-11-08 | 2015-03-04 | 济南大学 | Preparation method and application of gastric cancer antigen electrogenerated chemiluminescence sensor based on PP<y>-NH2GO-Ag2Se@CdSe |
| CN104880456A (en) * | 2015-05-22 | 2015-09-02 | 济南大学 | Preparation method and application of electrochemiluminescence immunosensor constructed on basis of GO/MWCNTs-COOH/Au @ CeO2 |
-
2016
- 2016-03-25 CN CN201610175925.9A patent/CN105842460B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104391117A (en) * | 2014-11-08 | 2015-03-04 | 济南大学 | Preparation method and application of gastric cancer antigen electrogenerated chemiluminescence sensor based on PP<y>-NH2GO-Ag2Se@CdSe |
| CN104880456A (en) * | 2015-05-22 | 2015-09-02 | 济南大学 | Preparation method and application of electrochemiluminescence immunosensor constructed on basis of GO/MWCNTs-COOH/Au @ CeO2 |
Non-Patent Citations (2)
| Title |
|---|
| FAYINGLI ET AL: "An ultrasensitive sandwich-type electrochemical immunosensor based on signal amplification strategy of gold nanoparticles functionalized magneti cmulti-walled carbon nanotubes loaded with lead ions", 《BIOSENSORS AND BIOELECTRONICS》 * |
| JIANXIU LI ET AL: "A label-free electrochemiluminescence immunosensor based on KNb03-Au nanoparticles@Bi2S3 for the detection of prostate specific antigen", 《BIOSENSORS AND BIOELECTRONICS》 * |
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|---|---|---|---|---|
| CN106501239A (en) * | 2016-10-17 | 2017-03-15 | 济南大学 | A kind of preparation method and application of the Electrochemiluminescsensor sensor based on poly-dopamine Nano microsphere |
| CN108918856A (en) * | 2018-07-31 | 2018-11-30 | 济南大学 | A kind of preparation method and application of double MOFs material quenching type electrochemiluminescimmunosensor immunosensors |
| CN108918855A (en) * | 2018-07-31 | 2018-11-30 | 济南大学 | A kind of preparation method and application based on signal " on-off " type Electrochemiluminescsensor sensor that AgNCs is illuminator |
| CN108918855B (en) * | 2018-07-31 | 2021-11-09 | 济南大学 | Preparation method and application of signal 'on-off' type electrochemiluminescence sensor based on AgNCs as luminophor |
| CN113588752A (en) * | 2021-09-01 | 2021-11-02 | 集美大学 | Preparation method and application of electrochemiluminescence aptamer sensor |
| CN113588752B (en) * | 2021-09-01 | 2023-10-13 | 集美大学 | Preparation method and application of electrochemiluminescence aptamer sensor |
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| CN115557525B (en) * | 2022-03-23 | 2023-11-03 | 黑龙江大学 | Method for preparing lanthanide metal oxide array material and application thereof |
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