CN103245656B - Preparation and application of alpha fetoprotein and carcino-embryonic antigen electrochemiluminescence sensor - Google Patents
Preparation and application of alpha fetoprotein and carcino-embryonic antigen electrochemiluminescence sensor Download PDFInfo
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Abstract
The invention provides preparation and application of an alpha fetoprotein and carcino-embryonic antigen electrochemiluminescence sensor, and belongs to the technical fields of nano function material, clinical analysis, a bioseneor technology and electrochemistry. The characteristics that platinum nanoparticle @meso-porous silicon @ graphene nanocomposite (PtNPs@m-Si@GS) is strong in conductivity, good in stability, large in specific surface area, good in biocompatibility, strong in catalytic activity and the like are utilized in preparation; an alpha fetoprotein second antibody (anti-alpha fetoprotein (AFP)) and a carcino-embryonic antigen secondary antibody (anti-carcino-embryonicantigen (CEA)) are marked, so as to prepare the marked second antibodies Ru-PtNPs@M-Si@GS/anti-AFP and luminol-PtNPs@M-Si@GS/anti-CEA; and the sensitivity of the sensor is obviously improved. Compared with other single-channel electrode sensors, alpha fetoprotein and carcino-embryonic antigen can be simultaneously detected on a same electrode at one time; the detection efficiency is obviously improved; and the alpha fetoprotein and carcino-embryonic antigen electrochemiluminescence sensor has important scientific significance and application value on clinical early diagnosis of hepatic carcinoma.
Description
Technical field
The invention belongs to nano-functional material, clinical analysis, biosensor technique and technical field of electrochemistry, preparation and the application of a kind of alpha-fetoprotein and carcinomebryonic antigen Electrochemiluminescsensor sensor are provided.
Background technology
The early diagnosis of tumour, early treatment are the keys that improves cancer patient's survival rate.The detection of tumor markers can reflect the biological characteristics of tumour, all significant to guiding treatment, auxiliary diagnosis, judging prognosis.Single tumor markers possibility susceptibility, accuracy are not high enough, and organ specificity is strong not.Joint-detection can make up the deficiency that single index detects, and has improved susceptibility, accuracy and the specificity of diagnosing tumor.
Liver cancer is life-threatening common disease, and early detection, early diagnosis are basis and the prerequisites of early treatment, is also the important guarantee that effectively improves the liver cancer patient survival rate, improves its prognosis.Because the early stage volume of cancer knurl is small, liver is hidden again in the upper abdomen deep, has rib to do barrier, adopt the means such as B ultrasonic, CT scan all to be difficult to early detection, and liver has powerful compensation, in early days often without clinical symptoms, bring difficulty also to the early diagnosis of liver cancer.
Joint-detection serum alpha-fetoprotein (AFP) and carcinomebryonic antigen (CEA) tumor markers not only can be used for the early diagnosis of liver cancer, and contribute to the antidiastole of primary carcinoma of liver.
Along with the fast development of nanometer technology in recent years, various inorganic nano materials as carbon nano-tube, metal nanoparticle, metal oxide, semiconductor and Graphene etc. for the structure of electrochemiluminescimmunosensor immunosensor.
Graphene (GrapheneSheets, GS) is the two-dimensional structure of a kind of individual layer, sheet, becomes in recent years the focus that theory and practice is paid close attention to.Mesoporous material is also paid close attention to widely due to large specific surface area and good biocompatibility.In addition, metal nano material is due to its unique character and advantage, as good conductivity, catalytic activity is high, specific surface area is large, and is widely used in the enhanced sensitivity aspect of electrochemical sensor.
The present invention has made single electrode binary channels electrochemiluminescimmunosensor immunosensor, and the mesoporous gold of decorated nanometer on working electrode at first is used for the primary antibodie of immobilized alpha-fetoprotein and carcinomebryonic antigen.
Adopt the double-antibody sandwich immunoassay method, two anti-labeling methods are inquired into.Adopt nano platinum particle@mesoporous silicon@graphene nanocomposite material as two anti-label carriers, tris (bipyridine) ruthenium and luminol are used for respectively that labelled AFP two is anti-and carcinomebryonic antigen two is anti-, add triethanolamine in end liquid, under different voltage, tris (bipyridine) ruthenium reacts with triethanolamine and produces electrogenerated chemiluminescence signal 1, luminol and dissolved oxygen DO produce electrogenerated chemiluminescence signal 2, prepare function admirable, detect alpha-fetoprotein and carcinomebryonic antigen binary channels electrochemiluminescimmunosensor immunosensor highly sensitive the time.
The present invention adopts a glass-carbon electrode, in conjunction with the electrogenerated chemiluminescence technology, realizes realizing binary channels mensuration on a glass-carbon electrode; The binding immunoassay technology, improved the selectivity of sensor; The method is saved time, and is a kind of quick, special, sensitive detection method.
Summary of the invention
One of purpose of the present invention is to provide the preparation method of a kind of alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor.
Two of purpose of the present invention is by prepared electrochemiluminescimmunosensor immunosensor, for detect alpha-fetoprotein and carcinomebryonic antigen simultaneously.
Technical scheme of the present invention, comprise the following steps.
1. the preparation method of alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor, is characterized in that, comprises the following steps:
(1) preparation of nano platinum particle@mesoporous silicon@graphene nanocomposite material (PtNPs@M-Si@GS)
By 6 ~ 10 mL, 2.66 mg/mL graphene oxides, 0.4 ~ 0.6 g cetyl trimethyl ammonium bromide (CTAB), 20 mg NaOH and 42 mL ultrapure waters mix, ultrasonic 30 ~ 50 min; 35 ~ 45 ℃ of lower magnetic forces stir, and dropwise add tetraethyl orthosilicate (TEOS) ethanolic solution, react 10 ~ 12 h; Add the hydrazine hydrate that 80 mL, massfraction are 85%, 65 ~ 75 ℃ of heating 3 h, ethanol centrifuge washing three times; Product is dissolved in acetone and stirs 24 h, centrifugal three times; Product is dissolved in 50 mL ethanol, adds 150 ~ 250 μ L 3-aminopropyl triethoxysilanes (APTES), stir 10 ~ 12 h, centrifuging; Product is dissolved in to 42.5 mL water, obtains amination mesoporous silicon@graphene nanocomposite material (M-Si@GS).
3 ~ 5 mL M-Si@GS aqueous solution and 40 ~ 60 mL nano platinum particle solution are mixed, stir 2 ~ 4 h, centrifuging twice, vacuum drying, obtain PtNPs@M-Si@GS.
Described TEOS ethanolic solution is to make in the ethanol that is dissolved in 1.6 mL by 350 ~ 450 μ L TEOS.
(2) preparation of mark two anti-(Ru-PtNPs@M-Si@GS/anti-AFP and luminol-PtNPs@M-Si@GS/anti-CEA) solution
The PtNPs@M-Si@GS of 0.5 ~ 1.5 mg is joined respectively to 1 ~ 2 mL, 1 * 10
-3mol/L tris (bipyridine) ruthenium (Ru (bpy)
3cl
2) solution and 1 ~ 2 mL, 1 * 10
-3in mol/L luminol (luminol) solution, ultrasonic 1 ~ 3 h, stir 6 ~ 10 h, and centrifuging obtains two anti-label Ru-PtNPs@M-Si@GS and luminol-PtNPs@M-Si@GS.
Prepared two anti-label Ru-PtNPs@M-Si@GS are joined in 1 mL, 5 ~ 10 μ g/mL alpha-fetoprotein antibody (anti-AFP) solution, concussion hatching 20 ~ 24 h under 4 ℃, centrifugal removal supernatant, the PBS buffer solution that adds 1 mL, pH=7.0 ~ 7.8, obtain in refrigerator that the anti-Ru-PtNPs@of mark two M-Si@GS/anti-AFP solution is stored in 4 ℃ standby.
Prepared two anti-label luminol-PtNPs@M-Si@GS are added in 1 mL, 5 ~ 10 μ g/mL carcinomebryonic antigen antibody (anti-CEA) solution, concussion hatching 20 ~ 24 h under 4 ℃, centrifugal removal supernatant, the PBS buffer solution that adds 1 mL, pH=7.0 ~ 7.8, obtain the anti-luminol-PtNPs@of mark two M-Si@GS/anti-CEA solution, be stored in the refrigerator of 4 ℃ standby.
(3) preparation method of alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor
1) on the surface of working electrode, modify one deck nano-porous gold film, under room temperature, naturally dry.
2) 1) in drip 5 ~ 10 μ L antibody mixed solutions on the surface of the working electrode that obtains, hatch 2 ~ 3 h under room temperature, then with ultrapure water, clean, dry.
Described antibody mixed solution is 5 ~ 10 μ g/mL containing the concentration of alpha-fetoprotein antibody and carcinomebryonic antigen antibody.
3) wash away with ultrapure water described alpha-fetoprotein antibody and the carcinomebryonic antigen antibody do not had on crosslinked, the bovine serum albumin solution that the dropping massfraction is 0.5% ~ 1%, place 1 ~ 2 h under 37 ℃, with the PBS buffer solution washing of pH=7.0 ~ 7.8, dry, store for future use under 4 ℃, make alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor.
2. alpha-fetoprotein described above and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor, detect in the time of for alpha-fetoprotein and carcinomebryonic antigen, and step is as follows:
(1) alpha-fetoprotein of concentration known and carcinomebryonic antigen solution are joined in the PBS buffer solution of 40 ~ 60 μ L, pH=7.0 ~ 7.8, make the antigen mixed solution, get 10 ~ 15 μ L antigen mixed solutions and drip on the working electrode that is coated onto prepared electrochemiluminescimmunosensor immunosensor, place 1 ~ 2 h.
(2) Ru-PtNPs@M-Si@GS/anti-AFP solution and luminol-PtNPs@M-Si@GS/anti-CEA solution equal-volume are mixed, make the anti-mixed solution of mark two, drip and be coated with 5 ~ 7 μ L mark two anti-mixed solutions to working electrode surface, wash away anti-ly in conjunction with upper mark two after 1 ~ 2 h with the PBS buffer solution of pH=7.0 ~ 7.8, obtain the working electrode of assembling.
(3) by contrast electrode, the working electrode of electrode and assembling is connected on electrogenerated chemiluminescence equipment, the PBS buffer solution that adds the pH=7.0 of 5 ~ 15 mL, 20 ~ 30 mmol/L triethanolamine saturations of the air ~ 7.8 in electrolytic tank, apply cyclical voltage by cyclic voltammetry to the working electrode of assembling; Ru-PtNPs@M-Si@GS/anti-AFP and luminol-PtNPs@M-Si@GS/anti-CEA produce light signal respectively under different voltage, according to the light signal strength of gained electrogenerated chemiluminescence and the relation between alpha-fetoprotein and carcinomebryonic antigen concentration of standard solution, drawing curve.
(4) testing sample solution is replaced to the standard solution of alpha-fetoprotein and carcinomebryonic antigen, detected according to the method for drafting of the working curve of described alpha-fetoprotein and carcinomebryonic antigen.
(5) for the sensing range of alpha-fetoprotein (AFP) and carcinomebryonic antigen (CEA): AFP concentration is at 0.01 ~ 25 ng/mL, and the CEA antigen concentration is at 0.005 ~ 20 ng/mL, and AFP detects and is limited to 2.5 pg/mL, and CEA detects and is limited to 1.0 pg/mL.
Described alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor, raw materials all can be bought in chemical reagents corporation or biopharmaceutical company.
The present invention possesses following advantage:
(1) when the present invention has realized alpha-fetoprotein and carcinomebryonic antigen on a glass-carbon electrode, detect.
(2) the present invention adopts PtNPs@M-Si@GS to strengthen the electrogenerated chemiluminescence signal, thereby improves the sensitivity of sensor.
(3) the present invention compares with other single-channel electrodes sensor, can be on same electrode once property detect alpha-fetoprotein and carcinomebryonic antigen simultaneously, significantly improved detection efficiency, the preparation of this immunosensor has important scientific meaning and using value to clinical early diagnosis liver cancer.
Embodiment
Further illustrate the present invention below in conjunction with embodiment.
embodiment 1the preparation of PtNPs@M-Si@GS
(1) by 6 mL, 2.66 mg/mL graphene oxides, 0.4 g cetyl trimethyl ammonium bromide (CTAB), 20 mg NaOH and 42 mL ultrapure waters mix, ultrasonic 30 min; 35 ℃ of lower magnetic forces stir, and dropwise add tetraethyl orthosilicate (TEOS) ethanolic solution, react 10 h; Add the hydrazine hydrate that 80 mL, massfraction are 85%, 65 ℃ of heating 3 h, ethanol centrifuge washing three times; Product is dissolved in acetone and stirs 24 h, centrifugal three times; Product is dissolved in 50 mL ethanol, adds 150 μ L 3-aminopropyl triethoxysilanes (APTES), stir 10h, centrifuging; Product is dissolved in to 42.5 mL water, obtains amination mesoporous silicon@graphene nanocomposite material (M-Si@GS).
(2) 3 mL M-Si@GS aqueous solution and 40 mL nano platinum particle solution are mixed, stir 2 h, centrifuging twice, vacuum drying, obtain PtNPs@M-Si@GS.
Described TEOS ethanolic solution is to make in the ethanol that is dissolved in 1.6 mL by 350 μ L TEOS.
embodiment 2the preparation of PtNPs@M-Si@GS
(1) by 8 mL, 2.66 mg/mL graphene oxides, 0.5 g cetyl trimethyl ammonium bromide (CTAB), 20 mg NaOH and 42 mL ultrapure waters mix, ultrasonic 40 min; 40 ℃ of lower magnetic forces stir, and dropwise add tetraethyl orthosilicate (TEOS) ethanolic solution, react 11 h; Add the hydrazine hydrate that 80 mL, massfraction are 85%, 70 ℃ of heating 3 h, ethanol centrifuge washing three times; Product is dissolved in acetone and stirs 24 h, centrifugal three times; Product is dissolved in 50 mL ethanol, adds 200 μ L 3-aminopropyl triethoxysilanes (APTES), stir 11 h, centrifuging; Product is dissolved in to 42.5 mL water, obtains amination mesoporous silicon@graphene nanocomposite material (M-Si@GS).
(2) 4 mL M-Si@GS aqueous solution and 50 mL nano platinum particle solution are mixed, stir 3 h, centrifuging twice, vacuum drying, obtain PtNPs@M-Si@GS.
Described TEOS ethanolic solution is to make in the ethanol that is dissolved in 1.6 mL by 400 μ L TEOS.
embodiment 3the preparation of PtNPs@M-Si@GS
(1) by 10 mL, 2.66 mg/mL graphene oxides, 0.6 g cetyl trimethyl ammonium bromide (CTAB), 20 mg NaOH and 42 mL ultrapure waters mix, ultrasonic 50 min; 45 ℃ of lower magnetic forces stir, and dropwise add tetraethyl orthosilicate (TEOS) ethanolic solution, react 12 h; Add the hydrazine hydrate that 80 mL, massfraction are 85%, 75 ℃ of heating 3 h, ethanol centrifuge washing three times; Product is dissolved in acetone and stirs 24 h, centrifugal three times; Product is dissolved in 50 mL ethanol, adds 250 μ L 3-aminopropyl triethoxysilanes (APTES), stir 12 h, centrifuging; Product is dissolved in to 42.5 mL water, obtains amination mesoporous silicon@graphene nanocomposite material (M-Si@GS).
(2) 5 mL M-Si@GS aqueous solution and 60 mL nano platinum particle solution are mixed, stir 4 h, centrifuging twice, vacuum drying, obtain PtNPs@M-Si@GS.
Described TEOS ethanolic solution is to make in the ethanol that is dissolved in 1.6 mL by 450 μ L TEOS.
embodiment 4the preparation of mark two anti-(Ru-PtNPs@M-Si@GS/anti-AFP and luminol-PtNPs@M-Si@GS/anti-CEA) solution
(1) the PtNPs@M-Si@GS of 0.5 mg is joined respectively to 1 mL, 1 * 10
-3mol/L tris (bipyridine) ruthenium (Ru (bpy)
3cl
2) solution and 1 mL, 1 * 10
-3in mol/L luminol (luminol) solution, ultrasonic 1h, stir 6 h, and centrifuging obtains two anti-label Ru-PtNPs@M-Si@GS and luminol-PtNPs@M-Si@GS.
(2) prepared two anti-label Ru-PtNPs@M-Si@GS are joined in 1 mL, 5 μ g/mL alpha-fetoprotein antibody (anti-AFP) solution, concussion hatching 20 h under 4 ℃, centrifugal removal supernatant, the PBS buffer solution that adds 1 mL, pH=7.0, obtain in refrigerator that the anti-Ru-PtNPs@of mark two M-Si@GS/anti-AFP solution is stored in 4 ℃ standby.
(3) prepared two anti-label luminol-PtNPs@M-Si@GS are added in 1 mL, 5 μ g/mL carcinomebryonic antigen antibody (anti-CEA) solution, concussion hatching 20 h under 4 ℃, centrifugal removal supernatant, the PBS buffer solution that adds 1 mL, pH=7.0, obtain the anti-luminol-PtNPs@of mark two M-Si@GS/anti-CEA solution, be stored in the refrigerator of 4 ℃ standby.
embodiment 5the preparation of mark two anti-(Ru-PtNPs@M-Si@GS/anti-AFP and luminol-PtNPs@M-Si@GS/anti-CEA) solution
(1) the PtNPs@M-Si@GS of 1.0 mg is joined respectively to 1.5 mL, 1 * 10
-3mol/L tris (bipyridine) ruthenium (Ru (bpy)
3cl
2) solution and 1.5 mL, 1 * 10
-3in mol/L luminol (luminol) solution, ultrasonic 2 h, stir 8 h, and centrifuging obtains two anti-label Ru-PtNPs@M-Si@GS and luminol-PtNPs@M-Si@GS.
(2) prepared two anti-label Ru-PtNPs@M-Si@GS are joined in 1 mL, 8 μ g/mL alpha-fetoprotein antibody (anti-AFP) solution, concussion hatching 22 h under 4 ℃, centrifugal removal supernatant, the PBS buffer solution that adds 1 mL, pH=7.5, obtain in refrigerator that the anti-Ru-PtNPs@of mark two M-Si@GS/anti-AFP solution is stored in 4 ℃ standby.
(3) prepared two anti-label luminol-PtNPs@M-Si@GS are added in 1 mL, 8 μ g/mL carcinomebryonic antigen antibody (anti-CEA) solution, concussion hatching 22 h under 4 ℃, centrifugal removal supernatant, the PBS buffer solution that adds 1 mL, pH=7.5, obtain the anti-luminol-PtNPs@of mark two M-Si@GS/anti-CEA solution, be stored in the refrigerator of 4 ℃ standby.
embodiment 6the preparation of mark two anti-(Ru-PtNPs@M-Si@GS/anti-AFP and luminol-PtNPs@M-Si@GS/anti-CEA) solution
(1) the PtNPs@M-Si@GS of 1.5 mg is joined respectively to 2 mL, 1 * 10
-3mol/L tris (bipyridine) ruthenium (Ru (bpy)
3cl
2) solution and 2 mL, 1 * 10
-3in mol/L luminol (luminol) solution, ultrasonic 3 h, stir 10 h, and centrifuging obtains two anti-label Ru-PtNPs@M-Si@GS and luminol-PtNPs@M-Si@GS.
(2) prepared two anti-label Ru-PtNPs@M-Si@GS are joined in 1 mL, 10 μ g/mL alpha-fetoprotein antibody (anti-AFP) solution, concussion hatching 24 h under 4 ℃, centrifugal removal supernatant, the PBS buffer solution that adds 1 mL, pH=7.8, obtain in refrigerator that the anti-Ru-PtNPs@of mark two M-Si@GS/anti-AFP solution is stored in 4 ℃ standby.
(3) prepared two anti-label luminol-PtNPs@M-Si@GS are added in 1 mL, 10 μ g/mL carcinomebryonic antigen antibody (anti-CEA) solution, concussion hatching 24 h under 4 ℃, centrifugal removal supernatant, the PBS buffer solution that adds 1 mL, pH=7.8, obtain the anti-luminol-PtNPs@of mark two M-Si@GS/anti-CEA solution, be stored in the refrigerator of 4 ℃ standby.
embodiment 7the preparation method of alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor
(1) on the surface of working electrode, modify one deck nano-porous gold film, under room temperature, naturally dry.
(2) drip 5 μ L antibody mixed solutions on the surface of the working electrode obtained in (1), under room temperature, hatching 2 h, then clean with ultrapure water, dries.
Described antibody mixed solution is 5 μ g/mL containing the concentration of alpha-fetoprotein antibody and carcinomebryonic antigen antibody.
(3) wash away with ultrapure water described alpha-fetoprotein antibody and the carcinomebryonic antigen antibody do not had on crosslinked, the bovine serum albumin solution that the dropping massfraction is 0.5%, place 1 h under 37 ℃, with the PBS buffer solution washing of pH=7.0, dry, store for future use under 4 ℃, make alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor.
embodiment 8the preparation method of alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor
(1) on the surface of working electrode, modify one deck nano-porous gold film, under room temperature, naturally dry.
(2) drip 7 μ L antibody mixed solutions on the surface of the working electrode obtained in (1), under room temperature, hatching 2.5 h, then clean with ultrapure water, dries.
Described antibody mixed solution is 8 μ g/mL containing the concentration of alpha-fetoprotein antibody and carcinomebryonic antigen antibody.
(3) wash away with ultrapure water described alpha-fetoprotein antibody and the carcinomebryonic antigen antibody do not had on crosslinked, the bovine serum albumin solution that the dropping massfraction is 0.7%, place 1.5 h under 37 ℃, with the PBS buffer solution washing of pH=7.5, dry, store for future use under 4 ℃, make alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor.
embodiment 9the preparation method of alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor
(1) on the surface of working electrode, modify one deck nano-porous gold film, under room temperature, naturally dry.
(2) drip 10 μ L antibody mixed solutions on the surface of the working electrode obtained in (1), under room temperature, hatching 3 h, then clean with ultrapure water, dries.
Described antibody mixed solution is 10 μ g/mL containing the concentration of alpha-fetoprotein antibody and carcinomebryonic antigen antibody.
(3) wash away with ultrapure water described alpha-fetoprotein antibody and the carcinomebryonic antigen antibody do not had on crosslinked, the bovine serum albumin solution that the dropping massfraction is 1%, place 2 h under 37 ℃, with the PBS buffer solution washing of pH=7.8, dry, store for future use under 4 ℃, make alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor.
embodiment 10in the time of alpha-fetoprotein and carcinomebryonic antigen, detect
(1) alpha-fetoprotein of concentration known and carcinomebryonic antigen solution are joined in the PBS buffer solution of 40 μ L, pH=7.0, make the antigen mixed solution, get 10 μ L antigen mixed solutions and drip on the working electrode that is coated onto prepared electrochemiluminescimmunosensor immunosensor, place 1 h.
(2) Ru-PtNPs@M-Si@GS/anti-AFP solution and luminol-PtNPs@M-Si@GS/anti-CEA solution equal-volume are mixed, make the anti-mixed solution of mark two, drip and be coated with 5 μ L mark two anti-mixed solutions to working electrode surface, wash away anti-ly in conjunction with upper mark two after 1 h with the PBS buffer solution of pH=7.0, obtain the working electrode of assembling.
(3) by contrast electrode, the working electrode of electrode and assembling is connected on electrogenerated chemiluminescence equipment, the PBS buffer solution that adds the pH=7.0 of 5 mL, the 20 mmol/L triethanolamine saturations of the air in electrolytic tank, apply cyclical voltage by cyclic voltammetry to the working electrode of assembling; Ru-PtNPs@M-Si@GS/anti-AFP and luminol-PtNPs@M-Si@GS/anti-CEA produce light signal respectively under different voltage, according to the light signal strength of gained electrogenerated chemiluminescence and the relation between alpha-fetoprotein and carcinomebryonic antigen concentration of standard solution, drawing curve.
(4) testing sample solution is replaced to the standard solution of alpha-fetoprotein and carcinomebryonic antigen, detected according to the method for drafting of the working curve of described alpha-fetoprotein and carcinomebryonic antigen.
(5) for the sensing range of alpha-fetoprotein (AFP) and carcinomebryonic antigen (CEA): AFP concentration is at 0.01 ~ 25 ng/mL, and the CEA antigen concentration is at 0.005 ~ 20 ng/mL, and AFP detects and is limited to 2.5 pg/mL, and CEA detects and is limited to 1.0 pg/mL.
(6) in the time of for 1# blood serum sample alpha-fetoprotein (AFP) and carcinomebryonic antigen (CEA), detect, adopt using standard samples recovery, every kind of sample Parallel testing 5 times, its relative standard deviation is lower than 2.8%, the recovery is 91.1% ~ 108%, and the preci-sion and accuracy of detection method of the present invention is all satisfactory.
embodiment 11in the time of alpha-fetoprotein and carcinomebryonic antigen, detect
(1) alpha-fetoprotein of concentration known and carcinomebryonic antigen solution are joined in the PBS buffer solution of 50 μ L, pH=7.5, make the antigen mixed solution, get 13 μ L antigen mixed solutions and drip on the working electrode that is coated onto prepared electrochemiluminescimmunosensor immunosensor, place 1.5 h.
(2) Ru-PtNPs@M-Si@GS/anti-AFP solution and luminol-PtNPs@M-Si@GS/anti-CEA solution equal-volume are mixed, make the anti-mixed solution of mark two, drip and be coated with 6 μ L mark two anti-mixed solutions to working electrode surface, 1.5 wash away anti-ly in conjunction with upper mark two after h with the PBS buffer solution of pH=7.5, obtain the working electrode of assembling.
(3) by contrast electrode, the working electrode of electrode and assembling is connected on electrogenerated chemiluminescence equipment, the PBS buffer solution that adds the pH=7.5 of 10 mL, the 25 mmol/L triethanolamine saturations of the air in electrolytic tank, apply cyclical voltage by cyclic voltammetry to the working electrode of assembling; Ru-PtNPs@M-Si@GS/anti-AFP and luminol-PtNPs@M-Si@GS/anti-CEA produce light signal respectively under different voltage, according to the light signal strength of gained electrogenerated chemiluminescence and the relation between alpha-fetoprotein and carcinomebryonic antigen concentration of standard solution, drawing curve.
(4) testing sample solution is replaced to the standard solution of alpha-fetoprotein and carcinomebryonic antigen, detected according to the method for drafting of the working curve of described alpha-fetoprotein and carcinomebryonic antigen.
(5) in the time of for 2# blood serum sample alpha-fetoprotein (AFP) and carcinomebryonic antigen (CEA), detect, adopt using standard samples recovery, every kind of sample Parallel testing 5 times, its relative standard deviation is all lower than 1.5%, the recovery is 95.21% ~ 106 %, and the preci-sion and accuracy of detection method of the present invention is all satisfactory.
embodiment 12in the time of alpha-fetoprotein and carcinomebryonic antigen, detect
(1) alpha-fetoprotein of concentration known and carcinomebryonic antigen solution are joined in the PBS buffer solution of 60 μ L, pH=7.8, make the antigen mixed solution, get 15 μ L antigen mixed solutions and drip on the working electrode that is coated onto prepared electrochemiluminescimmunosensor immunosensor, place 2 h.
(2) Ru-PtNPs@M-Si@GS/anti-AFP solution and luminol-PtNPs@M-Si@GS/anti-CEA solution equal-volume are mixed, make the anti-mixed solution of mark two, drip and be coated with 7 μ L mark two anti-mixed solutions to working electrode surface, wash away anti-ly in conjunction with upper mark two after 2 h with the PBS buffer solution of pH=7.8, obtain the working electrode of assembling.
(3) by contrast electrode, the working electrode of electrode and assembling is connected on electrogenerated chemiluminescence equipment, the PBS buffer solution that adds the pH=7.8 of 15 mL, the 30 mmol/L triethanolamine saturations of the air in electrolytic tank, apply cyclical voltage by cyclic voltammetry to the working electrode of assembling; Ru-PtNPs@M-Si@GS/anti-AFP and luminol-PtNPs@M-Si@GS/anti-CEA produce light signal respectively under different voltage, according to the light signal strength of gained electrogenerated chemiluminescence and the relation between alpha-fetoprotein and carcinomebryonic antigen concentration of standard solution, drawing curve.
(4) testing sample solution is replaced to the standard solution of alpha-fetoprotein and carcinomebryonic antigen, detected according to the method for drafting of the working curve of described alpha-fetoprotein and carcinomebryonic antigen.
(5) in the time of for 3# blood serum sample alpha-fetoprotein (AFP) and carcinomebryonic antigen (CEA), detect, adopt using standard samples recovery, every kind of sample Parallel testing 5 times, its relative standard deviation is all lower than 4.5%, the recovery is 94.8% ~ 104 %, and the preci-sion and accuracy of detection method of the present invention is all satisfactory.
Claims (2)
1. the preparation method of alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor, is characterized in that, comprises the following steps:
(1) preparation of nano platinum particle@mesoporous silicon@graphene nanocomposite material (PtNPs@M-Si@GS)
By 6 ~ 10 mL, 2.66 mg/mL graphene oxides, 0.4 ~ 0.6 g cetyl trimethyl ammonium bromide (CTAB), 20 mg NaOH and 42 mL ultrapure waters mix, ultrasonic 30 ~ 50 min; 35 ~ 45 ℃ of lower magnetic forces stir, and dropwise add tetraethyl orthosilicate (TEOS) ethanolic solution, react 10 ~ 12 h; Add the hydrazine hydrate that 80 mL, massfraction are 85%, 65 ~ 75 ℃ of heating 3 h, ethanol centrifuge washing three times; Product is dissolved in acetone and stirs 24 h, centrifugal three times; Product is dissolved in 50 mL ethanol, adds 150 ~ 250 μ L 3-aminopropyl triethoxysilanes (APTES), stir 10 ~ 12 h, centrifuging; Product is dissolved in to 42.5 mL water, obtains amination mesoporous silicon@graphene nanocomposite material (M-Si@GS) aqueous solution;
3 ~ 5 mL amination mesoporous silicon@graphene nanocomposite material (M-Si@GS) aqueous solution and 40 ~ 60 mL nano platinum particle solution are mixed, stir 2 ~ 4 h, twice of centrifuging, vacuum drying, obtain nano platinum particle@mesoporous silicon@graphene nanocomposite material (PtNPs@M-Si@GS);
Described TEOS ethanolic solution is to make in the ethanol that is dissolved in 1.6 mL by 350 ~ 450 μ L TEOS;
(2) preparation of Ru-PtNPs@M-Si@GS/anti-AFP mark two anti-solution and luminol-PtNPs@M-Si@GS/anti-CEA mark two anti-solution
The PtNPs@M-Si@GS of 0.5 ~ 1.5 mg is joined respectively to 1 ~ 2 mL, 1 * 10
-3mol/L tris (bipyridine) ruthenium (Ru (bpy)
3cl
2) solution and 1 ~ 2 mL, 1 * 10
-3in mol/L luminol (luminol) solution, ultrasonic 1 ~ 3 h, stir 6 ~ 10 h, and centrifuging obtains Ru-PtNPs@M-Si@GS bis-anti-labels and luminol-PtNPs@M-Si@GS bis-anti-labels;
Prepared Ru-PtNPs@M-Si@GS bis-anti-labels are joined in 1 mL, 5 ~ 10 μ g/mL alpha-fetoprotein antibody (anti-AFP) solution, concussion hatching 20 ~ 24 h under 4 ℃, centrifugal removal supernatant, the PBS buffer solution that adds 1 mL, pH=7.0 ~ 7.8, obtain in refrigerator that Ru-PtNPs@M-Si@GS/anti-AFP mark two anti-solution are stored in 4 ℃ standby;
Prepared luminol-PtNPs@M-Si@GS bis-anti-labels are added in 1 mL, 5 ~ 10 μ g/mL carcinomebryonic antigen antibody (anti-CEA) solution, concussion hatching 20 ~ 24 h under 4 ℃, centrifugal removal supernatant, the PBS buffer solution that adds 1 mL, pH=7.0 ~ 7.8, obtain luminol-PtNPs@M-Si@GS/anti-CEA mark two anti-solution, be stored in the refrigerator of 4 ℃ standby;
(3) preparation method of alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor
1) on the surface of working electrode, modify one deck nano-porous gold film, under room temperature, naturally dry;
2) 1) in drip 5 ~ 10 μ L antibody mixed solutions on the surface of the working electrode that obtains, hatch 2 ~ 3 h under room temperature, then with ultrapure water, clean, dry;
Described antibody mixed solution is 5 ~ 10 μ g/mL containing the concentration of alpha-fetoprotein antibody and carcinomebryonic antigen antibody;
3) wash away with ultrapure water described alpha-fetoprotein antibody and the carcinomebryonic antigen antibody do not had on crosslinked, the bovine serum albumin solution that the dropping massfraction is 0.5% ~ 1%, place 1 ~ 2 h under 37 ℃, with the PBS buffer solution washing of pH=7.0 ~ 7.8, dry, store for future use under 4 ℃, make alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor.
2. alpha-fetoprotein and carcinomebryonic antigen electrochemiluminescimmunosensor immunosensor as claimed in claim 1, detect in the time of for alpha-fetoprotein and carcinomebryonic antigen, and step is as follows:
(1) alpha-fetoprotein of concentration known and carcinomebryonic antigen solution are joined in the PBS buffer solution of 40 ~ 60 μ L, pH=7.0 ~ 7.8, make the antigen mixed solution, get 10 ~ 15 μ L antigen mixed solutions and drip on the working electrode that is coated onto prepared electrochemiluminescimmunosensor immunosensor, place 1 ~ 2 h;
(2) Ru-PtNPs@M-Si@GS/anti-AFP mark two anti-solution and the anti-solution equal-volume of luminol-PtNPs@M-Si@GS/anti-CEA mark two are mixed, make the anti-mixed solution of mark two, drip and be coated with 5 ~ 7 μ L mark two anti-mixed solutions to working electrode surface, wash away anti-ly in conjunction with upper mark two after 1 ~ 2 h with the PBS buffer solution of pH=7.0 ~ 7.8, obtain the working electrode of assembling;
(3) by contrast electrode, the working electrode of electrode and assembling is connected on electrogenerated chemiluminescence equipment, the PBS buffer solution that adds the pH=7.0 of 5 ~ 15 mL, 20 ~ 30 mmol/L triethanolamine saturations of the air ~ 7.8 in electrolytic tank, apply cyclical voltage by cyclic voltammetry to the working electrode of assembling; Ru-PtNPs@M-Si@GS/anti-AFP mark two is anti-and luminol-PtNPs@M-Si@GS/anti-CEA mark two is anti-produces light signal respectively under different voltage, according to the light signal strength of gained electrogenerated chemiluminescence and the relation between alpha-fetoprotein and carcinomebryonic antigen concentration of standard solution, drawing curve;
(4) testing sample solution is replaced to the standard solution of alpha-fetoprotein and carcinomebryonic antigen, detected according to the method for drafting of the working curve of described alpha-fetoprotein and carcinomebryonic antigen.
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