CN106198699A - Prepare two kind of two anti-conjugate and for detection alpha-fetoprotein and the method for carcinoembryonic antigen simultaneously - Google Patents
Prepare two kind of two anti-conjugate and for detection alpha-fetoprotein and the method for carcinoembryonic antigen simultaneously Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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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
- G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The present invention relates to medical science tumor markers detection field, specifically a kind of use 1,1 ' ferrocene dicarboxylic acid/gold@platinum and thionine/gold@platinum prepare two kind of two anti-conjugate and the method simultaneously alpha-fetoprotein and carcinoembryonic antigen being carried out specific detection thereof respectively.Use 1,1 ' ferrocene dicarboxylic acid/gold@platinum and thionine/gold@platinum prepare two kind of two anti-conjugate respectively, method AFP and CEA detected simultaneously for the electrochemical immunosensor built, the glass-carbon electrode of modification is put in the antibody mixed solution of alpha-fetoprotein and carcinoembryonic antigen, then the most specific binding avtive spot is closed, alpha-fetoprotein antigen and the carcinoembryonic antigen mixture modified glassy carbon electrode of variable concentrations is used successively after taking out glass-carbon electrode, antigen is combined with antibody specificity, is built into sandwich immunosensor；Sandwich immunosensor is used differential pulse voltammetry DPV detection alpha-fetoprotein and the concentration of carcinoembryonic antigen.
Description
Technical field
The present invention relates to medical science tumor markers detection field, specifically a kind of 1,1 ' ferrocene dicarboxylic acid/gold@platinum
(Fc/Au@Pt) nano-complex and the preparation of thionine/gold@platinum (Thi/Au@Pt) nano-complex and simultaneously to alpha-fetoprotein
The method carrying out specific detection with carcinoembryonic antigen.
Background technology
Modern oncology thinks that malignant tumor is the disease of a kind of general, in addition to indivedual less morbidities of organ such as heart,
Almost all may occur in which at each organ of human body, tissue, and part cancer is from falling ill until late period is without obvious subjective symptoms, thus
Difficulty is added to early diagnosis.In consideration of it, it is intended that by the early diagnosis that the detection of tumor markers is realized cancer
And treatment.The high-sensitivity detection of tumor markers had higher researching value to detection and the treatment of cancer.
(Freedland S., 2011. Cancer 117,1,123 1135.). but, to single tumor-marker analyte detection due to
Specificity between tumor markers and tumor is relatively low, thus faces huge challenge.(Chikkaveeraiah B.V.,
Bhirde A.A., Morgan N.Y., Eden H.S., Chen X.Y., 2012. ACS Nano 6,6541 6546.)
Such as, carcinoembryonic antigen (CEA) is related with pulmonary carcinoma, cancer of pancreas, hepatocarcinoma, breast carcinoma and colorectal carcinoma, and alpha-fetoprotein (AFP)
Relevant with hepatocarcinoma and ovarian cancer.Some researchs prove that detection Diagnostic Value of Several Serum Tumor Markers can effectively improve detection sensitivity simultaneously
And specificity, and totle drilling cost can be reduced, improve testing efficiency.(Hu M., Yan J., He Y., Lu H., Weng
L., Song S., Fan C., Wang L. ACS Nano 4 (2009) 488 494.) detection side to tumor markers
Method mainly has euzymelinked immunosorbent assay (ELISA) (Wan, Y., Qi P., Zhang D., Wu J.J., Wang Y. 2012.
Biosens.Bioelectron. 33,69 74. and Jia, C.P., Zhong, X.Q., Hua, B., Liu, M.Y.,
Jing, F.X., Lou, X.H., Yao, S.H., Xiang, J.Q., Jin, Q.H., Zhao, J.L. 2009.
Biosens.Bioelectron. 24,2836 2841.), Chemiluminescence immunoassay (Zhang A.M., Xiang H.K.,
Zhang X., Guo W.W., Yuan E.H., Huang C.S., Jia N.Q., 2016. Biosens.
Bioelectron. 75,206 212. and Xu S.J., Liu Y., Wang T.H., and Li J.H. 2011.Anal.
Chem., 83 (10), 3,817 3823.) and fluorescent immune method (Xie, Q.F., Weng X.H., Lu L.J., Lin
Z.Y., Xu X.W., Fu C.L.2015. Biosens. Bioelectron. 77,46 50. and Hua, W.H.,
Liua, Y.S., Yang, H.B., Zhou, X.Q., Li, C.M. 2011. Biosens. Bioelectron.26,
3683 3687.) etc., although these methods have sensitive, accurate, specificity high, but it is in environmental-protection, operation speed
The aspects such as degree, detection by quantitative are respectively arranged with weak point;Despite have been achieved with automatization, but due to instrumentation, special agent
Expensive, the most still can not be in laboratory popularization and application.Thus develop new immunoassay technology, reduce testing cost, add
Fast detection speed and online ability have become problem demanding prompt solution to adapt to extensive examination.In order to meet modern biotechnology detection
Demand, immunosensor arises at the historic moment.It is to be combined with specific immune response by high-sensitive sensing technology, and monitoring is anti-
The biosensor of antigen-antibody reaction, have quick, sensitive, selectivity is high, the advantage such as easy and simple to handle, is widely used in clinical inspection
Survey and diagnosis.Wherein, electrochemical immunosensor, due to its device miniaturization, Simplified analysis process, measured process automation etc.
Particular advantages has become as the powerful method of detection tumor marker.
The structure of nano material is the key issue of electrochemical immunosensor, and nano material has the strongest energy of adsorption
Power, good capacity of orientation and bio-compatibility, and can effectively solve the fixing of sensitive material and regeneration issues, greatly improve
The sensitivity of sensor and stability, can shorten the detection time and realize high flux and detect in real time.
Select preparation 1,1 ' ferrocene dicarboxylic acid/gold@platinum (Fc/Au@Pt) nano-complex and thionine/gold@platinum (Thi/ herein
Au@Pt) nano-complex is used as label and modifies two kind two respectively and resist to prepare electrochemical immunosensor, and for the most right
Two kinds of tumor markers (as a example by first embryo protein AFP and CEA) carry out specific detection.Two kinds of nano-complex materials
The preparation method of material is simple, quick, low cost, and the immunosensor as label has preferable selectivity, stability
And repeatability.
Summary of the invention
The technical problem to be solved is: how to provide one quickly to examine alpha-fetoprotein and carcinoembryonic antigen simultaneously
The nano material surveyed, this material can realize under normal conditions detecting alpha-fetoprotein and carcinoembryonic antigen simultaneously, and have
Relatively low detection limit, and the detection of actual blood serum sample can be used for.
The technical solution adopted in the present invention is: use 1,1 ' ferrocene dicarboxylic acids/gold@platinum and thionine/gold@platinum respectively
Prepare two kind of two anti-conjugate, carry out according to the steps:
Step one, by cobalt nitrate Co (NO3)2·6H2O and polyvinylpyrrolidone PVP is added to the water dissolving, at pure nitrogen gas bar
Vibrate under part 15min, is subsequently adding sodium borohydride NaBH4, stir 30min under nitrogen atmosphere, it is ensured that NaBH4Total overall reaction is complete
Finishing, solution is become dark-brown from colourless, illustrates to define cobalt nano-particle CoNP in solutionS；
Step 2, gold chloride HAuCl4 · 4H2O solution and potassium chloroplatinate K2PtCl6Solution is added dropwise to generate CoNPS?
In the solution of grain, stir 15min so that CoNPSWith HAuCl4、K2PtCl6Fully reaction.Close nitrogen, be centrifuged off Co mould
Plate, i.e. obtains the solution dissolved with Au@Pt nanoshell nuclear material；
Step 3,2-ethylaminoethanol acid solution is joined in prepared Au@Pt nano material aqueous solution, oscillating reactions under room temperature
5h, is subsequently added into 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and N-hydroxy-succinamide NHS solid
Body granule at room temperature reacts 30min, is then added to by 1,1 ' ferrocene dicarboxylic acid FcDC solution in above-mentioned solution under room temperature
Overnight shaking, prepares the solution of Fc/Au@Pt nano-complex；
Step 4, thioglycolic acid solution is joined prepared Au Pt nano material aqueous solution, oscillating reactions 5h under room temperature,
It is subsequently added into 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and N-hydroxy-succinamide NHS solid
Grain at room temperature reacts 30min, then thionine Thi solution is added to overnight at room temperature vibration in above-mentioned solution, prepares Thi/Au@
The solution of Pt nano-complex；
Step 5, by two anti-Ab of AFP and CEA2It is added separately to step 3 and is prepared by step 4
In Fc/Au@Pt nano-complex, Thi/Au@Pt nano-complex, night incubation 12h in the environment of 4 DEG C, to generating product
Centrifugal, after phosphate buffer solution PBS, eccentric cleaning has respectively obtained Fc/Au@Pt/Ab2With Thi/Au@Pt/Ab2Two kinds
Two anti-conjugates.
As a kind of optimal way: in step one, Co (NO3)2·6H2The amount of O is 6.85-8.22a milligram, and PVP is
100-120a milligram, the two is dissolved in the distilled water of 50-60a milliliter simultaneously, NaBH4The amount of solution is 10-12a milliliter, concentration
It is 1 mg/ml, in step 2, HAuCl4 · 4H2The amount of O is 80-96a microlitre, and concentration is 25 mM/ls,
K2PtCl6The amount of solution is 320-385a microlitre, and concentration is 25 mM/ls, step 3, the amount of 2-ethylaminoethanol aqueous acid
For 1-1.2a milliliter, concentration is 100 mM/ls, and the concentration of Au@Pt nano material aqueous solution is 3.5 mM/ls, measures and is
1-1.2a milliliter, the addition of EDC is 19.17-23a milligram, and the amount of NHS is 11.51-13.81a milligram, 1,1 '-ferrocene two
The amount of formic acid is 2-2.4a milliliter, and concentration is 100 mM/ls, and in step 4, the amount of thioglycolic acid solution is 1-1.2a milli
Rising, concentration is 100 mM/ls, and the concentration of Au@Pt nano material aqueous solution is 3.5 mM/ls, measures as 1-1.2 a milli
Rising, the addition of EDC is 19.17-23a milligram, and the amount of NHS is 11.51-13.81a milligram, and the amount of thionine is 2-2.4a milliliter,
Concentration is 100 mM/ls, and in step 5, alpha-fetoprotein two is anti-and the anti-concentration of carcinoembryonic antigen two is all 1 micromoles per liter, adds
Entering amount is all 250-400a microlitre, and the concentration of Fc/Au@Pt nano-complex and Thi/Au@Pt nano-complex is respectively 3 mmoles
You/liter, amount is all 1-1.2a milliliter, and a is positive integer.
1,1 ' ferrocene dicarboxylic acid/gold@platinum and thionine/gold@platinum is used to prepare two kind of two anti-conjugate respectively, for structure
The method that AFP and CEA are detected by the electrochemical immunosensor built simultaneously: will with graphene oxide/
The glass-carbon electrode that Au composite GO/Au modifies is put in the antibody mixed solution of alpha-fetoprotein and carcinoembryonic antigen, hatches 6-at 4 DEG C
12 hours, then take out glass-carbon electrode and be placed in bovine serum albumin BSA to hatch 2-6 hour and close the most specific binding active sites
Point, uses alpha-fetoprotein antigen and the carcinoembryonic antigen mixture modified glassy carbon electrode of variable concentrations successively, makes after taking out glass-carbon electrode
Antigen is combined with antibody specificity, then by the glass-carbon electrode modified at Fc/Au@Pt/Ab2With Thi/Au@Pt/Ab2Two kinds
Hatch at the mixture of two anti-conjugates keeps 25 DEG C 0.5-1 hour, then clean 2-5 time by PBS solution, be built into sandwich
Immunosensor；Sandwich immunosensor is used differential pulse voltammetry DPV detection alpha-fetoprotein and the concentration of carcinoembryonic antigen.
As a kind of optimal way: the glass-carbon electrode carrying out modifying with GO/Au nano-particle refers to use glass-carbon electrode
Al2O3Powder is polished, the most respectively at deionized water, and acetone, ultrasonic cleaning in ethanol, finally GO/Au nano-particle is dripped to glass
On carbon electrode.
As a kind of optimal way: alpha-fetoprotein antigen and carcinoembryonic antigen modified glassy carbon electrode with variable concentrations are successively
Refer to glass-carbon electrode is all respectively 0.01 nanograms/milliliter, 0.05 nanograms/milliliter at alpha-fetoprotein and carcinoembryonic antigen, 0.1 is received successively
Grams per milliliter, 0.5 nanograms/milliliter, 1 nanograms/milliliter, 5 nanograms/milliliter, 10 nanograms/milliliter, 50 nanograms/milliliter, 80 nanograms/in the least
In the mixture risen, at 25 DEG C, hatch 0.5-1 hour.
As a kind of optimal way: sandwich immunosensor is used differential pulse voltammetry DPV detection alpha-fetoprotein and
The concentration of carcinoembryonic antigen mixture refers to, by sandwich immunosensor at hydrogen peroxide H2O2Concentration is 0.25 mM/l, phosphorus
Acid concentration is 0.2 mM/l, PH be 7 solution to be measured in survey to 0.8V-0.6 with the sweep speed of 100 mV/s
Examination, sandwich immunosensor is non-linear adsorption to antigen, and the Concentration Testing scope of alpha-fetoprotein antigen and carcinoembryonic antigen is all divided
Be not 0.01 nanograms/milliliter to 80 nanograms/milliliter, in this range, the logarithm of alpha-fetoprotein concentration is linear with current signal
Dependency, corresponding linear equation is y=-0.79157lg (x)-2.57736, and wherein, x is the concentration of alpha-fetoprotein antigen, single
Position be nanograms/milliliter, y be detection current signal, unit is microampere, can get equally 0.01 nanograms/milliliter to 80 nanograms/
In the detection range of milliliter, the logarithm of carcinoembryonic antigen concentration and the linear dependency of current signal strength, corresponding linear correlation
Equation is y '=-0.78802lg (x ')-3.65705, and wherein, x ' is the concentration of carcinoembryonic antigen, and unit is nanograms/milliliter, and y ' is
The current signal of detection, unit is microampere.
The invention has the beneficial effects as follows: 1,1,1 ' ferrocene dicarboxylic acids/gold@platinum and thionine/two kind of two anti-conjugation of gold@platinum
The preparation of thing mixture, alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) are detected by the electrochemical immunosensor of structure simultaneously
Method.This electrochemical immunosensor has preferable sensitivity, and it is fast to detect speed, and accuracy rate is high, detects alpha-fetoprotein
There is preferable specificity.
Accompanying drawing explanation
Fig. 1 is GO/Au transmission electron microscope (TEM) figure (50nm)；
Fig. 2 is Au@Pt transmission electron microscope (TEM) figure (100nm)；
Fig. 3 is Fc/Au@Pt transmission electron microscope (TEM) figure (100nm)；
Fig. 4 is Thi/Au@Pt transmission electron microscope (TEM) figure (200nm)；
Fig. 5 differential pulse voltammetry DPV detection alpha-fetoprotein and the electric current of the concentration of carcinoembryonic antigen and potential energy diagram；
Fig. 6 uses the electric current of the concentration of differential pulse voltammetry DPV detection alpha-fetoprotein and the concentration logarithmic diagram of alpha-fetoprotein；
Fig. 7 uses the electric current of the concentration of differential pulse voltammetry DPV detection carcinoembryonic antigen and the concentration logarithmic diagram of alpha-fetoprotein.
Detailed description of the invention
1,1 ' ferrocene dicarboxylic acid/gold@platinum and thionine/gold@platinum is used to prepare two kind of two anti-conjugate respectively, for structure
The method that alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) are detected by the electrochemical immunosensor built simultaneously, according to following step
Suddenly carry out
Step one, by 6.85-8.22 milligram cobalt nitrate (Co (NO3)2·6H2And 100-120 milligram polyvinylpyrrolidone O)
(PVP) joining in 50-60 milliliter water and dissolve, vibrate under the conditions of pure nitrogen gas 15min.Being subsequently adding 10-12 milliliter, concentration is 1
Sodium borohydride (the NaBH of mg/ml4), stir 30min under nitrogen atmosphere, it is ensured that NaBH4Total overall reaction is complete, solution by
Colourless become dark-brown, illustrate solution defines cobalt nano-particle (CoNPS).
Step 2, by 80-96 microlitre, concentration is the gold chloride (HAuCl of 25 mM/ls4 · 4H2O) solution and
320-385 microlitre, concentration is the potassium chloroplatinate (K of 25 mM/ls2PtCl6) solution be added dropwise to generate CoNPSSolution
In, stir 15min so that CoNPSWith HAuCl4、K2PtCl6Fully reaction.Close nitrogen, be centrifuged off Co template, i.e. obtain
The solution of Au@Pt nanoshell nuclear material, Fig. 2 is the TEM collection of illustrative plates to Au@Pt nanoshell nuclear material, it appeared that define in figure
Diameter probably has the Au@Pt nanocluster of 100-150nm, and nano-cluster size is homogeneous, good dispersion.
Step 3, by 1-1.2 milliliter, concentration is that the 2-ethylaminoethanol acid solution of 100 mM/ls joins prepared 1-
1.2 milliliters, concentration is the Au@Pt nano material aqueous solution of 3.5 mM/ls, oscillating reactions 5h under room temperature, is centrifuged with secondary water
After cleaning three times soluble in water, be subsequently added into 1-(3-the dimethylamino-propyl)-3-ethyl carbodiimide salt of 19.17-23 milligram
N-hydroxy-succinamide (NHS) solid particle of hydrochlorate (EDC) and 11.51-13.81 milligram at room temperature reacts 30min.So
After 1,1 ' ferrocene dicarboxylic acid (FcDC) is added to overnight at room temperature vibration in above-mentioned solution, prepare Fc/Au@Pt nano combined
The solution of thing, Fig. 3 is the transmission electron microscope picture of Fc/Au@Pt nano-complex, and under the interconnection function of FcDC, nanocluster occurs
Assemble, form bigger nano-cluster.
Step 4, by 1-1.2 milliliter, concentration is that the thioglycolic acid solution of 100 mM/ls joins prepared 1-
1.2 milliliters, concentration is the Au@Pt nano material aqueous solution of 3.5 mM/ls, oscillating reactions 5h under room temperature, is centrifuged with secondary water
Clean three times soluble in water, be subsequently added into 1-(3-the dimethylamino-propyl)-3-ethyl carbodiimide hydrochloride of 19.17-23 milligram
N-hydroxy-succinamide (NHS) solid particle of salt (EDC) and 11.51-13.81 milligram at room temperature reacts 30min.Then
Thionine being added to overnight at room temperature vibration in above-mentioned solution, prepares Thi/Au@Pt nano-complex, Fig. 3 is Thi/Au@Pt nanometer
The transmission electron microscope picture of complex, under the interconnection function of Thi, nanocluster there occurs gathering, forms bigger nano-cluster.
Step 5, by two anti-Ab of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA)2(two anti-Ab2Concentration be all 1 micromole/
Rising, addition is all 250-400 microlitre) it is added separately to step 3 and 1-1.2 milliliter prepared by step 4, concentration is 3 mmoles
You/liter Fc/Au@Pt nano-complex 1-1.2 milliliter, concentration is in the Thi/Au@Pt nano-complex of 3 mM/ls,
Hatch 12h in the environment of 4 DEG C, be centrifuged generating product, after phosphate buffer solution PBS, the centrifugal Fc/Au@Pt/ obtained
Ab2Two anti-conjugates and Thi/Au@Pt/Ab2Two anti-conjugates.
1,1 ' ferrocene dicarboxylic acid/gold@platinum and thionine/gold@platinum is used to prepare two kind of two anti-conjugate respectively, for structure
The method that alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) are detected by the electrochemical immunosensor built simultaneously, first by glass carbon electricity
Pole aluminium sesquioxide Al2O3Powder is polished, the most respectively at deionized water, and acetone, ultrasonic cleaning in ethanol, GO/Au nanometer
Grain be attached on glass-carbon electrode, then the glass-carbon electrode carrying out modifying with GO/Au nano-particle is put into alpha-fetoprotein antibody and
In the antibody mixed solution of carcinoembryonic antigen, hatch 6-12 hour at 4 DEG C, then take out glass-carbon electrode and be placed on bovine serum albumin BSA
In hatch 2-6 hour and close the most specific binding avtive spot, take out after glass-carbon electrode successively with the first tire egg of variable concentrations
Bai Kangyuan and carcinoembryonic antigen modified glassy carbon electrode refer to protect in the alpha-fetoprotein and carcinoembryonic antigen mixture of variable concentrations successively
Hold and at 25 DEG C, hatch 0.5-1 hour, two kinds of antigen concentration in the mixture be all respectively 0.01 nanograms/milliliter, 0.05 nanogram/
Milliliter, 0.1 nanograms/milliliter, 0.5 nanograms/milliliter, 1 nanograms/milliliter, 5 nanograms/milliliter, 10 nanograms/milliliter, 50 nanograms/in the least
Rise, 80 nanograms/milliliter so that antigen is combined with antibody specificity, then by the glass-carbon electrode modified in Fc/Au@Pt nanometer
Complex, Thi/Au@Pt nano-complex Ab2Hatch 0.5-1 hour at the mixture of conjugate keeps 25 DEG C, then use
PBS solution is cleaned 2-5 time, is built into sandwich immunosensor；Sandwich immunosensor use differential pulse voltammetry DPV examine
Survey alpha-fetoprotein and the concentration of carcinoembryonic antigen.(using the differential pulse voltammetry DPV of CHI-660E electrochemical workstation) detection
Alpha-fetoprotein antigen and the concentration of carcinoembryonic antigen；Sandwich immunosensor is used differential pulse voltammetry DPV detection AFP and
The concentration of CEA refers to, by sandwich immunosensor at hydrogen peroxide H2O2Concentration is 0.25 mM/l, phosphoric acid concentration is 0.2
MM/l, PH be 7 solution to be measured in carry out testing (such as Fig. 5)-0.6 to 0.8V with the sweep speed of 100 mV/s, folder
Heart immunosensor is nonlinear adsorption to AFP and CEA, when the concentration of alpha-fetoprotein is at 0-0.1 pg/ml, it was observed that blank
The current signal of buffer approximates-1 microampere, and when the concentration of alpha-fetoprotein is in 0.01 nanograms/milliliter, the current signal obtained is opened
Beginning more than-1 microampere, along with the increase of alpha-fetoprotein concentration, current signal is also being gradually increased, the concentration inspection of alpha-fetoprotein antigen
Survey scope be 0.01 nanograms/milliliter to 80 nanograms/milliliter, as shown in Figure 6, in this range, the logarithm of alpha-fetoprotein concentration with
The linear dependency of current signal, its linearly dependent coefficient square is 0.994, and corresponding linear equation is y=-0.79157lg
X ()-2.57736, wherein, x is the concentration of alpha-fetoprotein antigen, and unit is nanograms/milliliter, and y is the current signal of detection, unit
It it is microampere.In like manner, when the concentration of carcinoembryonic antigen is at 0-0.1 pg/ml, it was observed that the current signal approximation-2 of plain buffer
Microampere, when the concentration of alpha-fetoprotein is in 0.01 nanograms/milliliter, the current signal obtained starts more than-2 microamperes, available cancer
The detection range of embryonal antigen concentration is 0.01 nanograms/milliliter to 80 nanograms/milliliter, as it is shown in fig. 7, the logarithm of carcinoembryonic antigen concentration
Being y '=-0.78802lg (x ')-3.65705 with the linear relationship of current signal, linearly dependent coefficient square is 0.993, first
The lowest detectable limit of fetoprotein and carcinoembryonic antigen is respectively 2.6 pg/ml and 3.2 pg/ml(Signal to noise ratio is 3), with it
Its detection method is compared, and the electrochemical immunosensor of structure has relatively low detection limit and wider detection range (Fig. 6 and Tu
In 7, R is the linearly dependent coefficient of linear regression, R2Be linearly dependent coefficient square, n represent be experiment number).
Alpha-fetoprotein antigen concentration is as shown in the table with the corresponding relation of current signal
Carcinoembryonic antigen concentration is as shown in the table with the corresponding relation of current signal
In order to detect selectivity and the specificity of targeted immune sensor in this experiment, we have employed recovery experiment and test
Card, is simultaneously introduced CEA and AFP of variable concentrations in hyclone sample, by the response rate of CEA and AFP in detection sample
With selectivity and the specificity of detection targeted immune sensor, testing result is as shown in the table:
Sequence number standard volume (ng/mL) yield (ng/mL) response rate (%)
CEA AFP CEA AFP CEA AFP
1 60 60 57.04 65.11 95 108
2 30 30 29.73 27.84 99.1 92.8
3 10 10 9.45 9.83 94.5 98.3
4 5 5 4.82 5.34 96.4 107
5 1 1 0.988 0.965 98.8 96.5
6 0.5 0.5 0.492 0.493 98.4 98.5
7 0.1 0.1 0.0968 0.0952 96.8 95.2
8 0.05 0.05 0.0502 0.0485 100.3 97
9 0.01 0.01 0.0091 0.0011 91.3 106
By upper table it can be seen that hyclone adds variable concentrations CEA and AFP antigen, the response rate of CEA 94.5%～
The zone of reasonableness of 100.3%, illustrates that other albumen in serum measure interference to CEA the least, and the response rate of AFP is 92.8～108
In the range of, this targeted immune sensor is preferable to two kinds of tumor markers selectivity specificitys.
Specificity
In order to verify the specificity of immunosensor, CEA and the AFP antigen mixture of 1ng/mL and the interfering material of 100ng/mL
(ascorbic acid, bovine serum albumin, glucose, carbamide, prostate specific antigen) mixes, and detects electrochemical signals,
Finding to compare with the antigen mixture of CEA and AFP of pure 1ng/mL, the current intensity change that interfering material causes, less than 3%, is demonstrate,proved
Bright 1,1 ' ferrocene dicarboxylic acids/gold@platinum (Fc/Au@Pt) nano-complex and thionine/gold@platinum (Thi/Au@Pt) are nano combined
The electrochemical immunosensor that thing builds has stronger specificity to alpha-fetoprotein and carcinoembryonic antigen.
Claims (6)
1. using 1,1 ' ferrocene dicarboxylic acid/gold@platinum and thionine/gold@platinum to prepare two kind of two anti-conjugate respectively, its feature exists
In carrying out according to the steps:
Step one, by cobalt nitrate Co (NO3)2·6H2O and polyvinylpyrrolidone PVP is added to the water dissolving, at pure nitrogen gas bar
Vibrate under part 15min, is subsequently adding sodium borohydride NaBH4, stir 30min under nitrogen atmosphere, it is ensured that NaBH4Total overall reaction is complete
Finishing, solution is become dark-brown from colourless, illustrates to define cobalt nano-particle CoNP in solutionS；
Step 2, gold chloride HAuCl4·4H2O solution and potassium chloroplatinate K2PtCl6Solution is added dropwise to generate cobalt nano-particle
CoNPSSolution in, stir 15min so that cobalt nano-particle CoNPSWith gold chloride HAuCl4, potassium chloroplatinate K2PtCl6The most anti-
Should, close nitrogen, be centrifuged off cobalt Co template, i.e. obtain the solution dissolved with Au@Pt nanoshell nuclear material；
Step 3,2-ethylaminoethanol acid solution is joined in prepared Au@Pt nano material aqueous solution, oscillating reactions under room temperature
5h, is subsequently added into 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and N-hydroxy-succinamide NHS solid
Body granule at room temperature reacts 30min, is then added to by 1,1 ' ferrocene dicarboxylic acid FcDC solution in above-mentioned solution under room temperature
Overnight shaking, prepares the solution of Fc/Au@Pt nano-complex；
Step 4, thioglycolic acid solution is joined prepared Au Pt nano material aqueous solution, oscillating reactions 5h under room temperature,
It is subsequently added into 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC and N-hydroxy-succinamide NHS solid
Grain at room temperature reacts 30min, then thionine Thi solution is added to overnight at room temperature vibration in above-mentioned solution, prepares Thi/Au@
The solution of Pt nano-complex；
Step 5, by two anti-Ab of AFP and CEA2It is added separately to step 3 and is prepared by step 4
In Fc/Au@Pt nano-complex, Thi/Au@Pt nano-complex, night incubation 12h in the environment of 4 DEG C, to generating product
Centrifugal, after phosphate buffer solution PBS, eccentric cleaning has respectively obtained Fc/Au@Pt/Ab2With Thi/Au@Pt/Ab2Two kinds
Two anti-conjugates.
Use 1,1 ' ferrocene dicarboxylic acid/gold@platinum the most according to claim 1 and thionine/gold@platinum prepare two kinds respectively
Two anti-conjugates, it is characterised in that: in step one, Co (NO3)2·6H2The amount of O is 6.85-8.22a milligram, and PVP is 100-
120a milligram, the two is dissolved in the distilled water of 50-60a milliliter simultaneously, NaBH4The amount of solution is 10-12a milliliter, and concentration is 1 milli
Grams per milliliter, in step 2, HAuCl4·4H2The amount of O is 80-96a microlitre, and concentration is 25 mM/ls, K2PtCl6Solution
Amount is 320-385a microlitre, and concentration is 25 mM/ls, step 3, and the amount of 2-ethylaminoethanol aqueous acid is 1-1.2a milliliter,
Concentration is 100 mM/ls, and the concentration of Au@Pt nano material aqueous solution is 3.5 mM/ls, measures as 1-1.2a milliliter, EDC
Addition be 19.17-23a milligram, the amount of NHS is 11.51-13.81a milligram, 1, the amount of 1 '-ferrocene dicarboxylic acid is 2-
2.4a milliliter, concentration is 100 mM/ls, and in step 4, the amount of thioglycolic acid solution is 1-1.2a milliliter, and concentration is 100
MM/l, the concentration of Au@Pt nano material aqueous solution is 3.5 mM/ls, measures as 1-1.2 a milliliter, the addition of EDC
Amount is 19.17-23a milligram, and the amount of NHS is 11.51-13.81a milligram, and the amount of thionine is 2-2.4a milliliter, and concentration is 100 millis
Mol/L, in step 5, alpha-fetoprotein two is anti-and the anti-concentration of carcinoembryonic antigen two is all 1 micromoles per liter, and addition is all
250-400a microlitre, the concentration of Fc/Au@Pt nano-complex and Thi/Au@Pt nano-complex is respectively 3 mM/ls, amount
Being all 1-1.2a milliliter, a is positive integer.
3. utilize claim 1 to use 1,1 ' ferrocene dicarboxylic acids/gold platinum and thionine/gold platinum to prepare two kind two respectively to resist altogether
Yoke thing, the method that AFP and CEA are detected by the electrochemical immunosensor for building simultaneously, it is special
Levy and be: the glass-carbon electrode modified with graphene oxide/Au composite GO/Au is put into the antibody of alpha-fetoprotein and carcinoembryonic antigen
In mixed solution, hatch 6-12 hour at 4 DEG C, then take out glass-carbon electrode and be placed in bovine serum albumin BSA and hatch 2-6 hour envelope
Close the most specific binding avtive spot, after taking out glass-carbon electrode, use alpha-fetoprotein antigen and the carcinoembryonic antigen of variable concentrations successively
Mixture modified glassy carbon electrode so that antigen is combined with antibody specificity, then by the glass-carbon electrode modified at Fc/Au@Pt/
Ab2With Thi/Au@Pt/Ab2Hatch at the mixture of two kind of two anti-conjugate keeps 25 DEG C 0.5-1 hour, then molten with PBS
Liquid cleans 2-5 time, is built into sandwich immunosensor；Sandwich immunosensor use differential pulse voltammetry DPV detect first
Fetoprotein and the concentration of carcinoembryonic antigen.
Use 1,1 ' ferrocene dicarboxylic acid/gold@platinum the most according to claim 3 and thionine/gold@platinum prepare two kinds respectively
Two anti-conjugates, the side that AFP and CEA are detected by the electrochemical immunosensor for building simultaneously
Method, it is characterised in that: the glass-carbon electrode carrying out modifying with GO/Au nano-particle refers to glass-carbon electrode Al2O3Powder is polished,
The most respectively at deionized water, acetone, ultrasonic cleaning in ethanol, finally GO/Au nano-particle is dripped on glass-carbon electrode.
Use 1,1 ' ferrocene dicarboxylic acid/gold@platinum the most according to claim 3 and thionine/gold@platinum prepare two kinds respectively
Two anti-conjugates, the side that AFP and carcinoembryonic antigen (CEA) are detected by the electrochemical immunosensor for building simultaneously
Method, it is characterised in that: alpha-fetoprotein antigen and carcinoembryonic antigen modified glassy carbon electrode with variable concentrations refer to glass carbon electricity successively
Pole successively alpha-fetoprotein and carcinoembryonic antigen be all respectively 0.01 nanograms/milliliter, 0.05 nanograms/milliliter, 0.1 nanograms/milliliter,
0.5 nanograms/milliliter, 1 nanograms/milliliter, 5 nanograms/milliliter, 10 nanograms/milliliter, 50 nanograms/milliliter, the mixing of 80 nanograms/milliliter
In thing, at 25 DEG C, hatch 0.5-1 hour.
Utilization use 1,1 ' ferrocene dicarboxylic acid/gold@platinum the most according to claim 3 and thionine/gold@platinum are prepared respectively
Two kind of two anti-conjugate, AFP and CEA are detected by the electrochemical immunosensor for building simultaneously
Method, it is characterised in that: sandwich immunosensor use differential pulse voltammetry DPV detection alpha-fetoprotein and carcinoembryonic antigen mix
The concentration of compound refers to, by sandwich immunosensor at hydrogen peroxide H2O2Concentration is 0.25 mM/l, phosphoric acid concentration is
0.2 mM/l, PH be 7 solution to be measured in test to 0.8V-0.6 with the sweep speed of 100 mV/s, sandwich exempt from
Epidemic disease sensor is non-linear adsorption to antigen, and the Concentration Testing scope of alpha-fetoprotein antigen and carcinoembryonic antigen is 0.01 to receive the most respectively
Grams per milliliter is to 80 nanograms/milliliter, in this range, and the logarithm of alpha-fetoprotein concentration and the linear dependency of current signal are corresponding
Linear equation be y=-0.79157lg (x)-2.57736, wherein, x is the concentration of alpha-fetoprotein antigen, and unit is nanogram/in the least
Rising, y is the current signal of detection, and unit is microampere, can get the detection in 0.01 nanograms/milliliter to 80 nanograms/milliliter equally
In the range of, the logarithm of carcinoembryonic antigen concentration and the linear dependency of current signal strength, corresponding linear correlation equation be y '=-
0.78802lg (x) '-3.65705, wherein, x ' is the concentration of carcinoembryonic antigen, and unit is nanograms/milliliter, and y ' is the electric current of detection
Signal, unit is microampere.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107677719A (en) * | 2017-09-07 | 2018-02-09 | 桂林电子科技大学 | A kind of method based on graphene, thionine and aptamer detection alpha-fetoprotein |
| CN110082415A (en) * | 2019-05-30 | 2019-08-02 | 吉林大学 | A kind of optical electro-chemistry detection probe, preparation method and applications based on conjugated polymer nanoparticle |
| CN110702759A (en) * | 2019-10-08 | 2020-01-17 | 西北大学 | ZIF-8 composite material electrochemical immunosensor for detecting alpha fetoprotein and preparation method and application thereof |
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| CN113325179A (en) * | 2021-04-14 | 2021-08-31 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Immunochromatographic test strip based on Au @ Pt enzyme and preparation method thereof |
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| CN102507953B (en) * | 2011-10-20 | 2013-04-17 | 济南大学 | Preparation method of electrochemistry immunosensor for determining alpha fetoprotein |
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| CN103245656B (en) * | 2013-04-25 | 2014-01-08 | 济南大学 | Preparation and application of alpha fetoprotein and carcino-embryonic antigen electrochemiluminescence sensor |
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| CN107677719A (en) * | 2017-09-07 | 2018-02-09 | 桂林电子科技大学 | A kind of method based on graphene, thionine and aptamer detection alpha-fetoprotein |
| CN107677719B (en) * | 2017-09-07 | 2019-10-18 | 桂林电子科技大学 | A method of alpha-fetoprotein is detected based on graphene, thionine and aptamer |
| CN110082415A (en) * | 2019-05-30 | 2019-08-02 | 吉林大学 | A kind of optical electro-chemistry detection probe, preparation method and applications based on conjugated polymer nanoparticle |
| CN110702759A (en) * | 2019-10-08 | 2020-01-17 | 西北大学 | ZIF-8 composite material electrochemical immunosensor for detecting alpha fetoprotein and preparation method and application thereof |
| CN110702759B (en) * | 2019-10-08 | 2021-04-27 | 西北大学 | ZIF-8 composite material electrochemical immunosensor for detecting alpha fetoprotein and preparation method and application thereof |
| CN112379098A (en) * | 2020-11-05 | 2021-02-19 | 集美大学 | ELISA detection method for mimic enzyme-labeled antibody of histamine content in aquatic product |
| CN113325179A (en) * | 2021-04-14 | 2021-08-31 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Immunochromatographic test strip based on Au @ Pt enzyme and preparation method thereof |
| CN113325179B (en) * | 2021-04-14 | 2024-02-27 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Immunochromatography test strip based on Au@Pt enzyme and preparation method thereof |
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