CN105044083A - Method for preparing electrochemiluminescence immunosensor for alpha fetoprotein based on Au-g-C3N4 nanocomposite and application - Google Patents
Method for preparing electrochemiluminescence immunosensor for alpha fetoprotein based on Au-g-C3N4 nanocomposite and application Download PDFInfo
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Abstract
The invention relates to the technical field of electrochemiluminescence immunosensors, in particular to a method for preparing an electrochemiluminescence immunosensor for alpha fetoprotein based on an Au-g-C3N4 nanocomposite and application. A dispensing method is adopted to modify the Au-g-C3N4 nanocomposite to the surface of an electrode to serve as a luminescent material and an antibody capturing substrate, and the excellent electrical conductivity and catalytic performances of gold nanoparticles can effectively improve light-emitting signals of the sensor. Based on good specificity between antigen-antibodies, the sensor is used for detecting alpha fetoproteins, and the alpha fetoproteins are detected according to the difference of the electrochemiluminescence strength of the alpha fetoproteins with different concentrations.
Description
Technical field
The present invention relates to electrochemiluminescimmunosensor immunosensor technical field, particularly relate to a kind of based on Au-g-C
3n
4the preparation method of the electrochemiluminescimmunosensor immunosensor of the alpha-fetoprotein of nano composite material and application.Be specifically related to a kind of g-C
3n
4nanometer sheet (g-C
3n
4nSs) as luminescent material, have the preparations and applicatio of the electrochemiluminescimmunosensor immunosensor of conduction and catalytic action concurrently using Au as antibody capture substrate simultaneously.
Background technology
Alpha-fetoprotein (AFP) is a kind of liver cancer marker, in normal human serum, the content of AFP is not higher than 20 μ g/L, but when liver cell generation canceration, its content in serum will sharply increase, and a kind of method therefore developing easy, quick, high-sensitive detection AFP is to the early diagnosis of liver cancer and differentiate significant.
The immunization method of current mensuration AFP has a lot, as enzyme-linked immuno assay, and fluoroimmunoassay, radiommunoassay etc.Wherein enzyme-linked immuno assay and fluoroimmunoassay cost higher, and there is the problem that kit is difficult to preserve, and the reappearance of radioimmunoassay is poor.It is simple that electrochemiluminescimmunosensor immunosensor has equipment, and easy and simple to handle, highly sensitive, selectivity is good, and antijamming capability is strong, low cost and other advantages.Therefore the present invention has prepared one based on (Au-g-C
3n
4nano composite material) Au-g-C
3n
4the electrochemiluminescimmunosensor immunosensor of the alpha-fetoprotein of NHs, combines immunological method with electrochemiluminescdetection detection method, realizes the detection to AFP by the specific binding of height between antigen-antibody.
The present invention utilizes drop-coating by Au-g-C
3n
4nHs modifies glassy carbon electrode surface, due to g-C
3n
4the film forming that NSs is good, therefore without the need to carrying out other process to electrode and decorative material.At this, the g-C in compound
3n
4nSs is as luminescent material, and Au, as antibody capture substrate, also plays simultaneously and promotes that electron transmission and catalytic action effectively enhance electrogenerated chemiluminescence intensity.The method creates good electrogenerated chemiluminescence intensity in testing process, may be used for the analysis of AFP.It is low that the electrochemiluminescimmunosensor immunosensor built in the present invention has cost, good stability, and preparation process is simple, and sensitivity advantages of higher, effectively overcomes the deficiency of current AFP detection method.
Summary of the invention
An object of the present invention is with Au-g-C
3n
4nHs is base material, constructs a kind of simple and quick, good stability, highly sensitive electrochemiluminescimmunosensor immunosensor.
Two of object of the present invention is the detections this electrochemiluminescimmunosensor immunosensor being used for AFP.
Technical scheme of the present invention is:
1. one kind based on Au-g-C
3n
4the preparation method of the electrochemiluminescimmunosensor immunosensor of the alpha-fetoprotein of nano composite material
(1) with the Al of 1.0 μm, 0.3 μm, 0.05 μm
2o
3burnishing powder diameter of polishing successively is the glass-carbon electrode (GCE) of 4mm, and ultrasonic cleaning 3min in ethanol and ultrapure water respectively, nitrogen dries up, and gets 6 ~ 10 μ LAu-g-C
3n
4nHs dispersant liquid drop is coated onto electrode surface, dries film forming under room temperature, rinses with PBS (pH7.4) buffer solution with the Au-g-C removing non-bonding
3n
4nHs obtains Au-g-C
3n
4nHs/GCE;
(2) alpha-fetoprotein antibody (anti-AFP) standard solution getting 50 μ L1.0 ~ 2.5 μ g/mL drips and is coated onto electrode surface, in 4 DEG C of refrigerators, hatch 12h, rinse removing physisorption with PBS (pH7.4) buffer solution and obtain anti-AFP/Au-g-C
3n
4nHs/GCE;
(3) get 50 μ L massfractions be 1 ~ 3% bovine serum albumin(BSA) (BSA) solution drip and be coated onto electrode surface, 1h is hatched at 37 DEG C, with closed nonspecific binding site, rinse electrode surface with PBS (pH7.4) buffer solution and obtain BSA/anti-AFP/Au-g-C
3n
4nHs/GCE;
(4) drip 50 μ L concentration be a series of variable concentrations of 0.001 ~ 5ng/mL alpha-fetoprotein antigen standard solution be used for and antibody specific recognition, 60min is hatched at 37 DEG C, electrode surface is rinsed with PBS (pH7.4) buffer solution, obtained a kind of based on Au-g-C
3n
4electrochemiluminescimmunosensor immunosensor (the AFP/BSA/anti-AFP/Au-g-C of the alpha-fetoprotein of NHs
3n
4nHs/GCE).
2. above-mentioned Au-g-C
3n
4the preparation of NHs dispersion liquid
(1) g-C
3n
4the preparation of NSs
Take 10g urea in crucible in muffle furnace with 50 DEG C/min temperature programme to 550 DEG C, calcining at constant temperature 2.5h obtains pale yellow powder g-C
3n
4.Take obtained g-C
3n
4100mg, in conical flask, adds 100mL ultrapure water, then ultrasonic 16h, and gained solution is centrifugal under 6000r/s, removes the g-C do not disperseed
3n
4, collect supernatant, steam at 60 DEG C of backspins and obtain milky g-C
3n
4nSs dispersion liquid;
(2) preparation of gold nano solution A u
Measure the ultrapure water of 97mL in conical flask, under agitation add the HAuCl of 1mL0.01mol/L
4with the 0.03mol/L disodium citrate of 1mL, sodium borohydride to the solution dropwise dripping 1mL0.047mol/L afterwards becomes bright claret, then stirs the gold nano solution A u of 15min;
(3) Au-g-C
3n
4the preparation of NHs dispersion liquid
The gold nano solution A u pipetting 5mL joins 5mL at stirring condition and obtains g-C
3n
4in NSs dispersion liquid, strong stirring 60min, dilutes with 1mL ultrapure water water and obtains Au-g-C again after centrifuge washing
3n
4nHs dispersion liquid.
The detection of 3.AFP
(1) with prepared immunosensor for working electrode, Ag/AgCl electrode is contrast electrode, and platinum electrode is to electrode; Use MPI-B type multiparameter chemiluminescence analysis test macro to test, the high pressure of photomultiplier is set to 600V, and sweep interval is-1.1 ~ 0V, and sweep velocity is 100mV/s;
(2) in the electrolytic cell of 10mLPBS (pH7.4) buffer solution containing 0.1mol/L potassium persulfate, the electrogenerated chemiluminescence intensity of the electrode of 0.001 ~ 5ng/mL a series of variable concentrations AFP standard solution and non-specific binding AFP is detected, drawing curve by MPI-B type multiparameter chemiluminescence analysis test macro;
(3) AFP standard solution is replaced by testing sample solution to detect.
Useful achievement of the present invention
(1) the present invention is with g-C
3n
4nSs is luminescent material, and the electrochemiluminescimmunosensor immunosensor utilizing the optical property of its excellence to build has higher luminous signal;
(2) the present invention does not need to introduce any link agent, using Au as antibody capture substrate, can be combined with antibody on the one hand, antibody is fixed on electrode surface, the opposing party promotes that electron transmission and catalysis potassium persulfate produce more electron hole, effectively enhances electrogenerated chemiluminescence intensity;
(3) the present invention base material Au-g-C used
3n
4nHs has good film forming, without the need to doing special processing, greatly simplify electrode production process;
(4) electrochemiluminescimmunosensor immunosensor prepared of the present invention is for the detection of AFP, and simple to operate, the range of linearity is wide, and detection limit is low, can realize simple, quick, the highly sensitive detection to AFP.The range of linearity is 0.001 ~ 5ng/mL, detects and is limited to 0.0005ng/mL.
accompanying drawing illustrates:
Fig. 1 is the electrogenerated chemiluminescence intensity map of variable concentrations AFP;
Fig. 2 is relative electrogenerated chemiluminescence intensity and the lg of variable concentrations AFP
clinear Fit Chart.
Wherein, the concentration representing AFP respectively by the electrogenerated chemiluminescence intensity map of a to i in Fig. 1 is 0,0.001,0.005,0.01,0.05,0.1,0.5,1,5ng/mL.
embodiment:
In order to understand the present invention better, describe technical scheme of the present invention in detail with instantiation below, but the present invention is not limited thereto.
Embodiment 1 one kinds is based on Au-g-C
3n
4the preparation method of the electrochemiluminescimmunosensor immunosensor of the alpha-fetoprotein of NHs
(1) with the Al of 1.0 μm, 0.3 μm, 0.05 μm
2o
3burnishing powder diameter of polishing successively is the GCE of 4mm, and ultrasonic cleaning 3min in ethanol and ultrapure water respectively, nitrogen dries up, and gets 6 μ LAu-g-C
3n
4nHs dispersant liquid drop is coated onto electrode surface, dries film forming under room temperature, rinses with PBS (pH7.4) buffer solution with the Au-g-C removing non-bonding
3n
4nHs obtains Au-g-C
3n
4nHs/GCE;
(2) the anti-AFP standard solution getting 50 μ L1 μ g/mL drips and is coated onto electrode surface, in 4 DEG C of refrigerators, hatch 12h, rinses removing physisorption obtain anti-AFP/Au-g-C with PBS (pH7.4) buffer solution
3n
4nHs/GCE;
(3) get 50 μ L massfractions be 1% BSA solution drip and be coated onto electrode surface, at 37 DEG C, hatch 1h, with closed nonspecific binding site, rinse electrode surface with PBS (pH7.4) buffer solution and obtain BSA/anti-AFP/Au-g-C
3n
4nHs/GCE;
(4) drip 50 μ L concentration be a series of variable concentrations of 0.001 ~ 5ng/mL alpha-fetoprotein antigen standard solution be used for and antibody specific recognition, 60min is hatched at 37 DEG C, electrode surface is rinsed with PBS (pH7.4) buffer solution, obtained a kind of based on Au-g-C
3n
4electrochemiluminescimmunosensor immunosensor (the AFP/BSA/anti-AFP/Au-g-C of the AFP of NHs
3n
4nHs/GCE).
Embodiment 2 one kinds is based on Au-g-C
3n
4the preparation method of the electrochemiluminescimmunosensor immunosensor of the alpha-fetoprotein of NHs
(1) with the Al of 1.0 μm, 0.3 μm, 0.05 μm
2o
3burnishing powder diameter of polishing successively is the GCE pole of 4mm, and ultrasonic cleaning 3min in ethanol and ultrapure water respectively, nitrogen dries up, and gets 8 μ LAu-g-C
3n
4nHs dispersant liquid drop is coated onto electrode surface, dries film forming under room temperature, rinses with PBS (pH7.4) buffer solution with the Au-g-C removing non-bonding
3n
4nHs obtains Au-g-C
3n
4nHs/GCE;
(2) the anti-AFP standard solution getting 50 μ L2.0 μ g/mL drips and is coated onto electrode surface, in 4 DEG C of refrigerators, hatch 12h, rinses removing physisorption obtain anti-AFP/Au-g-C with PBS (pH7.4) buffer solution
3n
4nHs/GCE;
(3) get 50 μ L massfractions be 2% BSA solution drip and be coated onto electrode surface, at 37 DEG C, hatch 1h, with closed nonspecific binding site, rinse electrode surface with PBS (pH7.4) buffer solution and obtain BSA/anti-AFP/Au-g-C
3n
4nHs/GCE;
(4) drip 50 μ L concentration be a series of variable concentrations of 0.001 ~ 5ng/mL alpha-fetoprotein antigen standard solution be used for and antibody specific recognition, 50min is hatched at 37 DEG C, electrode surface is rinsed with PBS (pH7.4) buffer solution, obtained a kind of based on Au-g-C
3n
4electrochemiluminescimmunosensor immunosensor (the AFP/BSA/anti-AFP/Au-g-C of the AFP of NHs
3n
4nHs/GCE).
Embodiment 3 one kinds is based on Au-g-C
3n
4the preparation method of the electrochemiluminescimmunosensor immunosensor of the alpha-fetoprotein of NHs
(1) with the Al of 1.0 μm, 0.3 μm, 0.05 μm
2o
3burnishing powder diameter of polishing successively is the GCE of 4mm, and ultrasonic cleaning 3min in ethanol and ultrapure water respectively, nitrogen dries up, and gets 10 μ LAu-g-C
3n
4nHs dispersant liquid drop is coated onto electrode surface, dries film forming under room temperature, rinses with PBS (pH7.4) buffer solution with the Au-g-C removing non-bonding
3n
4nHs obtains Au-g-C
3n
4nHs/GCE;
(2) the anti-AFP standard solution getting 50 μ L2.5 μ g/mL drips and is coated onto electrode surface, in 4 DEG C of refrigerators, hatch 12h, rinses removing physisorption obtain anti-AFP/Au-g-C with PBS (pH7.4) buffer solution
3n
4nHs/GCE;
(3) get 50 μ L massfractions be 3% BSA solution drip and be coated onto electrode surface, at 37 DEG C, hatch 1h, with closed nonspecific binding site, rinse electrode surface with PBS (pH7.4) buffer solution and obtain BSA/anti-AFP/Au-g-C
3n
4nHs/GCE;
(4) drip 50 μ L concentration be a series of variable concentrations of 0.001 ~ 5ng/mL alpha-fetoprotein antigen standard solution be used for and antibody specific recognition, 60min is hatched at 37 DEG C, electrode surface is rinsed with PBS (pH7.4) buffer solution, obtained a kind of based on Au-g-C
3n
4electrochemiluminescimmunosensor immunosensor (the AFP/BSA/anti-AFP/Au-g-C of the alpha-fetoprotein of NHs
3n
4nHs/GCE).
The above-mentioned Au-g-C of embodiment 4
3n
4the preparation of NHs dispersion liquid
(1) g-C
3n
4the preparation of NSs
Take 10g urea in crucible in muffle furnace with 50 DEG C/min temperature programme to 550 DEG C, calcining at constant temperature 2.5h obtains pale yellow powder g-C
3n
4.Take obtained g-C
3n
4100mg, in conical flask, adds 100mL ultrapure water, then ultrasonic 16h, and gained solution is centrifugal under 6000r/s, removes the g-C do not disperseed
3n
4, collect supernatant, steam at 60 DEG C of backspins and obtain milky g-C
3n
4nSs dispersion liquid;
(2) preparation of gold nano solution A u
Measure the ultrapure water of 97mL in conical flask, under agitation add the HAuCl of 1mL0.01mol/L
4with the 0.03mol/L disodium citrate of 1mL, sodium borohydride to the solution dropwise dripping 1mL0.047mol/L afterwards becomes bright claret, then stirs the gold nano solution A u of 15min;
(3) Au-g-C
3n
4the preparation of NHs dispersion liquid
The gold nano solution A u pipetting 5mL joins 5mL at stirring condition and obtains g-C
3n
4in NSs dispersion liquid, strong stirring 60min, dilutes with 1mL ultrapure water water and obtains Au-g-C again after centrifuge washing
3n
4nHs dispersion liquid.
The detection of embodiment 5AFP
(1) with prepared immunosensor for working electrode, Ag/AgCl electrode is contrast electrode, and platinum electrode is to electrode; Use MPI-B type multiparameter chemiluminescence analysis test macro to test, the high pressure of photomultiplier is set to 600V, and sweep interval is-1.1 ~ 0V, and sweep velocity is 100mV/s;
(2) in the electrolytic cell of 10mLPBS (pH7.4) buffer solution containing 0.1mol/L potassium persulfate, detected by MPI-B type multiparameter chemiluminescence analysis test macro the electrode of 0.001 ~ 5ng/mL a series of variable concentrations AFP standard solution electrogenerated chemiluminescence intensity (
i) and non-specific binding AFP electrode electrogenerated chemiluminescence intensity (
i 0), the results are shown in Figure 1, according to the electrogenerated chemiluminescence intensity of gained and the relation of AFP concentration, drawing curve;
(3) obtain relative electrogenerated chemiluminescence intensity (
i 0-
i)/
i 0with the logarithm (lg of AFP concentration
c) linear relationship see Fig. 2, as shown in Figure 2, AFP in the concentration range of 0.001 ~ 5ng/mL, (
i 0-
i)/
i 0with lg
cpresent good linear correlation, linear equation be (
i 0-
i)/
i 0=0.18579lg
c+ 2.29231, linearly dependent coefficient is 0.9936, detects and is limited to 0.0005ng/mL;
(4) AFP standard solution is replaced by testing sample solution to detect.
Claims (3)
1. one kind based on Au-g-C
3n
4the preparation method of the electrochemiluminescimmunosensor immunosensor of the alpha-fetoprotein of nano composite material, is characterized in that, comprises the following steps:
(1) with the Al of 1.0 μm, 0.3 μm, 0.05 μm
2o
3burnishing powder diameter of polishing successively is the glass-carbon electrode (GCE) of 4mm, and ultrasonic cleaning 3min in ethanol and ultrapure water respectively, nitrogen dries up, and gets 6 ~ 10 μ LAu-g-C
3n
4nano composite material (Au-g-C
3n
4nHs) dispersant liquid drop is coated onto electrode surface, dries film forming under room temperature, rinses with PBS (pH7.4) buffer solution with the Au-g-C removing non-bonding
3n
4nHs obtains Au-g-C
3n
4nHs/GCE;
(2) alpha-fetoprotein antibody (anti-AFP) standard solution getting 50 μ L1 ~ 2.5 μ g/mL drips and is coated onto electrode surface, in 4 DEG C of refrigerators, hatch 12h, rinse removing physisorption with PBS (pH7.4) buffer solution and obtain anti-AFP/Au-g-C
3n
4nHs/GCE;
(3) get 50 μ L massfractions be 1 ~ 3% bovine serum albumin(BSA) (BSA) solution drip and be coated onto electrode surface, 1h is hatched at 37 DEG C, with closed nonspecific binding site, rinse electrode surface with PBS (pH7.4) buffer solution and obtain BSA/anti-AFP/Au-g-C
3n
4nHs/GCE;
(4) drip 50 μ L concentration be a series of variable concentrations of 0.001 ~ 5ng/mL alpha-fetoprotein antigen standard solution be used for and antibody specific recognition, 60min is hatched at 37 DEG C, electrode surface is rinsed with PBS (pH7.4) buffer solution, obtained a kind of based on Au-g-C
3n
4electrochemiluminescimmunosensor immunosensor (the AFP/BSA/anti-AFP/Au-g-C of the alpha-fetoprotein of NHs
3n
4nHs/GCE).
2. one according to claim 1 is based on Au-g-C
3n
4the preparation method of the electrochemiluminescimmunosensor immunosensor of the alpha-fetoprotein of NHs, described Au-g-C
3n
4nHs dispersion liquid, is characterized in that, making step is as follows:
(1) g-C
3n
4nanometer sheet (g-C
3n
4nSs) preparation
Take 10g urea in crucible in muffle furnace with 50 DEG C/min temperature programme to 550 DEG C, calcining at constant temperature 2.5h obtains pale yellow powder g-C
3n
4;
Take obtained g-C
3n
4100mg, in conical flask, adds 100mL ultrapure water, then ultrasonic 16h, and gained solution is centrifugal under 6000r/s, removes the g-C do not disperseed
3n
4, collect supernatant, steam at 60 DEG C of backspins and obtain milky g-C
3n
4nSs dispersion liquid;
(2) preparation of gold nano solution A u
Measure the ultrapure water of 97mL in conical flask, under agitation add the HAuCl of 1mL0.01mol/L
4with the 0.03mol/L disodium citrate of 1mL, sodium borohydride to the solution dropwise dripping 1mL0.047mol/L afterwards becomes bright claret, then stirs the gold nano solution A u of 15min;
(3) Au-g-C
3n
4the preparation of NHs dispersion liquid
The gold nano solution A u pipetting 5mL joins 5mL at stirring condition and obtains g-C
3n
4in NSs dispersion liquid, strong stirring 60min, dilutes with 1mL ultrapure water water and obtains Au-g-C again after centrifuge washing
3n
4nHs dispersion liquid.
3. the one prepared of preparation method according to claim 1 is based on Au-g-C
3n
4the electrochemiluminescimmunosensor immunosensor of the alpha-fetoprotein of NHs, is characterized in that, for the detection of AFP, detecting step is as follows:
(1) with prepared immunosensor for working electrode, Ag/AgCl electrode is contrast electrode, and platinum electrode is to electrode; Use MPI-B type multiparameter chemiluminescence analysis test macro to test, the high pressure of photomultiplier is set to 600V, and sweep interval is-1.1 ~ 0V, and sweep velocity is 100mV/s;
(2) in the electrolytic cell of 10mLPBS (pH7.4) buffer solution containing 0.1mol/L potassium persulfate, the electrogenerated chemiluminescence intensity of the electrode of 0.001 ~ 5ng/mL a series of variable concentrations AFP standard solution and non-specific binding AFP is detected, drawing curve by MPI-B type multiparameter chemiluminescence analysis test macro;
(3) AFP standard solution is replaced by testing sample solution to detect.
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