CN106568973A - Preparation method and application of MoS2/Au-Pd compound-based procalcitonin electrochemical immunosensor - Google Patents
Preparation method and application of MoS2/Au-Pd compound-based procalcitonin electrochemical immunosensor Download PDFInfo
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Abstract
The invention relates to the field of electrochemical immunosensing technique, especially to a preparation method and application of a MoS2/Au-Pd compound-based label-free electrochemical immunosensor. H2O2 is used as an electrochemical probe, and a MoS2/Au-Pd compound is used as a substrate material. Based on good film forming capability and large specific surface area and excellent catalytic performance to H2O2, stability and sensitivity of an immunosensor are remarkably enhanced. The testing principle is to utilize current change before and after an antigen-antibody immunoreaction. When an antigen reacts with an antibody modified on an electrode, the formed immunocomplex is a non-electroactive substance and severely obstructs electron transfer, and current signal is reduced. The sensor is used for detection of procalcitonin. Linearity range for detection is 0.0001 ng/mL- 10 ng/mL, and detection limit is 0.05 pg/mL.
Description
Technical field
The present invention relates to electrochemistry immuno-sensing technical field, more particularly to a kind of to be based on MoS2The original of/Au-Pd things
Calcitonin(PCT)The preparation and application of electrochemical immunosensor.Specifically related to one kind is with H2O2 For electrochemical probe, MoS2/
Structure and application of the Au-Pd complex for the electrochemical immunosensor of base material.
Background technology
PCT belongs to peptide material before a kind of calcitonin, and without hormonal activity, it is due to serious thin that the level in serum is raised
Bacterium, funguses, the infection destruction of parasite and sepsis and MOFE and cause;In autoimmune, allergy and virus
During infection, content will not be raised.Therefore PCT can make diagnosis to antibacterial, fungus-caused systemic infection, be considered whole body
Property bacterium infection and sepsis auxiliary and Differential Diagnosiss conventional index.At present the detection meanss of PCT antigens are mainly put
Penetrate immune analysis method, double antibodies sandwich immunochemiluminescence method, gold colloidal colorimetry, transmission immune turbidimetry.These methods have
There are higher sensitivity and selectivity, but detection process needs expensive instrumentation, need complicated pre-treatment and operate numerous
Trivial, sample consumption is larger, is not suitable for quick detection.Electrochemical immunoanalytical has that specificity is good, selectivity is high, is easy to take
Band, cheap, simple operation and other advantages, it is possible to achieve the quick detection to sample.The present invention is based on electrochemical immunosensor
Advantage is prepared for a kind of based on MoS2The procalcitonin electrochemical immunosensor of/Au-Pd, realizes the sensitive inspection of procalcitonin
Survey.
The present invention utilizes MoS2/ Au-Pd composite modified glassy carbon electrodes, MoS2It is good two-dimensional sheet nano material,
Specific surface area is big, film forming ability is strong, can fix lot of antibodies as base material.Meanwhile, Au-Pd is used as fermentoid material pair
H2O2With excellent catalytic performance, by MoS2It is compound with Au-Pd, biocompatibility can be improved, strengthen conductive capability and catalysis
Performance, improves sensitivity and the stability of electrochemical immunosensor.The electrochemical immunosensor built in the present invention has
Preparation process is simple, low cost, good stability, the advantages of sensitivity is high, provides a kind of feasible method for PCT detections.
The content of the invention
An object of the present invention is with MoS2/ Au-Pd composites are base material, construct it is a kind of label-free, surely
It is qualitative good, the high electrochemical immunosensor of sensitivity;
The second object of the present invention is to adopt H2O2For electrochemical probe, by the specific binding between antigen-antibody and to electricity
The inhibition of son transmission realizes the Sensitive Detection to PCT.
The technical scheme is that:
1. it is a kind of to be based on MoS2The preparation method and application of the electrochemical immunosensor of/Au-Pd complex:
(1) with 1.0 μm, 0.3 μm, 0.05 μm of Al2O3Polishing powder is polished the glass-carbon electrode (GCE) of a diameter of 4 mm successively,
It is cleaned by ultrasonic 3 min respectively in ethanol and ultra-pure water, nitrogen is dried up, and takes 10 μ L MoS2/ Au-Pd composite dispersant liquid drops
It is coated onto under electrode surface, room temperature and dries film forming, is rinsed to remove the MoS not being bonded with PBS (pH 7.4) buffer solution2/Au-
Pd composites obtain Pd-Au/MoS2/GCE;
(2) procalcitonin antibody (anti-PCT) the standard solution drop coating of 10 μ L, 6 ~ 10 μ g/mL is taken to electrode surface, 4 DEG C
Lower incubated overnight, rinses to obtain anti-PCT/Pd-Au/MoS with PBS (pH 7.4) buffer solution2/GCE;
(3) take 10 μ L mass fractions be 1 ~ 3% bovine serum albumin (BSA) solution drop coating to electrode surface, incubate at 37 DEG C
Change 1 h, close the site of non-specific binding, electrode surface is rinsed with PBS (pH 7.4) buffer solution and obtain BSA/anti-
PCT/ Pd-Au/MoS2/GCE;
(4) 10 μ L concentration of Deca is used for a series of procalcitonin antigen standard solution of variable concentrations of 0.0001 ~ 10 ng/mL
In recognizing with antibody specificity, 60 min are incubated at 37 DEG C, electrode surface are rinsed with PBS (pH 7.4) buffer solution, be obtained
It is a kind of to be based on MoS2Electrochemical immunosensor (the PCT/BSA/anti-PCT/Pd-Au/MoS of/Au-Pd2/GCE)。
Above-mentioned MoS2The preparation of/Au-Pd dispersion liquids:
(1) preparation of Au-Pd alloy nanos dendrite
50.6 mg polyvidon (PVP) and 88.0 mg ascorbic acid (AA) are added in 5.0 mL deionized waters, are stirred molten
Solution.Then, 5mL is contained into 6.25 μm of ol HAuCl under stirring4With 6.25 μm of ol H2PdCl4Solution, with constant drop speed
(0.22 mL/min)It is added dropwise in bottle;1h is stirred at 25 DEG C, deep brown solution is formed, 10000 rpm are centrifuged 20 min, use
Milli-Q water three times, is re-dispersed into stand-by in 10 mL ultra-pure waters;
(2)MoS2The preparation of/Au-Pd dispersion liquids
Take the above-mentioned solution of 1mL to be added in the molybdenum bisuphide solution of 9mL 1mg/mL, persistently stir 48 h, under 10000 rpm
15 min are centrifuged, unconjugated alloy nano dendrite are removed, is re-dispersed in 10 mL ultra-pure waters, obtains MoS2/ Au-Pd disperses
Liquid.
The detection of PCT:
(1) PCT/BSA/anti-PCT/Pd-Au/MoS2/ GCE be working electrode, Ag/AgCl electrodes be reference electrode, platinum electricity
Extremely to electrode, H2O2 For electrochemical probe, tested using electrochemical workstation;
(2) a series of electrode of variable concentrations PCT standard solution is detected with i-t curve methods, the current potential of scanning is -0.4
V, records i-t curve charts, drawing curve linear according to the logarithm of the current value and PCT concentration of gained;
(3) testing sample solution replacement PCT standard solution is detected.
Beneficial effects of the present invention are:
(1) present invention is MoS2/ Au-Pd composites drop coating hinders electron transmission so as to change by protein to electrode surface
The mechanism detection PCT of power transformation intensity of flow, without enzyme, unmarked, greatly simplify electrode production process;
(2) present invention is with MoS2/ Au-Pd composites do substrate, and specific surface area is big, film forming ability is strong, not only can fix in a large number
Antibody, while to electrochemical probe H2O2There is good catalytic effect, effectively enhance the sensitive of electrochemical immunosensor
Degree;
(3) electrochemical immunosensor prepared by the present invention is used for the detection of PCT, simple to operate, range of linearity width, detection limit
It is low, it is possible to achieve to the simple, quick of AFP, Sensitive Detection.The range of linearity is 0.0001 ~ 10 ng/mL, detects and is limited to 0.05
pg/mL。
Description of the drawings:
I-t curve charts of the Fig. 1 for variable concentrations PCT;
Current intensity and lg of the Fig. 2 for variable concentrations PCTcLinear Fit Chart.
Wherein, the concentration of AFP is represented respectively as 0.0001,0.001,0.01,0.05 by 1 to 7 i-t curve charts in Fig. 1,
0.1,1,10 ng/mL.
Specific embodiment:
For a better understanding of the present invention, below with instantiation describing technical scheme in detail, but it is of the invention
It is not limited thereto.
Embodiment 1. is a kind of to be based on MoS2The preparation method of the electrochemical immunosensor of/Au-Pd complex:
(1) with 1.0 μm, 0.3 μm, 0.05 μm of Al2O3Polishing powder is polished the glass-carbon electrode (GCE) of a diameter of 4 mm successively,
It is cleaned by ultrasonic 3 min respectively in ethanol and ultra-pure water, nitrogen is dried up, and takes 6 μ L MoS2/ Au-Pd nano composite materials are disperseed
Drop is coated onto under electrode surface, room temperature and dries film forming, is rinsed to remove what is be not bonded with PBS (pH 7.4) buffer solution
MoS2/ Au-Pd obtains Au-Pd/MoS2/GCE;
(2) procalcitonin antibody (anti-PCT) the standard solution drop coating of 10 μ L, 6 μ g/mL is taken to electrode surface, incubate at 4 DEG C
Change overnight, anti-PCT/Pd-Au/MoS is rinsed to obtain with PBS (pH 7.4) buffer solution2/GCE;
(3) bovine serum albumin (BSA) solution drop coating that 10 μ L mass fractions are 1% is taken to electrode surface, hatch at 37 DEG C
1 h, closes the site of non-specific binding, rinses electrode surface with PBS (pH 7.4) buffer solution and obtains BSA/anti-
PCT/ Pd-Au/MoS2/GCE;
(4) 10 μ L concentration of Deca is used for a series of procalcitonin antigen standard solution of variable concentrations of 0.0001 ~ 10 ng/mL
In recognizing with antibody specificity, 60 min are incubated at 37 DEG C, electrode surface are rinsed with PBS (pH 7.4) buffer solution, be obtained
It is a kind of to be based on MoS2Electrochemical immunosensor (the PCT/BSA/anti-PCT/Pd-Au/MoS of/Au-Pd2/GCE)。
Embodiment 2. is a kind of to be based on MoS2The preparation method of the electrochemical immunosensor of/Au-Pd complex:
(1) with 1.0 μm, 0.3 μm, 0.05 μm of Al2O3Polishing powder is polished the glass-carbon electrode (GCE) of a diameter of 4 mm successively,
It is cleaned by ultrasonic 3 min respectively in ethanol and ultra-pure water, nitrogen is dried up, and takes 8 μ L MoS2/ Au-Pd nano composite materials are disperseed
Drop is coated onto under electrode surface, room temperature and dries film forming, is rinsed to remove what is be not bonded with PBS (pH 7.4) buffer solution
MoS2/ Au-Pd obtains Au-Pd/MoS2/GCE;
(2) procalcitonin antibody (anti-PCT) the standard solution drop coating of 10 μ L, 8 μ g/mL is taken to electrode surface, incubate at 4 DEG C
Change overnight, anti-PCT/Pd-Au/MoS is rinsed to obtain with PBS (pH 7.4) buffer solution2/GCE;
(3) bovine serum albumin (BSA) solution drop coating that 10 μ L mass fractions are 2% is taken to electrode surface, hatch at 37 DEG C
1 h, closes the site of non-specific binding, rinses electrode surface with PBS (pH 7.4) buffer solution and obtains BSA/anti-
PCT/ Pd-Au/MoS2/GCE;
(4) 10 μ L concentration of Deca is used for a series of procalcitonin antigen standard solution of variable concentrations of 0.0001 ~ 10 ng/mL
In recognizing with antibody specificity, 60 min are incubated at 37 DEG C, electrode surface are rinsed with PBS (pH 7.4) buffer solution, be obtained
It is a kind of to be based on MoS2Electrochemical immunosensor (the PCT/BSA/anti-PCT/Pd-Au/MoS of/Au-Pd2/GCE)。
Embodiment 3. is a kind of to be based on MoS2The preparation method of the electrochemical immunosensor of/Au-Pd complex:
(1) with 1.0 μm, 0.3 μm, 0.05 μm of Al2O3Polishing powder is polished the glass-carbon electrode (GCE) of a diameter of 4 mm successively,
It is cleaned by ultrasonic 3 min respectively in ethanol and ultra-pure water, nitrogen is dried up, and takes 10 μ L MoS2/ Au-Pd nano composite materials are disperseed
Drop is coated onto under electrode surface, room temperature and dries film forming, is rinsed to remove what is be not bonded with PBS (pH 7.4) buffer solution
MoS2/ Au-Pd obtains Au-Pd/MoS2/GCE;
(2) procalcitonin antibody (anti-PCT) the standard solution drop coating of 10 μ L, 10 μ g/mL is taken to electrode surface, at 4 DEG C
Incubated overnight, rinses to obtain anti-PCT/Pd-Au/MoS with PBS (pH 7.4) buffer solution2/GCE;
(3) bovine serum albumin (BSA) solution drop coating that 10 μ L mass fractions are 3% is taken to electrode surface, hatch at 37 DEG C
1 h, closes the site of non-specific binding, rinses electrode surface with PBS (pH 7.4) buffer solution and obtains BSA/anti-
PCT/ Pd-Au/MoS2/GCE;
(4) 10 μ L concentration of Deca is used for a series of procalcitonin antigen standard solution of variable concentrations of 0.0001 ~ 10 ng/mL
In recognizing with antibody specificity, 60 min are incubated at 37 DEG C, electrode surface are rinsed with PBS (pH 7.4) buffer solution, be obtained
It is a kind of to be based on MoS2Electrochemical immunosensor (the PCT/BSA/anti-PCT/Pd-Au/MoS of/Au-Pd2/GCE)。
4. MoS of embodiment2The preparation of/Au-Pd dispersion liquids:
(1) preparation of Au-Pd alloy nanos dendrite
50.6 mg polyvidon (PVP) and 88.0 mg ascorbic acid (AA) are added in 5.0 mL deionized waters, are stirred molten
Solution.Then, 5mL is contained into 6.25 μm of ol HAuCl under stirring4With 6.25 μm of ol H2PdCl4Solution, with constant drop speed
(0.22 mL/min)It is added dropwise in bottle;1h is stirred at 25 DEG C, deep brown solution is formed, 10000 rpm are centrifuged 20 min, use
Milli-Q water three times, is re-dispersed into stand-by in 10 mL ultra-pure waters;
(2)MoS2The preparation of/Au-Pd dispersion liquids
Take the above-mentioned solution of 1mL to be added in the molybdenum bisuphide solution of 9 mL, 1 mg/mL, persistently stir 48 h, 10000 rpm
15 min of lower centrifugation, remove unconjugated alloy nano dendrite, are re-dispersed in 10 mL ultra-pure waters, obtain MoS2/ Au-Pd point
Dispersion liquid.
The detection of 5. PCT of embodiment:
(1) PCT/BSA/anti-PCT/Pd-Au/MoS2/ GCE be working electrode, Ag/AgCl electrodes be reference electrode, platinum electricity
Extremely to electrode, H2O2 For electrochemical probe, tested using electrochemical workstation;
(2) a series of electrode of variable concentrations PCT standard solution is detected with i-t curve methods, the current potential of scanning is -0.4
V, records i-t curve charts, and acquired results are shown in Fig. 1, linear according to the logarithm of the current value and PCT concentration of gained, draws
Working curve;
(3) current value for obtainingIWith the logarithm (lg of PCT concentrationc) linear relationship see Fig. 2, as shown in Figure 2, PCT is 1.0
In pg/mL ~ 10ng/mL concentration ranges, IIt is in good linear correlation with the logarithm of PCT concentration, linear equation isI =
0.578lgc - 1.88,cIt is concentration, unit is g/mL, and linearly dependent coefficient is 0.973, detection line is 0.05 pg/mL;
(4) testing sample solution replacement PCT standard solution is detected.
Claims (3)
1. it is a kind of to be based on MoS2The preparation method and application of the electrochemical immunosensor of/Au-Pd complex, it is characterised in that bag
Include following steps:
(1) with 1.0 μm, 0.3 μm, 0.05 μm of Al2O3Polishing powder is polished the glass-carbon electrode (GCE) of a diameter of 4 mm successively,
It is cleaned by ultrasonic 3 min respectively in ethanol and ultra-pure water, nitrogen is dried up, and takes 10 μ L MoS2/ Au-Pd composite dispersant liquid drops
It is coated onto under electrode surface, room temperature and dries film forming, is rinsed to remove the MoS not being bonded with PBS (pH 7.4) buffer solution2/Au-
Pd composites obtain Pd-Au/MoS2/GCE;
(2) procalcitonin antibody (anti-PCT) the standard solution drop coating of 10 μ L, 6 ~ 10 μ g/mL is taken to electrode surface, 4 DEG C
Lower incubated overnight, rinses to obtain anti-PCT/Pd-Au/MoS with PBS (pH 7.4) buffer solution2/GCE;
(3) take 10 μ L mass fractions be 1 ~ 3% bovine serum albumin (BSA) solution drop coating to electrode surface, incubate at 37 DEG C
Change 1 h, close the site of non-specific binding, electrode surface is rinsed with PBS (pH 7.4) buffer solution and obtain BSA/anti-
PCT/ Pd-Au/MoS2/GCE;
(4) 10 μ L concentration of Deca is used for a series of procalcitonin antigen standard solution of variable concentrations of 0.0001 ~ 10 ng/mL
In recognizing with antibody specificity, 60 min are incubated at 37 DEG C, electrode surface are rinsed with PBS (pH 7.4) buffer solution, be obtained
It is a kind of to be based on MoS2Electrochemical immunosensor (the PCT/BSA/anti-PCT/Pd-Au/MoS of/Au-Pd2/GCE)。
2. according to claim 1 a kind of based on MoS2The preparation method of the electrochemical immunosensor of/Au-Pd complex
And application, described MoS2/ Au-Pd dispersion liquids, it is characterised in that making step is as follows:
(1) preparation of Au-Pd alloy nanos dendrite
50.6 mg polyvidon (PVP) and 88.0 mg ascorbic acid (AA) are added in 5.0 mL deionized waters, are stirred molten
Solution;Then, 5mL is contained into 6.25 μm of ol HAuCl under stirring4With 6.25 μm of ol H2PdCl4Solution, with constant drop speed
(0.22 mL/min)It is added dropwise in bottle;1h is stirred at 25 DEG C, deep brown solution is formed, 10000 rpm are centrifuged 20 min, use
Milli-Q water three times, is re-dispersed into stand-by in 10 mL ultra-pure waters;
(2)MoS2The preparation of/Au-Pd dispersion liquids
Take the above-mentioned solution of 1mL to be added in the molybdenum bisuphide solution of 9mL 1mg/mL, persistently stir 48 h, under 10000 rpm
15 min are centrifuged, unconjugated alloy nano dendrite are removed, is re-dispersed in 10 mL ultra-pure waters, obtains MoS2/ Au-Pd disperses
Liquid.
3. one kind that prepared by preparation method according to claim 1 is based on MoS2The electro-chemistry immunity of/Au-Pd complex is passed
Sensor, it is characterised in that for the detection of PCT, detecting step is as follows:
(1) PCT/BSA/anti-PCT/Pd-Au/MoS2/ GCE be working electrode, Ag/AgCl electrodes be reference electrode, platinum electrode
It is to electrode, H2O2 For electrochemical probe, tested using electrochemical workstation;
(2) a series of electrode of variable concentrations PCT standard solution is detected with i-t curve methods, the current potential of scanning is -0.4
V, records i-t curve charts, drawing curve linear according to the logarithm of the current value and PCT concentration of gained;
(3) testing sample solution replacement PCT standard solution is detected.
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CN108176393A (en) * | 2017-12-27 | 2018-06-19 | 肇庆市华师大光电产业研究院 | A kind of orderly, high density Ag-Al2O3-MoS2The preparation method of nanostructured |
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