CN108709922A - Polymer modified electrode for highly sensitive detection superoxide dismutase - Google Patents
Polymer modified electrode for highly sensitive detection superoxide dismutase Download PDFInfo
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Abstract
The present invention discloses a kind of polymer modified electrode for highly sensitive detection superoxide dismutase, utilize eATRP polymerizations, superoxide dismutase is then removed in electrode surface by polymer-modified there are superoxide dismutase, imprinted polymer modified electrode is made.The modified electrode is applicable as the electrochemical sensor of detection superoxide dismutase, has many advantages, such as high sensitivity, and detection speed is fast, and the range of linearity to the detection of superoxide dismutase standard solution is 1.0 × 10-7~ 100 mg/L, detection are limited to 6.7802 × 10-8mg/L（LOD,S/N=3）.
Description
Technical field
The present invention discloses a kind of electrochemistry trace sensor, especially it is a kind of prepare it is simple, save reagent, environmental protection
Polymer modified electrode for highly sensitive detection superoxide dismutase.
Background technology
Superoxide dismutase（Superoxide Dismutase, SOD）, alias orgotein is a kind of derived from life entity
Active material, the harmful substance that organism generates in metabolic processes can be eliminated, with anti-aging special-effect.Mesh
The common detection method of preceding superoxide dismutase has spectrophotometry, chemoluminescence method, electron spin resonance（ESR）Spectroscopic methodology
And utilize the methods of water-soluble tetrazolium salts detection.But above method exist detection sensitivity it is relatively low, detection limit it is higher and
The shortcomings of specificity is not strong.
Molecular engram is a kind of unique duplication accumulating method, can be identified " molecule key " by the lively manufacture that is described as
The technology of " artificial lock ".The core of molecular imprinting technology is molecularly imprinted polymer, is to pass through function monomer and target molecule
Non-covalent or covalent mode is copolymerized generation polymer, then is eluted target molecule by solvent, leaves in the polymer solely
Special " memory " hole, which can carry out reversible specific binding with the target molecule in mixture, in analysis
It is widely used.
There are many ways to preparing molecularly imprinted polymer, atom transfer radical polymerization（ATRP）Be realize " activity "/
The common method of controllable polymerization is to prepare one of effective means of molecularly imprinted polymer.Conventional ATRP methods are used for protein
There are two main difficulties for the preparation of MIP：（1）Lower valency metallic catalyst needed for polymerization is to the sensitivity such as air without easy to maintain；（2）
Catalyst has certain toxicity, and the post-processing work of common Removal of catalyst, complexant etc. to protein and other
Skill is more complicated.Various improved ATRP methods are constantly reported at present, e.g., Matyjaszewski in 2011 et al., it is proposed that
EATRP new methods, this method can make catalyst content needed for polymerization be reduced to 50 ppm, and can be by potential, electric current, electricity
Etc. the adjustings of conditions realize to polymerizeing more efficiently control；Silva et al. is in hemoglobin（Hemoglobin, Hb）Middle addition
Ascorbic acid reducing agent Hou [Fe in Hb（Ⅲ）It is reduced to Fe（Ⅱ）], it is found that Hb has ATRP catalytic activity, be successfully made
N-isopropyl acrylamide,（Ethylene glycol）Ethyl methacrylate,（Ethylene glycol）The ATRP polymerization of methyl ether methacrylate.
Electrochemical sensor is by working electrode, reference electrode and to the three-electrode system that electrode is constituted, and is a kind of utilization
Electrochemical signals change the device being detected to sample, and with high sensitivity, preparation is easy, at low cost, is easy to miniature
Change, be suitble to the features such as Site Detection, is the most ripe so far one of biosensor technique.So far, not related
It is applied to electrochemical sensor in the polymer modified electrode for preparing simple, saving reagent, environmental protection, is examined to high sensitivity
Survey the relevant report of superoxide dismutase.
Invention content
The present invention is provided a kind of for highly sensitive detection to solve the above-mentioned technical problem present in the prior art
The polymer modified electrode of superoxide dismutase.
Technical solution of the invention is：It is a kind of to detect the polymer-modified of superoxide dismutase for highly sensitive
Electrode, successively method in accordance with the following steps be made：
A. the naked gold electrode that will be handled well is impregnated 24 hours in the cysteine that 1mM is prepared by 0.1M PBS, is then taken out
Electrode is fully rinsed with ultra-pure water, removes the cysteine molecule of physical absorption, Au/Cys electrodes；
B. it is reference electrode using Au/Cys electrodes as working electrode, saturated calomel electrode, platinum filament is to electrode, by three electricity
Polar body system is placed in containing 5mM HAuCl4In solution, under the constant potential of -0.9V, Au/cys/Nano is made in electro-deposition 400s
Au electrodes；
C. it utilizes the bromine-containing compound of sulfydryl sealing end to modify clean gold electrode, obtains bromine-containing compound modified gold electrode；Then
0.025~0.5 moL/L acrylamides, 1~20mg/mL superoxide dismutases and 0.025~0.5 moL/L N, N- will be contained
The mixed solution of methylene-bisacrylamide is put into electrolytic cell, and at room temperature, bromine-containing compound modified gold electrode is inserted into
It states in mixed solution, while being inserted by Au/cys/Nano Au electrodes as working electrode, platinum filament as to electrode, saturation calomel electricity
The extremely three-electrode system of reference electrode applies constant potential 1.5h under 0.130V current potentials；
D. the bromine-containing compound modified gold electrode taking-up of polymer is cleaned with ultra-pure water deposition, in 30% hydrogenperoxide steam generator
Middle immersion 2h, then with naturally dry after ultrapure water, you can.
SOD is used not only to be used as template molecule but also prepared as catalyst progress eATRP reactions super for detecting in the present invention
The polymer modified electrode of superoxide dismutase can be quick, highly sensitive by the application of electrode in electrochemistry trace sensor
SOD is detected, the range of linearity to the detection of superoxide dismutase standard solution is 1.0 × 10-7~ 100 mg/L, detection are limited to
6.7802×10-8mg/L（LOD, S/N=3）, while having many advantages, such as to prepare simple, saving reagent, environmental protection.
Description of the drawings
Fig. 1 is in the polymer modified electrode preparation process of the embodiment of the present invention 1, and different modifying electrode is containing 5mM
[Fe CN)6]3-/4-+0.1 M KCl（PH 7.0 PBS）Cyclic voltammogram in solution.
Fig. 2 is the X-ray photoelectron spectroscopic analysis figure of the polymer modified electrode of the embodiment of the present invention 1.
Fig. 3 is the design sketch of the selectivity of the polymer modified electrode of the embodiment of the present invention 1.
Fig. 4 is the differential pulse voltammetry that the polymer modified electrode of the embodiment of the present invention 1 detects superoxide dismutase
Curve（A）And working curve（B）.
Specific implementation mode：
Embodiment 1：
The polymer modified electrode for highly sensitive detection superoxide dismutase of the present invention, successively in accordance with the following steps
Method is made：
A. the naked gold electrode that will be handled well is impregnated 24 hours in the cysteine that 1mM is prepared by 0.1M PBS, is then taken out
Electrode is fully rinsed with ultra-pure water, removes the cysteine molecule of physical absorption, Au/Cys electrodes；
B. it is reference electrode using Au/Cys electrodes as working electrode, saturated calomel electrode, platinum filament is to electrode, by three electricity
Polar body system is placed in containing 5mM HAuCl4In solution, under the constant potential of -0.9V, Au/cys/Nano is made in electro-deposition 400s
Au electrodes；
C. the bromine-containing compound of sulfydryl sealing end is utilized（Initiator）Clean gold electrode is modified, bromine-containing compound modification gold is obtained
Electrode；Then it will contain 0.1 moL/L acrylamides（Function monomer）, 4mg/mL superoxide dismutases and 0.1 moL/L N, N-
Methylene-bisacrylamide（Crosslinking agent）Mixed solution be put into electrolytic cell, at room temperature, by bromine-containing compound modified gold electrode
Be inserted into above-mentioned mixed solution, at the same be inserted by Au/cys/Nano Au electrodes as working electrode, platinum filament be to electrode, satisfy
Apply constant potential 1.5h under 0.130V current potentials with the three-electrode system that calomel electrode is reference electrode；
D. the bromine-containing compound modified gold electrode taking-up of polymer is cleaned with ultra-pure water deposition, in 30% hydrogenperoxide steam generator
Middle immersion 2h, then with naturally dry after ultrapure water, you can.
Fig. 1 is in the electrode production process of the embodiment of the present invention 1, and different modifying electrode is containing 5mM[Fe CN)6]3-/4-
+0.1 M KCl（PH 7.0 PBS）Cyclic voltammogram in solution.
In Fig. 1, curve 1 is the CV curves of naked gold electrode in the solution, which occurs a pair of reversible near 0.2 V
[Fe(CN)6]3-/4-Probe ion redox peaks.Curve 2 is gold electrode in bromine compounds（Initiator）CV curves after modification,
Peak current is less than curve 1, and probe ion arrival electrode surface is hindered after illustrating gold electrode surfaces self assembly initiator.Curve 3
It is after acrylamide polymerize, peak current is significantly lower than curve 2, illustrates, with after acrylamide polymerization, to be formed in electrode surface
One thick layer hinders probe ion to reach the polymer film of electrode surface, causes the peak current low.Curve 4 is eluted template point
Son（Superoxide dismutase）CV curves afterwards, peak current are apparently higher than curve 3, after removing superoxide dismutase, electricity
There is imprinted cavity in pole surface, is conducive to probe ion and reaches electrode surface, peak current is caused to increase.
Fig. 2 is the X-ray photoelectron spectroscopic analysis figure of the polymer modified electrode of the embodiment of the present invention 1.It can be with from Fig. 2
Find out and is successfully prepared polymer modified electrode.
For the selectivity of Study Polymer Melts modified electrode, with lysozyme in experiment（LYZ）（MW 14.4 kDa）, carbonic acid
Acid anhydride enzyme（CA, MW 29 kDa）, pepsin（MW 35 kDa）, hemoglobin（MW 65 kDa）As chaff interferent.Using difference
Divide pulse voltammetry（DPV）Polymer modified electrode using the embodiment of the present invention 1 and non-trace electrode（Preparation method and implementation
Example 1 is identical, and superoxide dismutase is not added with when only preparing）Measure same concentrations（1mg·L -1）Not homologous protein sound
The difference of induction signal, the results are shown in Figure 3.Polymer modified electrode of the invention detects superoxides discrimination as can be seen from Figure 3
It is 39.86 μ A to change enzyme response signal Δ I, is 5.84,12.56,14.80 and 9.20 times of CA, Hb, Lyz and PG respectively.As a result it shows
Show, it is more preferable to the selectivity of target protein superoxide dismutase.The selectivity of electrode can also by imprinting factor (β) into
Row assessment.Its accounting equation isβ=ΔI(MIP)/ΔI(NIP), wherein Δ I(NIP)It is response signal of the non-trace electrode to protein,
And Δ I(MIP)It is response signal of the polymer modified electrode to protein.As shown in figure 3, by equation calculation SOD, CA, Hb,
Lyz's and PGβValue is respectively 6.087,1.846,2.160,1.940 and 2.302.SOD'sβValue maximum shows the embodiment of the present invention
The ability of 1 polymer modified electrode detection SOD is significantly larger than non-trace electrode, shows 1 polymer-modified electricity of the embodiment of the present invention
Pole only has good selectivity to target protein.
Fig. 4 is the differential pulse voltammetry that the polymer modified electrode of the embodiment of the present invention 1 detects superoxide dismutase
Curve（A）And working curve（B）.
In Fig. 4 A, 1~10 corresponding superoxide dismutase concentration of curve is respectively 0,10-7, 10-6, 10-5, 10-4,
10-3, 10-2, 10-1, 1,10,100mg/L.With the increase differential pulse voltammetry curve peak of SOD concentration it can be seen from Fig. 4 A
Current reduction.This is because the embodiment of the present invention 1 combines the polymer modified electrode after superoxide dismutase, imprinted cavity
It is occupied by SOD, hinders probe ion and reach electrode surface, to reduce the differential pulse voltammetry curve peak point current of electrode,
SOD concentration is higher, and imprinted cavity is occupied also more, and peak point current declines more.Fig. 4 B are peak point current decaying（Letter
Number response, Δ I）With the relationship of SOD log concentrations.The embodiment of the present invention 1 combines superoxide dismutase as can be seen from Figure 4B
The concentration range of linearity of polymer modified electrode detection superoxide dismutase afterwards is 1.0 × 10-7~100mg/L.It is linear to return
It is Δ to return equationI（μA）=0.6563logC（mg/L）+ 35.386, related coefficient 0.9944.Find out from standard curve, detects
It is limited to（LOD, S/N=3）6.7802×10-8mg/L。
Embodiment 2：
The present invention detects the polymer modified electrode of superoxide dismutase for high sensitivity, successively side in accordance with the following steps
Method is made：
A. the naked gold electrode that will be handled well is impregnated 24 hours in the cysteine that 1mM is prepared by 0.1M PBS, is then taken out
Electrode is fully rinsed with ultra-pure water, removes the cysteine molecule of physical absorption, Au/Cys electrodes；
B. it is reference electrode using Au/Cys electrodes as working electrode, saturated calomel electrode, platinum filament is to electrode, by three electricity
Polar body system is placed in containing 5mM HAuCl4In solution, under the constant potential of -0.9V, Au/cys/Nano is made in electro-deposition 400s
Au electrodes；
C. the bromine-containing compound of sulfydryl sealing end is utilized（Initiator）Clean gold electrode is modified, bromine-containing compound modification gold is obtained
Electrode；Then it will contain 0.2 moL/L acrylamides（Function monomer）, 8mg/mL superoxide dismutases and 0.2 moL/L N, N-
Methylene-bisacrylamide（Crosslinking agent）Mixed solution be put into electrolytic cell, at room temperature, by bromine-containing compound modified gold electrode
Be inserted into above-mentioned mixed solution, at the same be inserted by Au/cys/Nano Au electrodes as working electrode, platinum filament be to electrode, satisfy
Apply constant potential 1.5h under 0.130V current potentials with the three-electrode system that calomel electrode is reference electrode；
D. the bromine-containing compound modified gold electrode taking-up of polymer is cleaned with ultra-pure water deposition, in 30% hydrogenperoxide steam generator
Middle immersion 2h, then with naturally dry after ultrapure water, you can.
Embodiment 3：
The polymer modified electrode for highly sensitive detection superoxide dismutase of the present invention, successively in accordance with the following steps
Method is made：
A. the naked gold electrode that will be handled well is impregnated 24 hours in the cysteine that 1mM is prepared by 0.1M PBS, is then taken out
Electrode is fully rinsed with ultra-pure water, removes the cysteine molecule of physical absorption, Au/Cys electrodes；
B. it is reference electrode using Au/Cys electrodes as working electrode, saturated calomel electrode, platinum filament is to electrode, by three electricity
Polar body system is placed in containing 5mM HAuCl4In solution, under the constant potential of -0.9V, Au/cys/Nano is made in electro-deposition 400s
Au electrodes；
C. the bromine-containing compound of sulfydryl sealing end is utilized（Initiator）Clean gold electrode is modified, bromine-containing compound modification gold is obtained
Electrode；Then it will contain 0.4 moL/L acrylamides（Function monomer）, 16mg/mL superoxide dismutases and 0.4 moL/L N,
N- methylene-bisacrylamides（Crosslinking agent）Mixed solution be put into electrolytic cell, at room temperature, by bromine-containing compound modify gold electricity
Pole is inserted into above-mentioned mixed solution, at the same be inserted by Au/cys/Nano Au electrodes as working electrode, platinum filament for electrode,
Saturated calomel electrode is the three-electrode system of reference electrode, under 0.130V current potentials, applies constant potential 1.5h；
D. the bromine-containing compound modified gold electrode taking-up of polymer is cleaned with ultra-pure water deposition, in 30% hydrogenperoxide steam generator
Middle immersion 2h, then with naturally dry after ultrapure water, you can.
Embodiment 4：
The polymer modified electrode for highly sensitive detection superoxide dismutase of the present invention, successively in accordance with the following steps
Method is made：
A. the naked gold electrode that will be handled well is impregnated 24 hours in the cysteine that 1mM is prepared by 0.1M PBS, is then taken out
Electrode is fully rinsed with ultra-pure water, removes the cysteine molecule of physical absorption, Au/Cys electrodes；
B. it is reference electrode using Au/Cys electrodes as working electrode, saturated calomel electrode, platinum filament is to electrode, by three electricity
Polar body system is placed in containing 5mM HAuCl4In solution, under the constant potential of -0.9V, Au/cys/Nano is made in electro-deposition 400s
Au electrodes；
C. the bromine-containing compound of sulfydryl sealing end is utilized（Initiator）Clean gold electrode is modified, bromine-containing compound modification gold is obtained
Electrode；Then it will contain 0.05 moL/L acrylamides（Function monomer）, 2mg/mL superoxide dismutases and 0.05moL/L N,
N- methylene-bisacrylamides（Crosslinking agent）Mixed solution be put into electrolytic cell, at room temperature, by bromine-containing compound modify gold electricity
Pole is inserted into above-mentioned mixed solution, at the same be inserted by Au/cys/Nano Au electrodes as working electrode, platinum filament for electrode,
Saturated calomel electrode is the three-electrode system of reference electrode, under 0.130V current potentials, applies constant potential 1.5h；
D. the bromine-containing compound modified gold electrode taking-up of polymer is cleaned with ultra-pure water deposition, in 30% hydrogenperoxide steam generator
Middle immersion 2h, then with naturally dry after ultrapure water, you can.
Embodiment 5：
The polymer modified electrode for highly sensitive detection superoxide dismutase of the present invention, successively in accordance with the following steps
Method is made：
A. the naked gold electrode that will be handled well is impregnated 24 hours in the cysteine that 1mM is prepared by 0.1M PBS, is then taken out
Electrode is fully rinsed with ultra-pure water, removes the cysteine molecule of physical absorption, Au/Cys electrodes；
B. it is reference electrode using Au/Cys electrodes as working electrode, saturated calomel electrode, platinum filament is to electrode, by three electricity
Polar body system is placed in containing 5mM HAuCl4In solution, under the constant potential of -0.9V, Au/cys/Nano is made in electro-deposition 400s
Au electrodes；
C. the bromine-containing compound of sulfydryl sealing end is utilized（Initiator）Clean gold electrode is modified, bromine-containing compound modification gold is obtained
Electrode；Then it will contain 0.025 moL/L acrylamides（Function monomer）, 1mg/mL superoxide dismutases and 0.025moL/L
N, N- methylene-bisacrylamide（Crosslinking agent）Mixed solution be put into electrolytic cell, at room temperature, by bromine-containing compound modify gold
Electrode is inserted into above-mentioned mixed solution, while being inserted by Au/cys/Nano Au electrodes as working electrode, platinum filament as to electricity
Pole, the three-electrode system that saturated calomel electrode is reference electrode apply constant potential 1.5h under 0.130V current potentials；
D. the bromine-containing compound modified gold electrode taking-up of polymer is cleaned with ultra-pure water deposition, in 30% hydrogenperoxide steam generator
Middle immersion 2h, then with naturally dry after ultrapure water, you can.
The experimental result of embodiment 2 ~ 5 is the same as embodiment 1.
Claims (1)
1. a kind of being used for the highly sensitive polymer modified electrode for detecting superoxide dismutase, successively side in accordance with the following steps
Method is made：
A. the naked gold electrode that will be handled well is impregnated 24 hours in the cysteine that 1mM is prepared by 0.1M PBS, is then taken out
Electrode is fully rinsed with ultra-pure water, removes the cysteine molecule of physical absorption, Au/Cys electrodes；
B. it is reference electrode using Au/Cys electrodes as working electrode, saturated calomel electrode, platinum filament is to electrode, by three electricity
Polar body system is placed in containing 5mM HAuCl4In solution, under the constant potential of -0.9V, Au/cys/Nano is made in electro-deposition 400s
Au electrodes；
C. it utilizes the bromine-containing compound of sulfydryl sealing end to modify clean gold electrode, obtains bromine-containing compound modified gold electrode；Then
0.025~0.5 moL/L acrylamides, 1~20mg/mL superoxide dismutases and 0.025~0.5 moL/L N, N- will be contained
The mixed solution of methylene-bisacrylamide is put into electrolytic cell, and at room temperature, bromine-containing compound modified gold electrode is inserted into
It states in mixed solution, while being inserted by Au/cys/Nano Au electrodes as working electrode, platinum filament as to electrode, saturation calomel electricity
The extremely three-electrode system of reference electrode applies constant potential 1.5h under 0.130V current potentials；
D. the bromine-containing compound modified gold electrode taking-up of polymer is cleaned with ultra-pure water deposition, in 30% hydrogenperoxide steam generator
Middle immersion 2h, then with naturally dry after ultrapure water, you can.
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Cited By (1)
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| CN109490389A (en) * | 2018-11-28 | 2019-03-19 | 辽宁师范大学 | The preparation method of the imprinted polymer modified electrode of high sensitivity detection myoglobins |
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