CN110658246A - Myoglobin modified electrode with hydrogen peroxide response function based on ionic liquid and preparation method thereof - Google Patents
Myoglobin modified electrode with hydrogen peroxide response function based on ionic liquid and preparation method thereof Download PDFInfo
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Abstract
The invention provides a myoglobin modified electrode with a hydrogen peroxide response function and based on ionic liquid and a preparation method thereof. The invention utilizes the glassy carbon electrode and the ionic liquid-clay composite modification layer to construct the clay/IL/Myb/GCE modification electrode, wherein the myoglobin (Myb) can be firmly adsorbed on the ionic liquid-clay modification film through electrostatic attraction, and the ionic liquid can effectively promote direct electrochemistry of the myoglobin. The electrode has good electrocatalysis performance on hydrogen peroxide, can be used for quantitative detection of hydrogen peroxide, has a linear range of 4.0-250.0 mu M for detecting hydrogen peroxide and a detection limit of 0.737 mu M, and can be used for further constructing a biosensor for detecting hydrogen peroxide.
Description
Technical Field
The invention belongs to the field of biochemical detection, and relates to a myoglobin modified electrode with a hydrogen peroxide response function and based on an ionic liquid, a preparation method of the myoglobin modified electrode and application of the myoglobin modified electrode in a hydrogen peroxide sensor.
Background
The research on the direct electrochemical behavior of proteins is helpful for understanding the complex electron transfer mechanism in biological systems, researching the relationship between biological structures and functions, and the like. In addition, the research of direct electrochemistry of the protein can lay a certain foundation for the development of the biosensor.
It is often difficult to achieve direct electron transfer between the protein and the electrode, and it is therefore necessary to find an effective promoter or supporting electrolyte to promote the electron transfer process. Since the ionic liquid has the characteristics of good conductivity, wide potential window and the like, the ionic liquid is widely applied to the research of direct electrochemistry of protein in recent years. In one aspect, the ionic liquid can act as a supporting electrolyte and promote direct electrochemistry between the electrode and the protein; on the other hand, ionic liquids can also be used for electrode modification. Compared with the conventional electrode, the ionic liquid modified electrode can more easily realize direct electrochemistry of a plurality of proteins.
The clay has the characteristics of good thermal stability, stable physical property, clear layered structure, good ion exchange performance and the like, is a common electrode modification material, and can be used for fixing protein.
Myoglobin (Myb) is a major protein in skeletal muscle and has the structure: a quadrivalent iron porphyrin is embedded in a lipophilic aperture, and is surrounded by eight alpha helices to form a sphere. Myoglobin plays an important role in the life process, being the oxygen storage protein in the tissue and transporting oxygen from the periphery of the cell to the mitochondria. In addition, myoglobin can also scavenge intracellular NO. So far, various materials including layered titanate nano-materials, MCNT, agar gel, clay and the like have been used for direct electrochemical research of myoglobin, and a specific electrode modification method is found to be applicable to biochemical detection, which is an important research subject in cash.
Disclosure of Invention
The invention aims to solve the problem of constructing a novel hydrogen peroxide biosensor and provides an ionic liquid-based myoglobin modified electrode with a hydrogen peroxide response function.
The myoglobin direct electrochemical hydrogen peroxide response electrode based on the ionic liquid mainly comprises a working electrode, a platinum electrode and a silver-silver chloride electrode, wherein the working electrode comprises a glassy carbon electrode, an ionic liquid-clay composite modification layer and a myoglobin modification layer.
The glassy carbon electrode adopts a three-electrode system, wherein a working electrode is an ionic liquid-clay composite membrane modified electrode, platinum is modified on the surface of a counter electrode, and silver-silver chloride is modified on the surface of a reference electrode.
The mass ratio of the ionic liquid-clay composite material on the surface of the working electrode to the myoglobin is 15: 4.
The invention also aims to provide a preparation method of the myoglobin modified electrode based on the ionic liquid with the hydrogen peroxide response function.
The method specifically comprises the following steps:
s1, preparing an ionic liquid-clay modification liquid: dispersing a certain amount of clay by using water, and then adding a proper amount of ionic liquid to obtain 7.5g/L ionic liquid-clay modification liquid with the ionic liquid volume percentage of 2%;
s2, preparation of clay/lL/Myb/GCE modified electrode: and (3) dripping 3 mu L of the ionic liquid-clay modified liquid on the surface of a glassy carbon electrode, naturally drying at room temperature, and dripping 2 mu L of 3g/L myoglobin solution on the surface of the glassy carbon electrode to obtain the clay/IL/Myb/GCE modified electrode.
The clay is a substance with a special layered structure, has good film-forming property and can be bonded with cations with electrochemical activity. The ionic liquid can exchange cations such as silicon, aluminum and the like in the clay through ion exchange effect and form a uniform and stable mixed membrane with the clay by combining with the steric exclusion effect of the clay lamellar structure. The myoglobin has an isoelectric point of 6.8, shows positive electricity in a buffer solution with pH of 5.8, and can be adsorbed on the surface of the clay-ionic liquid mixed membrane through electrostatic interaction.
According to the invention, clay film is used for fixing ionic liquid and myoglobin, the ionic liquid is added to promote direct electron transfer between protein and an electrode, myoglobin is mainly fixed on the surface of an ionic liquid-clay mixed film through electrostatic acting force, and when the maximum loading capacity of the film is exceeded, redundant myoglobin cannot be effectively fixed on the film and can be rapidly lost. Therefore, experiments are needed to confirm the ionic liquid ratio in the clone/IL/Myb modified membrane and the concentration of myoglobin on the clone/IL/Myb/GCE modified electrode.
Further, in the step S1, the ionic liquid is [ BMIM ]]+[BF4]-。
Further, the myoglobin solution in the step S2 is NaAc-HAc solution with pH 5.8.
The prepared clay/IL/Myb/GCE modified electrode is detected by XRD and ultraviolet-visible spectrum analysis.
As shown in the attached figure 1, XRD detection is carried out on the prepared clay/IL/Myb/GCE modified electrode, and the XRD pattern of the clay/IL/Myb modified film is obtained. The XRD pattern of the clay/IL/Myb modified membrane shows a 2 theta peak at 6.25 degrees, and the corresponding aperture is
And the pore diameter is smaller than that of the clay/Myb modified membrane, because the ionic liquid is connected to the edge of the clay layer through the embedding/falling/re-stacking process, and the myoglobin is connected with the ionic liquid through electrostatic acting force.
As shown in the attached figure 2, ultraviolet-visible spectrum detection is carried out on the prepared clay/IL/Myb/GCE modified electrode to obtain an ultraviolet-visible spectrum of the clay/IL/Myb modified film. The myoglobin in the clone/IL/Myb modified membrane has an absorption peak at 412.4nm, and the peak position of the myoglobin solution has no obvious shift, which indicates that the myoglobin in the clone/IL/Myb modified membrane well maintains the biological activity, namely the ionic liquid [ BMIM ] used in the invention]+[BF4]-Has no poison to myoglobin, can not cause degeneration, and has good reliability.
The prepared clay/IL/Myb/GCE modified electrode is tested by cyclic voltammetry.
As shown in fig. 3, the prepared clay/IL/Myb/GCE modified electrode was placed in 0.1M PBS solution (pH 6.0) and subjected to cyclic voltammetric detection at a sweep rate of 300 mV/s. A pair of clear quasi-reversible oxidation and reduction peaks (curve a) can be found on the cyclic voltammetry curve of the electrode, the peak potential of the oxidation peak is-0.256V, the peak potential of the reduction peak is-0.338V, and the potential difference is 82mV, which indicates that the addition of the ionic liquid can effectively promote the electron transfer between myoglobin and the electrode.
As shown in the attached figure 4, the prepared clay/IL/Myb/GCE modified electrode is placed in 0.1M PBS solution (pH is 7.0) and subjected to cyclic voltammetry detection, and the sweep rate is 100-1000 mV/s. When the sweep rate is increased from 100mV/s to 1000mV/s, the oxidation-reduction peak current of the myoglobin is also increased, and the two are in a linear relation, which indicates that the electrode process is a surface-controlled electrochemical process, and myoglobin molecules are embedded into an ionic liquid-clay mixed membrane in a multilayer form and participate in direct electron transfer. The electron transfer rate k of the myoglobin can be calculatedsIs (3.58 +/-0.12) s-1The ionic liquid-clay modified membrane prepared by the method is helpful for realizing faster electron transfer between myoglobin and electrodes, has good biocompatibility and can effectively maintain the bioactivity of the myoglobin.
The cyclic voltammetry is adopted to test the electrocatalytic performance of the prepared clay/IL/Myb/GCE modified electrode on hydrogen peroxide.
As shown in fig. 5, the prepared clay/IL/Myb/GCE modified electrode is put into 0.1M PBS solution (pH 7.0), and hydrogen peroxide solution is added sequentially, and the hydrogen peroxide concentration is: 0M, 0.039M, 0.078M, 0.117M, and sweep rate of 300 mV/s. With the continuous increase of the concentration of hydrogen peroxide, the reduction peak current of the electrode curve is continuously increased, and with the continuous decrease of the oxidation peak current, the electrode curve shows obvious catalytic reaction characteristics, so that the ionic liquid-clay modified membrane prepared by the method has obvious hydrogen peroxide electrocatalysis performance and higher affinity to hydrogen peroxide.
The prepared clay/IL/Myb/GCE modified electrode is tested by a chronoamperometry method.
As shown in fig. 6, the prepared clay/IL/Myb/GCE modified electrode is placed in 0.1M PBS solution (pH 7.0) with nitrogen introduced and oxygen removed, 25 μ L of 0.78mM hydrogen peroxide is continuously added under stirring, and a chronoamperometric test is performed, with a working potential of-0.15V and a pulse of 50 mV; the time interval was 50 s. With the addition of hydrogen peroxide, the reduction current also increases stepwise. As can be seen from the figure, the detection limit of the prepared clay/IL/Myb/GCE modified electrode is calculated to be 0.737 mu M when the current response value and the hydrogen peroxide concentration are in a linear relation within the range of 4.0-250.0 mu M of hydrogen peroxide concentration.
The relative standard deviation RSD of the electrode measured in the same concentration hydrogen peroxide solution for five times is less than 10%, and the fact that the clay/IL/Myb/GCE modified electrode prepared by the method has better reproducibility is proved, and 90% of the initial current can be maintained after the electrode is stored for one week at 4 ℃, and the electrode has better stability.
According to the invention, clay and ionic liquid are used to form a mixed membrane modified glassy carbon electrode, and myoglobin is adsorbed on the surface of the modified membrane through electrostatic interaction. The direct electrochemical behavior of the constructed modified electrode on myoglobin can prove that the electrode has the electrocatalysis effect on hydrogen peroxide, and can be further used for constructing a novel hydrogen peroxide biosensor.
The invention has the beneficial effects that:
(1) according to the invention, the glassy carbon electrode is modified with the ionic liquid-clay modification layer, myoglobin can be firmly adsorbed on the ionic liquid-clay modification film through electrostatic attraction, the ionic liquid can effectively promote direct electrochemistry of myoglobin, and the electrode has good reliability and stability.
(2) The electrode preparation method is simple and feasible, has low cost, and does not need special environment and large-scale instruments.
(3) The composite electrode prepared by the invention overcomes the characteristic that myoglobin is easy to inactivate, can be applied to quantitatively detecting hydrogen peroxide, and has the advantages of high detection speed, low detection limit and the like.
Drawings
The invention is further illustrated by means of the attached drawings, but the embodiments in the drawings do not constitute any limitation to the invention, and for a person skilled in the art, other drawings can be obtained on the basis of the following drawings without inventive effort.
FIG. 1 is an XRD pattern of a clay/IL/Myb/GCE modified electrode prepared by the present invention, wherein (a) clay, (b) clay-Myb, (c) clay/IL, (d) clay/IL/Myb;
FIG. 2 is a UV-VIS spectrum of a clone/IL/Myb/GCE modified electrode prepared according to the present invention, wherein (a) the clone/IL/Myb modified membrane, (b) Myb dissolved in NaAc-HAc (pH 5.8);
FIG. 3 is a cyclic voltammogram of the clone/IL/Myb/GCE modified electrode (a), clone/Myb/GCE modified electrode (b) prepared in accordance with the present invention in 0.1M PBS solution (pH 6.0) at a scan rate of 300 mV/s;
FIG. 4 is a cyclic voltammogram of a clay/IL/Myb/GCE modified electrode prepared according to the invention in a 0.1M PBS solution (pH 7.0), with a scanning speed of 100-1000 mV/s;
FIG. 5 is a cyclic voltammogram of the invention prepared by sequentially adding different concentrations of hydrogen peroxide to a 0.1M PBS solution (pH 7.0) at a scan rate of 300 mV/s;
FIG. 6 is a chronoamperometric assay of a clay/IL/Myb/GCE modified electrode prepared according to the present invention;
FIG. 7 is a schematic diagram of the clay/IL/Myb/GCE modified electrode prepared by the present invention.
Detailed Description
In order that the objects, aspects and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the following detailed description and the accompanying drawings.
Example 1
Preparing a clay/IL/Myb/GCE modified electrode:
(1) preparing an ionic liquid-clay modifying liquid: dispersing a certain amount of clay with water, and adding a proper amount of ionic liquid BMIM]+[BF4]-Obtaining 7.5g/L of ionic liquid-clay modifying liquid with the ionic liquid volume percentage of 2 percent;
(2) preparing a clay/IL/Myb/GCE modified electrode: polishing glassy carbon electrode with alumina of 1.0, 0.3 and 0.05 μm, and then ultrasonic cleaning in nitric acid, ethanol and ultrapure water of 1: 1 in sequence. And (3) dripping 3 mu L of the ionic liquid-clay modified liquid on the surface of a glassy carbon electrode, naturally drying at room temperature, and dripping 2 mu L of myoglobin solution of 3g/L on the surface of the glassy carbon electrode to obtain the clay/IL/Myb/GCE modified electrode.
The specifically prepared clay/IL/Myb/GCE modified electrode can be seen in figure 7.
Ultraviolet-visible spectrum tests are carried out on the clay/IL/Myb/GCE modified electrode prepared in the example 1 to obtain an attached figure 1, and the structural characteristics that myoglobin is connected with ionic liquid through electrostatic acting force in the composite electrode prepared in the example 1 are proved.
The ultraviolet-visible spectrum test was performed on the clay/IL/Myb/GCE modified electrode prepared in example 1 to obtain the attached FIG. 2, which demonstrates that the ionic liquid [ BMIM ] used in example 1]+[BF4]-Has no poison and harm to myoglobin, does not cause degeneration of myoglobin and has reliable performance.
Cyclic voltammetry tests are carried out on the clay/IL/Myb/GCE modified electrode prepared in example 1, and an attached figure 3 is obtained, so that the ionic liquid added in example 1 can effectively promote electron transfer between myoglobin and the electrode.
The method comprises the steps of carrying out hydrogen peroxide quantitative detection on the clay/IL/Myb/GCE modified electrode prepared in the embodiment 1 to obtain an attached figure 4-5, and proving that the composite electrode prepared in the embodiment 1 has electrocatalytic performance on hydrogen peroxide.
The time-lapse current test is carried out on the clay/IL/Myb/GCE modified electrode prepared in the example 1 to obtain an attached figure 6, and the linear range of the hydrogen peroxide detection of the composite electrode prepared in the example 1 is proved to be 4.0-250.0 mu M, and the electrode detection limit is 0.737 mu M.
Example 2
Preparing a clay/IL/Myb/GCE modified electrode:
(1) preparing an ionic liquid-clay modifying liquid: dispersing a certain amount of clay with water, and adding a proper amount of ionic liquid BMIM]+[BF4]-Obtaining 7.2g/L ionic liquid-clay modifying liquid with the ionic liquid volume percentage of 1.8 percent;
(2) preparing a clay/IL/Myb/GCE modified electrode: polishing glassy carbon electrode with alumina of 1.0, 0.3 and 0.05 μm, and then ultrasonic cleaning in nitric acid, ethanol and ultrapure water of 1: 1 in sequence. And (3) dripping 3 mu L of the ionic liquid-clay modified liquid on the surface of a glassy carbon electrode, naturally drying at room temperature, and dripping 2 mu L of 2.7g/L myoglobin solution on the surface of the glassy carbon electrode to obtain the clay/IL/Myb/GCE modified electrode.
Example 3
Preparing a clay/IL/Myb/GCE modified electrode:
(1) preparing an ionic liquid-clay modifying liquid: dispersing a certain amount of clay with water, and adding a proper amount of ionic liquid BMIM]+[BF4]-Obtaining 7.2g/L of ionic liquid-clay modifying liquid with the ionic liquid volume percentage of 1.6 percent;
(2) preparing a clay/IL/Myb/GCE modified electrode: polishing glassy carbon electrode with alumina of 1.0, 0.3 and 0.05 μm, and then ultrasonic cleaning in nitric acid, ethanol and ultrapure water of 1: 1 in sequence. And (3) dripping 3 mu L of the ionic liquid-clay modified liquid on the surface of a glassy carbon electrode, naturally drying at room temperature, and dripping 2 mu L of 2.5g/L myoglobin solution on the surface of the glassy carbon electrode to obtain the clay/IL/Myb/GCE modified electrode.
Comparative example 1
Preparing a clay/Myb/GCE modified electrode:
(1) preparing clay modifying liquid: dispersing a certain amount of clay with water to obtain clay modification liquid of 7.5 g/L;
(2) preparing a clay/Myb/GCE modified electrode: polishing glassy carbon electrode with alumina of 1.0, 0.3 and 0.05 μm, and then ultrasonic cleaning in nitric acid, ethanol and ultrapure water of 1: 1 in sequence. And (3) dripping 3 mu L of the clay modified liquid on the surface of a glassy carbon electrode, naturally drying at room temperature, and dripping 2 mu L of 3g/L myoglobin solution on the surface of the glassy carbon electrode to obtain the clay/Myb/GCE modified electrode.
The glassy carbon electrode of the above comparative example was the same as that of example 1.
The clone/IL/Myb/GCE modified electrode prepared in example 1 and the clone/Myb/GCE modified electrode prepared in comparative example 1 were placed in a 0.1M PBS solution (pH 6.0) and subjected to cyclic voltammetric measurements at a sweep rate of 300mV/s, giving figure 1. As can be seen from the figure, a pair of clear quasi-reversible redox peaks (curve a) can be found on the cyclic voltammetry curve of the clay/IL/Myb/GCE modified electrode, the peak potential of the oxidation peak is-0.256V, the peak potential of the reduction peak is-0.338V, and the potential difference is 82mV, while the curve of the clay/Myb/GCE modified electrode has a pair of redox peaks, which shows that myoglobin can realize direct electrochemistry on a clay modification layer, but the peak current of the curve is less than that of the clay/IL/Myb/GCE modified electrode, and proves that the addition of ionic liquid can effectively promote the electron transfer between myoglobin and the electrode.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single technical solution, and such description is for clarity only, and those skilled in the art should take the description as a whole, and the technical solutions in the embodiments may be combined appropriately to form other embodiments that those skilled in the art can understand. The technical details not described in detail in the present invention can be implemented by any of the prior arts in the field. In particular, all technical features of the invention which are not described in detail can be achieved by any prior art.
Claims (5)
1. The myoglobin modification electrode based on the ionic liquid and having the hydrogen peroxide response function is characterized by comprising a working electrode, a platinum electrode and a silver-silver chloride electrode, wherein the working electrode comprises a glassy carbon electrode, an ionic liquid-clay composite modification layer and a myoglobin modification layer.
2. The ionic liquid-based myoglobin modification electrode with hydrogen peroxide response function as claimed in claim 1, wherein the mass ratio of the ionic liquid-clay composite material on the surface of the working electrode to the myoglobin is 15: 4.
3. The method for preparing the myoglobin modification electrode based on ionic liquid with hydrogen peroxide response function according to claim 1, comprising the following steps:
s1, preparing an ionic liquid-clay modification liquid: dispersing a certain amount of clay by using water, and then adding a proper amount of ionic liquid to obtain 7.5g/L ionic liquid-clay modification liquid with the ionic liquid volume percentage of 2%;
s2, preparation of clone/IL/Myb/GCE modified electrode: and (3) dripping 3 mu L of the ionic liquid-clay modified liquid on the surface of a glassy carbon electrode, naturally drying at room temperature, and dripping 2 mu L of myoglobin solution of 3g/L on the surface of the glassy carbon electrode to obtain the clay/IL/Myb/GCE modified electrode.
4. The method for preparing an ionic liquid-based myoglobin-modified electrode having a hydrogen peroxide response function according to claim 3, wherein said ionic liquid is [ BMIM ] in step S1]+[BF4]-。
5. The method for preparing an ionic liquid-based myoglobin-modified electrode having a hydrogen peroxide response function according to claim 3, wherein said myoglobin solution in step S2 is prepared by dissolving myoglobin in NaAc-HAc solution having a pH of 5.8.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102645473A (en) * | 2012-04-18 | 2012-08-22 | 上海师范大学 | Kaolin nanotube/hemoglobin/ionic liquid nanocomposite film-modified electrode and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102645473A (en) * | 2012-04-18 | 2012-08-22 | 上海师范大学 | Kaolin nanotube/hemoglobin/ionic liquid nanocomposite film-modified electrode and preparation method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| HONG SUN: "Direct electrochemical and electrocatalytic properties of heme protein immobilized on ionic liquid-clay-nanoparticle-composite films", 《J POROUS MATER》 * |
| JIANBO LIU ET AL: "Investigation of the conformation changes of myoglobin by an electrochemical method and a biosensing application based on controlled protein unfolding", 《ANAL. METHODS》 * |
| JIN LOU ET AL: "Application of an ionic liquid-functionalized Mg2Al layered double hydroxide for the electrochemical myoglobin biosensor", 《IONICS》 * |
| 高瑞芳: "基于离子液体和纳米材料的氧化还原蛋白质修饰电极研究", 《中国博士学位论文全文数据库》 * |
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