CN113376233A - Novel liposome-based electrode constructed by horseradish peroxidase, preparation method and direct electrochemical application thereof - Google Patents
Novel liposome-based electrode constructed by horseradish peroxidase, preparation method and direct electrochemical application thereof Download PDFInfo
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- CN113376233A CN113376233A CN202110788012.5A CN202110788012A CN113376233A CN 113376233 A CN113376233 A CN 113376233A CN 202110788012 A CN202110788012 A CN 202110788012A CN 113376233 A CN113376233 A CN 113376233A
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Classifications
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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
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
-
- 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
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
Abstract
The invention discloses a novel liposome-based horseradish peroxidase-constructed electrode, a preparation method and direct electrochemical application thereof. The ionic liquid-based polymerized liposome/gold nanoparticles obtained by the invention are combined with HRP to construct a modified electrode, and the composite material has good conductivity and biocompatibility, so that the immobilized HRP almost retains the original structure and shows higher electric activity and electrocatalysis. The polysome-Au composite material can effectively promote direct electron transmission between the HRP and the surface of the electrode. In addition, the Nafion/HRP/polysome-Au/GCE modified electrode pair H2O2And NaNO2Shows better analysisAnd (6) detecting the performance.
Description
Technical Field
The invention relates to the technical field of electrochemistry, and particularly relates to an electrode constructed by novel liposome-based horseradish peroxidase, a preparation method and direct electrochemical application thereof.
Background
The electrochemical sensor is regarded as an effective analysis and detection method due to simple structure, high efficiency and sensitivity. Among them, the enzyme-based electrochemical biosensor has attracted people's attention in the field of sensors due to its high catalytic activity, specificity and biocompatibility. Horse radish peroxidase HRP can catalyze hydrogen peroxide or oxidize various nitrites, and is one of the most commonly used enzymes for constructing electrochemical biosensors. Direct electron transfer due to HRP on bare electrode is shielded by the protein shell around the HRP redox center. Furthermore, HRP is not biologically active and the native enzyme is difficult to reuse. Immobilization of enzymes on nanoparticles or nanomaterials is expected to solve these problems. Therefore, the construction of the immobilized enzyme layer can accelerate the electron transfer and retain the specific biological activity thereof. To date, a large number of nanomaterials have been used as matrix materials for immobilizing enzymes for the construction of electrochemical biosensors.
Ionic liquids, since they are composed entirely of ions, have negligible vapor pressures and a wide range of possible cations and anions, which means that the properties of ionic liquids can be easily controlled. The ionic liquid has good conductivity, nonvolatility, no combustion and wide electrochemical window, and the characteristics enable the ionic liquid to be widely applied to the fields of electrochemistry, electroanalytical chemistry and the like.
Liposomes, as a biomimetic nanomaterial with excellent properties, have attracted extensive attention in various biological applications, particularly in the field of electrochemical biosensors. Liposomes have a membrane structure similar to that of cell membranes because they are closed vesicles composed of phospholipid bilayers and having an aqueous phase inside. Thus providing a good biocompatible microenvironment for stabilizing the conformation of the immobilized enzyme. Therefore, the prepared liposome enzyme biosensor has good analysis performance.
In recent years, metal nanoparticles have been widely used in the manufacture of various biosensors due to their electron transport properties, biocompatibility, low cytotoxicity and optical properties. In addition, many works have shown that enzymes immobilized on gold nanoparticles can retain their biocatalytic and electrochemical activities.
Disclosure of Invention
In view of this, the invention discloses and provides a novel liposome-based horseradish peroxidase-constructed electrode, a preparation method and direct electrochemical application thereof. The polyester plastid/gold nanometer composite material is a novel functional nanometer material, and the chemical modification electrode constructed by the polyester plastid/gold nanometer composite material shows better electrochemical response, has good electrocatalytic performance, and is suitable for the analysis and detection of actual samples;
in a first aspect, the invention provides a preparation method of a novel liposome-based horseradish peroxidase-constructed modified electrode, which comprises the following steps:
preparing an ionic liquid-based polyester/gold nano composite material polysome-Au;
selecting a glassy carbon electrode, and pretreating the glassy carbon electrode;
step three, mixing a horse radish peroxidase solution HRP and a polysome/gold nano composite material polysome-Au solution, adding a Nafion solution into the mixture, and obtaining a final dispersion liquid; then, the dispersion liquid is dripped on a pretreated glassy carbon electrode to slowly evaporate redundant water; finally obtaining the modified electrode Nafion/HRP/polysome-Au/GCE constructed by the novel liposome-based horse radish peroxidase.
Preferably, the preparation of the ionic liquid-based polyester body/gold nano composite material in the first step comprises the following steps:
1) synthesizing an ionic liquid-based liposome, which consists of a hydrophilic head of cationic bromoimidazoline ionic liquid and a hydrophobic tail of two long-chain terminal alkenes;
2) synthesizing ionic liquid-based polysome, and carrying out thermal initiation cross-linking polymerization between hydrophobic tail olefins of lipid monomers to obtain the ionic liquid-based polysome;
3) preparing a polyester body/gold nano composite material polysome-Au: by utilizing the ion exchange property of ionic liquid in the polymerized liposome structure, the ionic liquid-based polyester liposome is combined with gold nanoparticles by an in-situ reduction method.
Preferably, the ionic liquid-based liposome concentration in the step 1) is 1.00mg/mL, and the specific preparation method comprises the following steps:
1) sequentially adding 2-methylimidazole, triethylamine and bromo-11-carbene into toluene, and reacting for 48 hours at 90 ℃; cooling to room temperature after the reaction is finished, carrying out suction filtration to remove amine salt solids, evaporating the obtained filtrate to dryness, washing the obtained filtrate by using normal hexane for multiple times, evaporating the solvent by using normal hexane again, recrystallizing the product by using an acetonitrile-ethyl acetate mixed solvent, and carrying out vacuum drying to obtain yellowish white powder, namely a liposome monomer;
and (3) dissolving the lipid monomer in deionized water, performing ultrasonic treatment for 1 hour, and completely dispersing to obtain a clear and transparent solution, namely the ionic liquid-based liposome.
Preferably, the step 2) of synthesizing the ionic liquid-based polyester plastid polysome specifically comprises: adding K to an ionic liquid-based liposome solution2S2O8Reacting for 24 hours at 100 ℃ under the protection of nitrogen to obtain milky white suspension; and cooling the milky white suspension to room temperature, centrifuging at 8000rpm for 20min, centrifuging and washing the obtained solid product with deionized water for three times, and freeze-drying to obtain the solid, namely the ionic liquid-based polyester plastid.
Preferably, the step 3) is specifically: dispersing the ionic liquid-based polyester in deionized water, and adding HAuCl dropwise under magnetic stirring4Stirring the solution at room temperature for 30 min;
after the reaction was complete, centrifugation was carried out at 8000rpm for 15min to remove excess HAuCl4Re-dispersing the solid product obtained by centrifugation in deionized water; dropwise adding a reducing agent NaBH4Reacting for 2h to obtain an orange dispersion, centrifuging at 8000rpm for 15min, and centrifuging and washing a solid-phase product for three times; and (5) freeze-drying to obtain the polyester body/gold nano composite.
Preferably, a glassy carbon electrode with a diameter of 3mm is selected, and the pretreatment method comprises the step of firstly using Al with a diameter of 0.3 μm and a diameter of 0.05 μm on a polishing cloth for the glassy carbon electrode2O3Powder polishing, then sequentiallyUltrasonically cleaning with deionized water, anhydrous alcohol and deionized water for 2 min.
Preferably, in the third step, the mixing volume ratio of the horseradish peroxidase solution HRP to the polysome/gold nano composite material polysome-Au solution is 1:1, the mixture is stirred for 10 minutes in a vortex mode and stored for 24 hours at the temperature of 4 ℃; adding an equal volume of Nafion solution into the mixture, and then stirring the obtained final dispersion solution for 5 minutes by vortex; then, the dispersion liquid is dripped on a pretreated glassy carbon electrode and covered by a beaker, so that the redundant water is slowly evaporated at the temperature of 4 ℃; obtaining the modified electrode Nafion/HRP/polysome-Au/GCE constructed by the novel liposome-based horse radish peroxidase.
The second aspect of the invention also provides a modified electrode Nafion/HRP/polysome-Au/GCE prepared by the preparation method.
In a third aspect, the invention also provides application of the modified electrode constructed by the novel liposome-based horseradish peroxidase, and the Nafion/HRP/polysome-Au/GCE modified electrode is used for H2O2And NaNO2Carrying out catalysis and analysis detection.
The invention has the beneficial effects that:
the invention combines the liposome and the gold nanoparticles by an in-situ reduction method to prepare the ionic liquid-based polyester liposome/gold nanoparticle composite with structural stability, biocompatibility and electron transmission. The electrode is used as an electrode material and combined with HRP to construct a chemically modified electrode. Electrochemical experiment results show that the composite material can effectively promote direct electron transfer between the HRP and the electrode.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a picture (a) of an ionic liquid-based liposome dispersed in an aqueous solution, a Zeta potential diagram (b) and a dynamic light scattering diagram (c) provided by an embodiment of the disclosure;
FIG. 2 is a picture (a) of an ionic liquid-based polysome dispersed in an aqueous solution, a Zeta potential diagram (b) and a dynamic light scattering diagram (c) according to an embodiment of the disclosure;
FIG. 3 shows a graph (a), a Zeta potential diagram (b) and dynamic light scattering (c) of an ionic liquid-based polysome-Au dispersed in an aqueous solution according to an embodiment of the disclosure;
FIG. 4 is an SEM photograph of (A) liposome, (B) polysome, and (C) polysome-Au provided by the disclosed embodiment of the present invention;
FIG. 5 is a TEM photograph of (A) liposome, (B) polysome, and (C) polysome-Au, provided in the disclosed embodiment of the present invention;
FIG. 6 is a diagram of the UV-VIS absorption spectra of HRP (a) and HRP/polysome-Au (b) provided in the examples of the disclosure;
FIG. 7 shows FT-IR spectra of HRP (a) and HRP/polysome-Au (b) according to the disclosed embodiment;
FIG. 8 shows the modification of the electrode at 5mM K for Bar GCE, polysome/GCE, polysome-Au/GCE, HRP/polysome/GCE, HRP/polysome-Au/GCE according to the disclosure of the present invention3Fe(CN)6/K4Fe(CN)6(1:1), 0.1M KCl is an impedance spectrum in a supporting electrolyte solution; wherein the frequency range is from 0.1Hz to 100 KHz, and the amplitude is 5 mV; the open circuit potential is 0.26V;
FIG. 9 shows Nafion/HRP/GCE (a), Nafion/HRP/liposome/GCE (b), Nafion/HRP/polysome/GCE (c), and Nafion/HRP/polysome-Au/GCE (d) modified electrodes on N2Cyclic voltammogram in saturated 0.1M PBS (pH 7.0); wherein the sweeping speed is as follows: 200 mV/s;
FIG. 10 shows the present inventionThe (A) Nafion/HRP/polysome-Au/GCE provided by the open embodiment is in N2Cyclic voltammograms from 100mV/s to 800mV/s in saturated PBS (pH 7.0); (B) a linear plot of peak current versus sweep rate;
FIG. 11 shows (A) Nafion/HRP/polysome-Au/GCE in N2Cyclic voltammogram in PBS solution at different pH of saturation. Sweeping speed: 200 mV/s; (B) graph of the relationship between the formula potential and the pH value; (C) a graph of the relationship between the reduction peak current and the pH value of the solution;
FIG. 12 shows (A) Nafion/HRP/polysome-Au/GCE in N2Cyclic voltammogram was continuously scanned for 50 cycles in saturated PBS (pH 7.0) solution at a scan rate: 200 mV/s; (B) scanning cycle number and corresponding peak current variation trend graph;
FIG. 13(A) shows a Nafion/HRP/polysome-Au/GCE modified electrode provided in the disclosed embodiment of the present invention in N2In saturated PBS (pH 7.0), on H2O2The catalytic cyclic voltammogram of (sweep rate: 200 mV/s); (B) Nafion/HRP/polysome/GCE modified electrode on N2Saturated PBS (pH 7.0) vs. H2O2The catalytic cyclic voltammogram of (sweep rate: 200 mV/s);
FIG. 14(A) shows the continuous addition of 50. mu. M H in 0.1M PBS (pH 7.0) to Nafion/HRP/polysome-Au/GCE and Nafion/HRP/polysome/GCE modified electrodes provided in the disclosed embodiments of the invention2O2A chronoamperometric graph of; potential: -0.38V; (B) electrocatalytic current and H2O2A graph of concentration dependence;
FIG. 15 shows a combination of Nafion/HRP/polysome-Au/GCE pairs with 1mM UA,1mM AA,1mM DA and 100M H2O2Amperometric response in 0.1M pH 7.0 PBS; potential: -0.38V;
FIG. 16(A) shows a Nafion/HRP/polysome-Au/GCE modified electrode provided in the disclosed embodiment of the present invention in N2NaNO in saturated PBS (pH 7.0)2The catalytic cyclic voltammogram of (sweep rate: 200 mV/s); (B) catalytic peak current and NaNO2A concentration correction curve graph; (C) Nafion/HRP/polysome/GCE modified electrode on N2In saturated PBS (pH 7.0), to NaNO2The catalytic cyclic voltammogram of (sweep rate: 200 mV/s); (D) catalytic peak current and NaNO2A concentration correction curve graph;
FIG. 17 shows an embodiment of the present disclosure in which Nafion/HRP/polysome-Au/GCE contains N2Cyclic voltammograms after the first day and 10 days of storage in saturated 0.1MPBS (pH 7.0).
Detailed Description
Reference will now be made in detail to the exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, like numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Rather, they are merely examples of systems consistent with certain aspects of the invention, as detailed in the appended claims.
Direct electrochemistry of enzymes has attracted considerable attention in the research of biological systems and electrochemical biosensors. However, direct electron transfer of the enzyme at the electrode is often difficult to observe because the active site of the enzyme is deeply buried in the protein matrix. Thus, various immobilization matrices and mediators are used to facilitate electron transfer of proteins.
The integration of the nano material in the biosensor obviously improves the sensitivity, detection limit, response time and other analysis characteristics of the biosensor. Therefore, the novel functional nano material is a key component of a plurality of enzyme-based electrochemical biosensors.
The ionic liquid-based poly and liposome/gold nanoparticle composite with good conductivity and biocompatibility is introduced into the construction of a biosensing interface;
first, the present embodiment provides a method for preparing a polyester body/gold nanocomposite, including the steps of:
step one, synthesizing an ionic liquid-based liposome;
optionally, the synthesis of the ionic liquid-based liposome comprises the following steps: 15.00mL of toluene was charged in a 50mL round-bottom flask, and 2-methylimidazole, triethylamine and bromo-11-carbene were sequentially added thereto and reacted at 90 ℃ for 48 hours. And after the reaction is finished, cooling to room temperature, carrying out suction filtration to remove amine salt solids, evaporating the obtained filtrate to dryness, washing the obtained filtrate by using normal hexane for multiple times, evaporating the solvent again, recrystallizing the product by using an acetonitrile-ethyl acetate mixed solvent, wherein the volume ratio of acetonitrile to ethyl acetate is about 1:3, and carrying out vacuum drying to obtain yellowish white powder, namely the liposome monomer. Dissolving 10.0mg of lipid monomer in 10.0mL of deionized water, performing ultrasonic treatment for 1 hour, and completely dispersing to obtain a clear and transparent solution, namely the ionic liquid-based liposome, with the concentration of 1.00 mg/mL. The ionic liquid-based liposome consists of a hydrophilic head of cationic bromoimidazoline ionic liquid and a hydrophobic tail of two long-chain terminal alkenes.
As can be seen from fig. 1(a), the liposome is a clear and transparent solution, and no obvious precipitation is observed, indicating that it has good water solubility, which is determined by the high hydrophilicity of the ionic liquid on the surface of the liposome. The unpolymerized liposome water solution has good stability and no obvious precipitation after one week of storage. As can be seen from FIG. 1(b), the Zeta potential of the liposomal liposome is +114mV and the positive surface charge of the unpolymerized liposome solution is due to the presence of the imidazolyl cation. The surface charge enables the liposome to have stronger electrostatic repulsion action, so that the liposome has better stability. As is clear from FIG. 1(c), the particle size of liposome was 234 nm.
Step two, synthesizing an ionic liquid-based polyester plastid;
alternatively, the polymerized liposomes are obtained by thermally initiated cross-linking polymerization between the hydrophobic tail olefins of the lipid monomers. The preparation method comprises placing 0.04g liposome solution 25.00mL into a two-neck bottle, adding 10.0mg K2S2O8And reacting for 24 hours at 100 ℃ under the protection of nitrogen to obtain milky white suspension. And cooling the suspension to room temperature, centrifuging at 8000rpm for 20min, centrifuging and washing the obtained solid product with deionized water for three times, and freeze-drying to obtain a solid, namely the polymerized liposome polysome.
As can be seen from fig. 2(a), polysome is a translucent solution, which shows that it can exist stably in water. As can be seen from FIG. 2(b), the Zeta potential of the polysome solution is +24.7mV, and the surface charge property of the polysome solution is similar to that of liposome, indicating that the surface charge property of the polymerized liposome is not changed by the radical polymerization. As can be seen from fig. 2(c), the average particle size of the polysome solution is 1755nm, and the increase in the polysome particle size relative to liposome is caused by aggregation of some particles due to polymerization.
Step three, preparing polyester body/gold nano composite
5.0mg of polysome was dispersed in 50.00mL of deionized water, and 5.00mL of HAuCl was added dropwise thereto under magnetic stirring4(10.00. mu.L of HAuCl4Dispersed in 20.00mL of deionized water), and stirred at room temperature for 30 min. After the reaction was complete, centrifugation was carried out at 8000rpm for 15min to remove excess HAuCl4The solid product obtained by centrifugation was redispersed in 10mL of deionized water. 1mL of NaBH as a reducing agent is added dropwise4(1.0 mg NaBH4Dispersing in 1mL deionized water) for 2h to obtain an orange dispersion, centrifuging at 8000rpm for 15min, and centrifuging and washing the solid-phase product with water for three times. And (5) freeze-drying to obtain the polysome/gold nano composite polysome-Au.
As shown in FIG. 3(a), polysome and HAuCl were found4Ion exchange followed by NaBH4The reduction gave a pale pink clear solution of polysome-Au, indicating that gold nanoparticles were attached to the polysome. As is clear from FIG. 3(b), the Zeta potential of the polysome-Au solution is +24.1mV, which is close to the potential of the polysome. As can be seen from FIG. 3(c), the average particle size of the polysome-Au solution is 1302nm, which proves that the polysome particle size modified by nano Au is not damaged.
From fig. 4(a), it is evident that the existence of irregular multi-layered sheet structure, the edge shape of which is irregular, is completely different from the inherent spherical shape of the unpolymerized liposome lipome in the aqueous solution, because the lipome structure has poor stability, and the multi-layered membrane structure is formed due to the fusion with the substrate in the dry state, so that the spherical vesicle structure is destroyed. However, in fig. 4(B), under the same conditions, the polymerized monomers are connected by the valence bond formed by polymerization, and the polysome has better structural stability and still maintains better spherical morphology in a dry state. Because the polymerization also causes partial agglomeration, the spheres have clear boundary and uniform size, and the particle size is about 200 nm. The structural stability not only enables the polysome to keep a spherical structure under a dry condition, but also enables the polysome to be resistant to in-situ reduction of gold on the surface of the polysome, for the first-step decoration of inorganic AuNPs on a liposome substrate, the AuNPs are loaded on the surface of the liposome by utilizing the ion exchange property of ionic liquid in the polysome structure and through the exchange reaction of chloroauric acid and the reduction reaction of sodium borohydride, and the polysome-Au nanospheres are prepared, the appearance of the nanospheres is shown in figure 4(C), the existence of the spherical vesicle structure can be clearly seen from the figure, and the particle size is consistent with that of the polysome and is about 200 nm. On one hand, due to the structural stability of the polymerized liposome, the morphology of the polymerized liposome after in-situ loading of AuNPs still keeps the stability of a spherical structure. On the other hand, the conductivity of the polysome-Au loaded with AuNPs in situ is increased, so that the SEM image is clear and stable.
As can be seen from the TEM image in fig. 5(a), the vesicle structure of the liposome is destroyed, indicating that the structure of the unpolymerized liposome is difficult to exist stably in a dry state. As is evident from FIG. 5(B), the polymerized liposome material with spherical vesicle structure is stably distributed in the visual field, and the surface is smooth, the boundary is obvious, and the particle size is distributed between 150-220 nm. It can be seen from fig. 5(C) that, through in-situ growth of AuNPs, it is observed that the spherical morphology of the liposome is well preserved, the morphology and size are consistent with those of polysome, but some black spots with deeper contrast appear on the originally smooth surface, and the black spots are in-situ generated AuNPs, the size is about 3-4nm, and the distribution is relatively uniform. In addition, AuNPs have better monodispersity, no agglomeration phenomenon is observed, which shows that polysome has a stabilizing effect on AuNPs growing in situ, and the generation of the monodisperse nanoparticles can effectively promote the transmission of electrons.
The embodiment also provides a preparation method of the Nafion/HRP/polysome-Au/GCE modified electrode, which comprises the following steps:
a solution of 7.5mg/mL HRP was mixed with a solution of 1.0mg/mL polysome-Au at a volume ratio of 1:1, the mixture was stirred with a vortex for 10 minutes, and stored at 4 ℃ for 24 hours.
After an equal volume of 1% Nafion solution was added to the mixture, the final dispersion was again vortexed for 5 minutes. Then, 7.0. mu.L of the above dispersion was dropped onto a pretreated Glassy Carbon Electrode (GCE), which was then covered with a beaker, and placed in a refrigerator to allow the excess water to evaporate slowly at 4 ℃. Obtaining the Nafion/HRP/polysome-Au/GCE modified electrode. Other modified electrodes, including Nafion/HRP/GCE, Nafion/HRP/liposome/GCE, Nafion/HRP/polysome/GCE, referred to later in this embodiment, were also prepared by the same method as described above.
Wherein the electrode pretreatment comprises: before use, glassy carbon electrode (diameter 3mm) was coated with Al (0.3 μm, 0.05 μm) on a polishing cloth2O3Polishing the powder, and then ultrasonically cleaning the powder for 2min by using deionized water, absolute ethyl alcohol and deionized water in sequence. And finally, taking potassium ferricyanide as a probe, and making a cyclic voltammetry curve at a sweep rate of 100mV/s within a potential range of-0.2-0.8 mV. When the potential difference between the oxidation peak and the reduction peak is less than 70mV, the electrode surface meets the requirements of activation and cleaning, and finally, high-purity nitrogen is used for blow-drying for later use.
The HRP/polysome-Au compound is characterized, and the possible structural change of the immobilized enzyme is researched by utilizing the ultraviolet-visible absorption spectrum because the storage of the self structure of the immobilized enzyme is very important for the construction of the biosensor. As can be seen from FIG. 6, the free HRP in curve a shows a distinct absorption peak at 403nm, which is typical of Soret bands of enzymes. In contrast, it can be observed from the curve b in the figure that an absorption peak also appears at 403nm in the HRP/polysome-Au complex, which is completely consistent with the Soret band of the natural HRP, indicating that the natural structure of the HRP is well preserved after immobilization.
FIG. 7 shows FT-IR spectra of HRP and HRP/polysome-Au, curve b in the figure illustrates that FTIR spectra of HRP/polysome-Au show similar absorption bands to that of the polysome-Au after further interaction with HRP. One absorption band is located at 1125cm-11390cm, due to C-H deformation vibration of imidazole-1C-H bending vibrations due to imidazole. Furthermore, it is located at 1661cm-1And 1539cm-1Can be attributed toShape of the infrared absorption bands of amides I (C ═ O) and II (N — H) in HRP. These absorption bands were essentially identical to those of free HRP (curve a), indicating that the secondary structure of the bound enzyme was not disturbed after immobilization.
Electrochemical experiments were performed using the above-described electrode with 0.1M Phosphate Buffer Solution (PBS) at pH 7.0 as the supporting electrolyte. All electrochemical experiments were performed using CHI620B electrochemical workstation for cyclic voltammetry, using a three-electrode system, an Ag/AgCl electrode as a reference electrode, a platinum electrode as a counter electrode, and a modified electrode Nafion/HRP/polysome-Au/GCE as a working electrode. Before the electrochemical experiment begins, 8.00mL of PBS buffer solution is put into a small beaker, high-purity nitrogen is used for deoxidizing for 30min, and if no special instruction exists, the nitrogen atmosphere is kept in the experimental process all the time.
FIG. 8Bar GCE, polysome/GCE, polysome-Au/GCE, HRP/polysome-Au/GCE modified electrode at 5mM K3Fe(CN)6/K4Fe(CN)6(1:1), wherein 0.1M KCl is an impedance spectrogram in a supporting electrolyte solution, the frequency range is from 0.1Hz to 100 KHz, and the amplitude is 5 mV; the open circuit potential was 0.26V. The electrochemical impedance spectrogram can confirm the charge transfer resistance of the material on the surface of the electrode, and the impedance spectrum results of different modified electrodes are shown as a linear part and a semicircular part, and the diameters of the linear part and the semicircular part correspond to the charge transfer resistance. As shown in fig. 8, the resistance values of the bare electrode, the polysome/GCE and the polysome-Au/GCE modified electrode are respectively 200 Ω, 775 Ω and 541 Ω, and the decrease of the resistance values in sequence shows that the polysome material has conductive property, and after Au is immobilized on the polysome, the conductivity is further improved. The resistance values of the HRP/polysome/GCE and the HRP/polysome-Au/GCE modified electrodes are reduced from 930 omega to 642 omega. The introduction of AuNPs shows that the polysome-Au composite material has better electron transport property, thereby being beneficial to promoting the direct electron transport between enzyme and the surface of the electrode. In addition, after the HRP is loaded, the resistance values of the HRP/polysome/GCE and the HRP/polysome-Au/GCE are increased, and the successful immobilization of the enzyme is further shown by the EIS result.
As can be seen from FIG. 9, in the potential scanning range of-0.8 to 0.2V, no electrochemical response exists on the Nafion/HRP/liposome/GCE modified electrode, which indicates that liposome cannot be used as an effective interface for direct electron transfer between the oxidoreductase and the surface of the electrode. A pair of weak oxidation and reduction peaks appear on Nafion/HRP/GCE and Nafion/HRP/polysome/GCE, Epa-0.305V and Epc-0.345V are read from curve c as oxidation peak potential and reduction peak potential, respectively, and the calculated potential is-0.325V, and the peak difference (Δ Ep) is 40 mV. The redox peak is due to direct electron transfer between the HRP and the electrode and corresponds to the Fe III/Fe II redox center of the HRP enzyme. Whereas a significant increase in the redox peak current can be observed on curve d. Therefore, the biocompatibility and the conductivity of the polysome-Au composite material can accelerate the electron transfer rate between the HRP and the surface of the electrode, so that the electrochemical signal is increased. From the curve d, the redox peak potentials were Epa ═ 0.307V and Epc ═ 0.349V, respectively, and the potential of the formula was calculated to be-0.328V, which was consistent with the results obtained above, indicating that the peak was also a characteristic peak of HRP. The Δ Ep is 42mV, which not only indicates better reversibility of Nafion/HRP/polysome-Au/GCE, but also indicates faster electron transfer. The results show that although direct electrochemical reaction can be realized after the HRP is immobilized by the polysome, the electron transfer reaction of the HRP can be further promoted after the gold nanoparticles are added.
According to faraday's law Q nFA Γ, where Γ: amount of surface coverage; q: the amount of electricity consumed by the reaction; n: electron transfer number, here taken to be 1; f is the Faraday constant 96493; a: electrode area. From this formula, the coverage (Γ) of the electrically active HRP on the electrode surface was estimated to be about 2.71X 10-11mol cm-2. This value is close to the theoretical coverage value of a single layer of HRP on the electrode surface (2.0X 10)-11mol·cm-2) The electrically active horseradish peroxidase was shown to be present in a monolayer state.
The embodiment synthesizes ionic liquid-based liposome monomers, prepares the ionic liquid-based polymerized liposome through free radical polymerization, and prepares the polysome-Au composite material by utilizing the ion exchange property of the ionic liquid in the polymerized liposome structure. Using dynamic light scattering, Zeta potential, scanning electrodesThe surface appearance, structure and properties of the composite material are characterized by the technologies of a mirror, a transmission electron microscope and the like. The ionic liquid-based polymerized liposome/gold nanoparticles and HRP are combined to construct a modified electrode, and the FT-IR and UV-vis technologies are utilized to investigate the properties of the enzyme composite material. And the direct electrochemical properties of the modified electrode are researched. The result shows that the electron transfer rate constant of the Nafion/HRP/polysome-Au/GCE modified electrode is 5.08s-1The detection limit was 11.56. mu.M, and the sensitivity was 0.15. mu.A.mM-1·cm-2Has better selectivity and stability to H2O2And NaNO2Has a wide linear detection range. In addition, the Nafion/HRP/polysome-Au/GCE modified electrode is used for H in a human serum sample2O2Has better analysis and detection performance. The details are as follows:
FIG. 10(A) shows cyclic voltammograms of a Nafion/HRP/polysome-Au/GCE electrode at scan rates of 100, 200, 300, 400, 500, 600, 700, and 800mV/s in 0.1M pH 7.0PBS saturated with nitrogen. The results of the study show that the anodic peak current and the cathodic peak current increase with increasing scan rate. As can be seen from fig. 10(B), the peak current has a good linear relationship with the sweep rate, in which the linear regression equation: anode: -0.13289-0.0076x, r-0.999; cathode: y 0.26045+0.00908x, r 0.999, thus indicating that the electrode reaction is a surface-controlled process.
In addition, the current ratio of Ipa/Ipc was about 1:1 at a scan rate of 200mV/s, indicating that the HRP electrochemical reaction at this electrode is quasi-reversible. Peak difference Δ Ep is 42mV, and n Δ Ep<The electron transfer coefficient (. alpha.) was estimated to be 0.5 at 200 mV. Electron transfer rate constant (k)s) Can be determined according to Laviron theory:
where n is the electron transfer number, F is the Faraday constant, θ is the scan rate, R is the gas constant, T is the Kelvin temperature, and m is a constant related to Δ Ep. K is obtained by calculationsIs 5.08s-1. The results show that the prepared electrode can promote the electron transfer between the HRP and the surface of the electrode.
As shown in FIG. 11(A), the negative shift of the peak potentials of the anode and the cathode of the Nafion/HRP/polysome-Au/GCE modified electrode occurred with the increase of the pH value, which indicates that the electrochemical behavior of HRP is affected by the pH value of PBS. And as can be seen from fig. 11(B), the potential of the formula decreases linearly as the pH increases from 5.5 to 8.0, with a slope of-57.46 mV/pH, which is close to the expected theoretical value of the reversible proton-coupled single electron transfer process of-58.6 mV/pH, indicating that the electron transfer between the electrode and HRP is accompanied by a single protonation. As shown in fig. 11(C), the maximum peak current occurs at pH 7.0. The result shows that the HRP has higher activity in a neutral pH solution, which is consistent with the research result of other HRP-based electrochemical sensors;
as shown in FIG. 12, by scanning at N at a scan rate of 200mV/s2Cycling stability of the developed biosensor was tested by conducting 50 potential cycles in 0.1M saturated phosphate buffer solution. As shown in FIG. 12(A), even if the electrode is continuously scanned for 50 times, the cyclic voltammetry curve of the Nafion/HRP/polysome-Au/GCE modified electrode has good stability and no obvious change of the formula potential. As can be seen from FIG. 12(B), after 50 consecutive scans, the redox peak current changes within 2%, which indicates that the modified electrode Nafion/HRP/polysome-Au/GCE has good stability.
FIGS. 13(A) and (B) show the continuous addition of H at a sweep rate of 200mV/s2O2CV response of Nafion/HRP/polysome-Au/GCE modified electrode and Nafion/HRP/polysome/GCE modified electrode, shown in FIGS. A and B, N at pH 7.02Saturated PBS buffer solution, with H2O2The electrochemical behavior of the HRP immobilized electrode is changed remarkably. The reduction peak current is obviously increased, two modified electrodes respectively have an obvious reduction peak at about-0.34V and-0.30V, and the oxidation peak current is gradually reduced until disappears. The results show H2O2Typical of electrocatalytic reduction processes. Obviously, H2O2Due to the electrocatalytic effect of HRP (FeII), it can be used inOriginal H2O2Simultaneously oxidized to HRP (feiii) and then rapidly reduced back to HRP Fe (II) by its direct electron transfer capability with the electrode. HRP to H2O2The mechanism of the catalytic process of (a) is as follows:
HRP[Heme(FeIII)]+H2O2→Compound I+H2O
Compound I+e-→Compound II
Compound II+e-→HRP[Heme(FeIII)]
when H is shown in FIG. 14(A)2O2The Nafion/HRP/polysome-Au/GCE all showed a transient increase in catalytic current when continuously added to the constantly stirred PBS buffer solution, indicating that the fabricated biosensor pair H2O2Quick response of the reduction. Although Nafion/HRP/polysome/GCE also showed catalytic current, with H2O2The concentration increases and the catalytic current gradually decreases until it disappears. According to FIG. 14(A), the two modified electrodes estimated detection limits were 7.39. mu.M and 11.56. mu.M, respectively, at a signal-to-noise ratio of 3. The sensitivity was 0.24. mu.A.M, respectively-1·cm-2And 0.15. mu.A.M-1·cm-2. Apparent Michaelis Menten constant
Is an indicator of the kinetics of enzyme substrates and is commonly used to assess the biological activity of immobilized enzymes.
The smaller the biological activity, the higher. When H is present2O2At high concentrations, a steady current was observed, showing the typical characteristics of Michaelis Menten kinetics. Determining the Nafion/HRP/polysome-Au/GCE modified electrode through a Lineweaver Burk equation
The value was about 48.63. mu.M.
To determine whether a possible interfering substance has an effect on the HRP biosensor, Nafion/HRP/polysome-Au/GCE was usedThe selectivity and the anti-interference capability of interference substances such as glucose (Glu), Ascorbic Acid (AA), Uric Acid (UA), Dopamine (DA) and the like are detected. As shown in FIG. 15, at N2Saturated pH 7.0PBS buffer 100M H was added2O2After that, the current response is significantly enhanced. When 10 times the concentration of interferents was injected, there was no change in current. And H2O2The resulting current changes are not affected by these interferents. Shows that the biosensor has good anti-interference capability on interfering substances and H2O2Good selectivity of the catalyst.
When NaNO is added as shown in FIGS. 16(A) and (C)2When PBS was added, a new reduction peak was observed at about 0.8V, indicating the reduction of NO produced by the nitrite disproportionation reaction. In addition, the peak current follows NaNO in PBS2The concentration increases. The electrocatalytic process can be described as follows:
3NO2 -+2H+→2NO+NO3 -+H2O
HRP-Fe(II)+NO→HRP-Fe(II)-NO
HRP-Fe(II)-NO+e-+2H+→HRP-Fe(II)+H2O+N2O
FIGS. 16(B) and (D) are NaNO for Nafion/HRP/polysome-Au/GCE and Nafion/HRP/polysome/GCE modified electrode pairs2Catalytic peak current and NaNO2Calibration curve of concentration. As can be seen from FIG. B, NaNO was present in the range of 10 mM-1400 mM2The concentration and the catalytic peak current have good linear relation, and the linear regression equation is that y is 0.0130x +2.370 (R)20.994), x and y each represent NaNO2Concentration (mM) and reduction peak current (. mu.A), as seen from FIG. D, NaNO was in the range of 10mM to 400mM2The concentration and the catalytic peak current have better linear relation, and the linear regression equation is that y is 0.0069x +3.037 (R)20.996), the detection limits of the two modified electrodes were 2.06mM and 3.88mM, respectively (S/N-3), and the sensitivities were 0.19 μ a · mM, respectively, from the slopes of the partial straight lines of the calibration curves-1·cm-2And 0.10. mu.A.mM-1·cm-2。
FIG. 17 investigates the electrocatalytic reaction of a Nafion/HRP/polysome-Au/GCE modified electrode after 10 days of storage. As shown, the CV curves of the Nafion/HRP/polysome-Au/GCE modified electrodes after 10 days of storage showed a slightly weaker current response compared to the original signal of the curve measured before storage. The peak current of the anode is reduced from 1.63 muA to 1.54 muA, and the reduction value range is within 5 percent, which indicates that the Nafion/HRP/polysome-Au/GCE modified electrode has acceptable storage stability.
H in human serum actual sample for Nafion/HRP/polysome-Au/GCE modified electrode2O2The performance of the analysis and detection of (2) was examined. The results of the analytical tests are shown in Table 2.1, and the recovery rate is between 96.3% and 103.6%. The obtained results show that the electrode is suitable for analyzing H in actual samples2O2This is attributable to its high sensitivity, high selectivity.
TABLE 2.1 Nafion/HRP/polysome-Au/GCE modified electrode pairs for H in real samples2O2Analysis of detection Performance
In the embodiment, an ionic liquid-based liposome monomer is synthesized, an ionic liquid-based polymerized liposome is prepared through free radical polymerization, and then the polysome-Au composite material is prepared by utilizing the ion exchange property of the ionic liquid in the polymerized liposome structure. Compared with unpolymerized liposome, the composite material provided by the invention not only has better structural stability, but also has good biocompatibility and electron transport property. In addition, due to the stabilizing effect of the surface ionic liquid, AuNPs are loaded on the polysome in a monodisperse form.
The ionic liquid-based liposome, the ionic liquid-based polymerized liposome and the ionic liquid-based polymerized liposome/gold nanoparticles are combined with HRP to construct a modified electrode, and the FT-IR and UV-vis technologies are utilized to investigate the properties of the enzyme composite material. Because the composite material has good conductivity and biocompatibility, the immobilized HRP almost retains the original structure and shows higher electric activity and electrocatalysisAnd (4) acting. And the modified electrode is directly examined in electrochemical properties. The result shows that the polysome-Au composite material can effectively promote direct electron transmission between the HRP and the surface of the electrode. In addition, the Nafion/HRP/polysome-Au/GCE modified electrode pair H2O2And NaNO2The method has the advantages of good analysis and detection performances, such as high sensitivity, wide linear detection range and low detection limit. Experimental results show that the method is suitable for immobilization of HRP, and has potential application prospects in construction of biosensors or bioreactors.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
Claims (9)
1. A preparation method of a novel liposome-based horseradish peroxidase-constructed modified electrode is characterized by comprising the following steps:
preparing an ionic liquid-based polyester/gold nano composite material polysome-Au;
selecting a glassy carbon electrode, and pretreating the glassy carbon electrode;
step three, mixing a horse radish peroxidase solution HRP and a polysome/gold nano composite material polysome-Au solution, adding a Nafion solution into the mixture, and obtaining a final dispersion liquid; then, the dispersion liquid is dripped on a pretreated glassy carbon electrode to slowly evaporate redundant water; finally obtaining the modified electrode Nafion/HRP/polysome-Au/GCE constructed by the novel liposome-based horse radish peroxidase.
2. The preparation method of the novel liposome-based horseradish peroxidase-constructed modified electrode according to claim 1, wherein the step one of preparing the ionic liquid-based polyester liposome/gold nanocomposite comprises the following steps:
1) synthesizing an ionic liquid-based liposome, which consists of a hydrophilic head of cationic bromoimidazoline ionic liquid and a hydrophobic tail of two long-chain terminal alkenes;
2) synthesizing ionic liquid-based polysome, and carrying out thermal initiation cross-linking polymerization between hydrophobic tail olefins of lipid monomers to obtain the ionic liquid-based polysome;
3) preparing a polyester body/gold nano composite material polysome-Au: by utilizing the ion exchange property of ionic liquid in the polymerized liposome structure, the ionic liquid-based polyester liposome is combined with gold nanoparticles by an in-situ reduction method.
3. The preparation method of the novel liposome-based horseradish peroxidase-constructed modified electrode according to claim 2, wherein the ionic liquid-based liposome concentration in the step 1) is 1.00mg/mL, and the specific preparation method comprises the following steps:
1) sequentially adding 2-methylimidazole, triethylamine and bromo-11-carbene into toluene, and reacting for 48 hours at 90 ℃; cooling to room temperature after the reaction is finished, carrying out suction filtration to remove amine salt solids, evaporating the obtained filtrate to dryness, washing the obtained filtrate by using normal hexane for multiple times, evaporating the solvent by using normal hexane again, recrystallizing the product by using an acetonitrile-ethyl acetate mixed solvent, and carrying out vacuum drying to obtain yellowish white powder, namely a liposome monomer;
and (3) dissolving the lipid monomer in deionized water, performing ultrasonic treatment for 1 hour, and completely dispersing to obtain a clear and transparent solution, namely the ionic liquid-based liposome.
4. The method for preparing the novel liposome-based horseradish peroxidase-constructed modified electrode according to claim 2, wherein the step 2) of synthesizing the ionic liquid-based polysome specifically comprises the following steps: adding K to an ionic liquid-based liposome solution2S2O8Reacting for 24 hours at 100 ℃ under the protection of nitrogen to obtain milky white suspension; cooling the milky white suspension to room temperature, centrifuging at 8000rpm for 20min,and centrifuging and washing the obtained solid product with deionized water for three times, and then freeze-drying to obtain the solid, namely the ionic liquid-based polyester plastid.
5. The method for preparing the novel liposome-based horseradish peroxidase-constructed modified electrode according to claim 2, wherein the step 3) is specifically as follows: dispersing the ionic liquid-based polyester in deionized water, and adding HAuCl dropwise under magnetic stirring4Stirring the solution at room temperature for 30 min;
after the reaction was complete, centrifugation was carried out at 8000rpm for 15min to remove excess HAuCl4Re-dispersing the solid product obtained by centrifugation in deionized water; dropwise adding a reducing agent NaBH4Reacting for 2h to obtain an orange dispersion, centrifuging at 8000rpm for 15min, and centrifuging and washing a solid-phase product for three times; and (5) freeze-drying to obtain the polyester body/gold nano composite.
6. The preparation method of the novel modified electrode constructed by the liposome-based horseradish peroxidase according to claim 1, wherein a glassy carbon electrode with the diameter of 3mm is selected, and the pretreatment method comprises the step of firstly using Al with the diameters of 0.3 μm and 0.05 μm on a polishing cloth for the glassy carbon electrode respectively2O3Polishing the powder, and then ultrasonically cleaning the powder for 2min by using deionized water, absolute ethyl alcohol and deionized water in sequence.
7. The preparation method of the novel liposome-based horse radish peroxidase-constructed modified electrode according to the claim 1, characterized in that in the third step, the mixing volume ratio of horse radish peroxidase solution HRP and the poly plastid/gold nanocomposite polysome-Au solution is 1:1, the mixture is stirred for 10 minutes by vortex and stored for 24 hours at 4 ℃; adding an equal volume of Nafion solution into the mixture, and then stirring the obtained final dispersion solution for 5 minutes by vortex; then, the dispersion liquid is dripped on a pretreated glassy carbon electrode and covered by a beaker, so that the redundant water is slowly evaporated at the temperature of 4 ℃; obtaining the modified electrode Nafion/HRP/polysome-Au/GCE constructed by the novel liposome-based horse radish peroxidase.
8. A modified electrode Nafion/HRP/polysome-Au/GCE prepared by the preparation method of claim 1.
9. The application of the modified electrode constructed by the novel liposome-based horseradish peroxidase is characterized in that the Nafion/HRP/polysome-Au/GCE modified electrode is used for H2O2And NaNO2Carrying out catalysis and analysis detection.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115656289A (en) * | 2022-05-16 | 2023-01-31 | 沈阳师范大学 | Preparation method of cross-linked polymeric ionic liquid material and application of cross-linked polymeric ionic liquid material to horseradish peroxidase modified electrode |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110233458A1 (en) * | 2010-03-19 | 2011-09-29 | EASTERN MICHIGAN UNIVERSITY Office of Research Development | Nanoparticle dispersions with ionic liquid-based stabilizers |
| CN103940874A (en) * | 2014-03-31 | 2014-07-23 | 西北师范大学 | Preparation of glassy carbon electrode modified by gold nanoparticles wrapped with ionic liquid and detection on cholesterol by glassy carbon electrode |
| CN105758917A (en) * | 2016-04-07 | 2016-07-13 | 海南师范大学 | Preparation and catalytic application of Nafion/horseradish peroxidase/tricobalt tetraoxide-graphene/ionic liquid carbon paste electrode |
| CN105784992A (en) * | 2016-05-13 | 2016-07-20 | 河南大学 | Method for constructing electrochemical immunosensor employing TNS-MWCNT (TiO2 nanosheet-multi walled carbon nanotube) composite material as antibody and tracing marker carrier |
| CN106053571A (en) * | 2016-05-25 | 2016-10-26 | 辽宁大学 | Preparation and application of ionic liquid based polymerization liposome-gold nano-particle compound |
| KR20200137666A (en) * | 2019-05-31 | 2020-12-09 | 한국과학기술연구원 | Method of detecting antigen using magnetic support based bio-sensor |
-
2021
- 2021-07-13 CN CN202110788012.5A patent/CN113376233A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110233458A1 (en) * | 2010-03-19 | 2011-09-29 | EASTERN MICHIGAN UNIVERSITY Office of Research Development | Nanoparticle dispersions with ionic liquid-based stabilizers |
| CN103940874A (en) * | 2014-03-31 | 2014-07-23 | 西北师范大学 | Preparation of glassy carbon electrode modified by gold nanoparticles wrapped with ionic liquid and detection on cholesterol by glassy carbon electrode |
| CN105758917A (en) * | 2016-04-07 | 2016-07-13 | 海南师范大学 | Preparation and catalytic application of Nafion/horseradish peroxidase/tricobalt tetraoxide-graphene/ionic liquid carbon paste electrode |
| CN105784992A (en) * | 2016-05-13 | 2016-07-20 | 河南大学 | Method for constructing electrochemical immunosensor employing TNS-MWCNT (TiO2 nanosheet-multi walled carbon nanotube) composite material as antibody and tracing marker carrier |
| CN106053571A (en) * | 2016-05-25 | 2016-10-26 | 辽宁大学 | Preparation and application of ionic liquid based polymerization liposome-gold nano-particle compound |
| KR20200137666A (en) * | 2019-05-31 | 2020-12-09 | 한국과학기술연구원 | Method of detecting antigen using magnetic support based bio-sensor |
Non-Patent Citations (2)
| Title |
|---|
| 张亚 等: "辣根过氧化物酶修饰电极在离子液体[EMIM]BF_4中的直接电化学", 《化学学报》 * |
| 郑瑜 等: "基于胆碱修饰层为基底的纳米金固定过氧化物酶修饰电极的研究", 《分析化学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115656289A (en) * | 2022-05-16 | 2023-01-31 | 沈阳师范大学 | Preparation method of cross-linked polymeric ionic liquid material and application of cross-linked polymeric ionic liquid material to horseradish peroxidase modified electrode |
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