KR20140090005A - Sensors for detecting Hydrogen Peroxidase based on nano structures - Google Patents

Abstract

The invention relates to a sensor for detecting hydrogen peroxide, comprising: (a) a metal oxide coupled onto a conducting oxide substrate; and (b) myoglobin coupled to the metal oxide. The sensor for detecting hydrogen peroxide of the invention can generate a direct electrochemical signal of myoglobin on a CeO_2ITO electrode to detect hydrogen peroxide in an analysis sample accurately and precisely without effect of intermediate compound. Moreover, minute amounts of hydrogen peroxide contained in the sample can be detected since the detection limit is far lower than the conventional sensor for detecting hydrogen peroxide, and the prompt sample analysis is possible due to the very short reaction time.

Description

[0001] The present invention relates to a sensor for detecting hydrogen peroxide using nanostructures,

The present invention relates to a sensor for detecting hydrogen peroxide using a nanostructure.

Electrochemical sensors are widely used in clinical, environmental and food-related materials because of their high sensitivity, high specificity, fast reactivity and low cost (1-3). To develop efficient electrochemical biosensors, catalytic processes and chemical properties of oxidoreductases / proteins as intermediates have been studied. Direct electrochemical interaction between the redox protein substrate and the prosthetic group on the surface of the electrode is very important for the development of a biosensor with high sensitivity and high specificity based on the mechanism of the biological redox reaction and the bioelectrocatalytic reaction (4-6). However, the direct electrochemical signal measurement of most redox proteins in uncoated electrodes is very difficult due to various factors. First, the electro-active site of the redox protein is located deep within the protein by an insulated protein bark, thereby greatly lengthening the distance between the electro-active site and the underlying electrode. Therefore, it is difficult to transport the electrons directly to the electrodes. Moreover, biological matrices become unstable when interacting with electrode surfaces (7, 8)

Second, when proteins are adsorbed onto the electrode surface, there is often a loss of electrochemical activity and bioactivity (9). An effective way to measure direct enzymatic electrochemical signals is to insert the enzyme into the cast film on the deformed electrode surface (10, 11). Efforts have been made to develop new support materials and methods in order to develop electrochemical reactions by immobilizing proteins on the electrode surface and developing highly sensitive, highly specific and durable electrochemical biosensors. Electron mediators, which are small, diffusible redox species, are often required to enable interaction between the protein redox region and the electrode surface (12,13). One alternative to developing a mediator-free biosensor is a chemically modified electrode (CME) that produces a more sensitive and specific membrane on the electrode surface, and CME itself allows direct electron transfer And provides an electronic mediation function (14,15). It is also important to immobilize the functional material on the electrode surface and to maintain the biological / chemical activity of the immobilized molecule in direct electrochemical signal measurement. CMEs made from porous materials are widely used in direct electron transfer studies of proteins because of their wide surface area, excellent biocompatibility, and specificity of non-toxic properties (16,17). The porous material provides a favorable microenvironment for the protein and improves the electron transfer between the electro-active site of the protein and the electrode surface.

Thin metal oxide films, which are nanostructures, can be used in a wide variety of applications, including their specific forms, particle size, porosity, and physical, electrical and magnetic properties in various fields such as catalysis, gas sensing and separation, power storage and generation, Has attracted attention due to its characteristics. The functional properties of nanoscopic metal oxide films have recently been highlighted in different fields such as photovoltaic (18), gas sensor (19) and biosensor (20). Over the past several decades, the properties of the porous and porous structure, such as the highly regular structure, ultra-high surface area, distribution of pores in the mesopore range, adjustable micropore size, Many efforts have been made to synthesize, identify, and apply a shaped porous material (mesoporous). There have been advances in the stability of mesoporous materials in the structural, structural and morphological control of mesoporous materials and in recent applications of catalysis, adsorption and sensing (21, 22).

Research based on nanostructured materials for sensor applications is a promising area of research. _{TiO 2 (23), MnO 2} (24), Fe 3 O 4 (25), ZnO (26) and a film of other similar nano-structures is H ₂ O ₂ and other biological compounds, such as 3,4-dihydroxy-phenyl It was used in a new-generation biosensor for detecting acetic acid, ascorbic acid, guanine, l-tyrosine, acetaminophen and? -NADH (27). The present inventors have studied the direct electron transfer of redox proteins using a porous ceria film which not only has a large surface area but also can deform the electrode surface. The present invention is the first to disclose direct electrochemical signals of heme proteins on metal oxide films of such modified electrodes.

In the present invention, the formation of a cerium oxide (CeO ₂ ) film on the surface of indium tin oxide (ITO) by electrodeposition, the direct electrochemical signal of myoglobin (Mb) on the surface of CeO ₂ / ITO, Were investigated. Although Mb and but as used in this sensing mechanism, a metal oxide film, the present invention is the first attempt for the selective detection of a CeO ₂ / ITO electrode Mb direct electrochemical signals and hydrogen peroxide on. The present inventors analyzed nanostructure films using a scanning electron microscope (SEM) and an atomic force microscope (AFM). The electrochemical characteristics of the Mb / CeO ₂ / ITO electrodes and their hydrogen peroxide sensing methods were evaluated by cyclic voltammetry CV), differential pulse voltammetry (DPV), and I / t measurements.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop a biosensor having excellent specificity and sensitivity to hydrogen peroxide. As a result, a nanoscale film of porous cerium dioxide (CeO ₂ ) having a large surface area was electrodeposited on an indium tin oxide (ITO) substrate, and a microglobin (Mb) , And then developed a biosensor that detects hydrogen peroxide by measuring direct electrochemical signals of Mb. By observing the electrochemical properties of Mb immobilized on a processed film using cyclic voltammetry and differential pulse voltammetry, the present inventors have found that the sensor of the present invention is an electrocatalyst of hydrogen peroxide (electrocatalytic) action of Mb without a separate electron mediator. That is, the sensor for detecting hydrogen peroxide of the present invention does not use any reagent and is not affected by the reagent-free intermediate compound, and thus shows excellent safety and high sensitivity in measuring hydrogen peroxide.

Accordingly, an object of the present invention is to provide a sensor for detecting hydrogen peroxide.

It is another object of the present invention to provide a method for detecting hydrogen peroxide.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method of manufacturing a semiconductor device, comprising: (a) a metal oxide bonded on a Conducting Oxide substrate; And (b) Myoblobin bound to the metal oxide.

The present inventors have made extensive efforts to develop a biosensor having excellent specificity and sensitivity to hydrogen peroxide. As a result, it has been found that a nano-structured film of porous cerium dioxide (CeO ₂ ) (Mb) was immobilized on the indium tin oxide (ITO) substrate, and then a direct electrochemical signal of Mb was measured to develop a biosensor for detecting hydrogen peroxide. By observing the electrochemical properties of Mb immobilized on a processed film using cyclic voltammetry and differential pulse voltammetry, the present inventors have found that the sensor of the present invention is an electrocatalyst of hydrogen peroxide (electrocatalytic) action of Mb without a separate electron mediator. That is, since the sensor for detecting hydrogen peroxide of the present invention does not use any reagent and is not influenced by the reagentless intermediate compound, it is confirmed that the sensor exhibits excellent safety and high sensitivity in measuring hydrogen peroxide.

The conductive oxide included in the sensor of the present invention may be coated on a substrate. For example, a solid substrate made of an organic polymer or inorganic materials may be used. Any substrate that can be used can be used regardless of the surface characteristics.

The organic polymer may be selected from the group consisting of a polyamide homopolymer or copolymer (e.g., nylon), a heat resistant plastic fluorinated polymer (e.g., polyvinylidene fluoride PVDF), a polyvinyl halide (E.g., polyvinyl chloride PVC), polysulfone, cellulose materials such as paper, nitrocellulose or cellulose acetate, polyolefins, polyacrylamides such as poly (N-isopropylacrylamide), polyglycolic acid (PGA) (PDA), poly-4-hydroxybutyrate, carbon, carbon nanotubes, colloids, and natural polymers such as polylactic acid (PLA), polyglycine 910 (Vicryl), polygluconate Inorganic materials such as glass, quartz, silica, silver, gold, aluminum, copper, other silicon-containing materials (e.g., , A silicon oxide or nitride), metal oxides (e.g., aluminum oxide), metal alloys, Si / SiO ₂ wafer, germanium and gallium arsenide.

The metal that can be used as a solid substrate in the present invention is not particularly limited, and any metal used in the art can be used. In addition, the shape, size, and chemical composition of the surface of the metal solid substrate used in the present invention are not particularly limited. As used herein, the term " metal solid phase substrate " has the meaning of covering all of the metals, metal oxides, and alloys. For example, the metal solid substrate that can be used in the present invention includes gold, silver, copper, platinum, copper, aluminum, an alloy of the above metals (for example, an alloy of gold and copper), or a metal oxide substrate. The metal solid substrate includes not only a solid substrate composed of a metal but also a substrate having a metal surface coated.

The solid substrate may be non-porous or have a certain degree of porosity. In the case of a porous material, the size of the pores may be nano or micro units.

According to a specific embodiment of the present invention, a conductive oxide electrode having a conductive oxide deposited on a glass substrate can be produced. The electrode can be formed on the surface of the substrate by a variety of methods known in the art and is preferably formed by electron beam evaporation, vacuum evaporation, thermal evaporation, spin coating, vacuum evaporation, sputtering sputtering, low pressure chemical vapor deposition, sol-gel synthesis, electroplating or electroless plating.

As used herein, the term 'transparent conductive oxide (TCO)' is one of conductive films that is optically transparent and has electrical conductivity. Conductive films used in photovoltaic cells are made of inorganic and organic materials. Typically, the inorganic film is comprised of a common conductive oxide layer such as phosphorous-tin oxide, fluorine-doped tin oxide, and zinc zinc oxide. The organic film was developed using a carbon nanotube network and graphene together with a polymer network such as poly (3,4-ethylindyoxythiophene and derivatives thereof) which can be produced to have high transparency to infrared rays. On average, this application uses a conductive electrode material that is superior to 80% transmission of incident light and has a conductivity higher than 10 ³ S / cm for efficient carrier transport.

Wherein the conductive oxide is selected from the group consisting of phosphorus-tin oxide, phosphorous-zinc oxide, tin oxide, zinc oxide, copper-aluminum oxide, copper-gallium oxide, copper- scandium oxide, copper-chromium oxide, Lt; / RTI > is at least one conductive oxide selected from the group consisting of silver-phosphorous oxides.

Phosphorus oxides are one of the most widely used transparent conductive oxides due to their electrical and optical transparency. In addition, it is easy to deposit into a thin film, and as with all transparent conductive films, increase in thickness and increase in the concentration of a charge carrier necessitate a trade-off between conductivity and transparency because it increases the conductivity while reducing the transparency of the material.

The indium tin oxide (ITO or tin-donor phosphorus oxide) of the present invention is a solid solution of phosphorus (III) oxide (In ₂ O ₃ ) and tin (IV) oxide (SnO ₂ ) 90% In ₂ O ₃ and 10% SnO ₂ . In ^{3 +} ion sites, electrons are displaced while Sn ^{4 +} ions are substituted, resulting in an increase in electron concentration and a low specific resistance. Since such a substitution process requires high energy, according to a common general technique in the art, when the substrate is deposited on a glass substrate, the substrate temperature is raised to a temperature of 300 DEG C or more so that tin ions can easily replace the phosphorus ion sites It is possible to fabricate a phosphorus-tin oxide electrode having a low resistance.

When the metal oxide is electrodeposited on the surface of the phosphorus-tin oxide substrate, the metal oxide may form a metal oxide film layer having a porous structure. When hydrogen peroxide is present in the sample to be analyzed, redox reaction occurs in the myoblobin by electrocatalytic activity of the hydrogen peroxide, and [Fe ^{3 +} and Fe ^{2 +} ], Myoblobin ), Hydrogen peroxide can be detected by measuring an electrochemical signal by electron transfer between a metal oxide and a conductive oxide.

The metal oxide electrodeposited on the surface of the phosphorus-tin oxide substrate may be any of various known metal oxides. Examples of the metal oxide include cerium oxide, copper oxide, titanium oxide, cobalt oxide, tungsten oxide, molybdenum oxide, Niobium, vanadium oxide, transition metal oxides or transition metal oxides can be used.

The electrodeposition or deposition of the metal oxide may be performed by a potentiostatic or constant potential method, a galvanostatic or constant current method, or a potential sweep method.

On the other hand, the surface of the metal oxide of the sensor for detecting hydrogen peroxide of the present invention binds myoglobin protein. The myoglobin may be directly bonded to the surface of the metal oxide or may be indirectly bonded by a solid electrolyte bonded to the surface of the metal oxide. The solid electrolyte is a material that serves as an electron transfer path between the metal oxide and myoglobin, for example, a polymer solid electrolyte or a metal atom. As the polymer solid electrolyte, various known materials can be used. For example, Nafion, PEO (polyethylene oxide), or PEG (polyethylene glycol) can be used as a solid electrolyte.

The myoglobin is a redox enzyme containing a heme group in which a redox reaction can take place, and a redox reaction of Fe ^{3 +} and Fe ^{2 +} occurs due to peroxide. The myoglobin may form one or two or more layers on the surface of the metal oxide.

On the other hand, one of the greatest features of the present invention is that it can detect even a very small amount of hydrogen peroxide in the sample. In addition, since hydrogen peroxide in the sample does not require an additional " reaction promoter " for causing an electron transfer reaction to directly act on the redox protein contained in the sensor of the present invention and shortening the reaction time, Hydrogen peroxide can be detected very accurately and quickly. According to a specific embodiment of the present invention, the minimum detection limit of the sensor of the present invention is 0.6 μM, and the minimum reaction time is 8-10 seconds, which depends on the state of the sample to be analyzed, the concentration of hydrogen peroxide in the sample, Differences can be shown. In addition, since the sensor for detecting hydrogen peroxide of the present invention reaches a steady state current within 10 seconds, the sample can be analyzed quickly.

The sensor of the present invention can be fabricated in a form including a myoglobin / metal oxide / conductive oxide capable of causing a redox reaction by hydrogen peroxide on the working electrode. In addition, the sensor of the present invention can be manufactured by using the conductive oxide, A counter electrode and a reference electrode may be additionally included in addition to the working electrode. The counter electrode may be platinum or platinum, and the reference electrode may be Ag / AgCl or Ag / AgCl / KCl _sat .

According to another aspect of the present invention, the present invention provides a hydrogen peroxide detection method comprising the steps of:

(a) preparing a sensor for detecting hydrogen peroxide of the present invention;

(b) contacting the sample to be analyzed with the sensor; And

(c) measuring an electrical signal generated in the step (b).

The hydrogen peroxide detection method of the present invention is based on the same technical idea as the hydrogen peroxide detection sensor of the present invention described above, and the description common to both of them is omitted in order to avoid the excessive complexity of the present specification.

When the sample is brought into contact with the sensor of the present invention, the oxidation-reduction reaction in the protein immobilized on the surface of the sensor occurs due to the electrocatalytic activity of the hydrogen peroxide contained in the sample, and the conductivity of the conductive metal oxide Hydrogen peroxide can be detected by measuring electrochemical signals by electrodeposited metal oxide and electron transfer between the proteins.

The electrocatalytic reduction mechanism of hydrogen peroxide is as follows:

Mb (Fe ^{3 +} ) + H ₂ O ₂ → Intermediate I (Fe ^{4 +} = O) + H ₂ O (i)

Intermediate I (Fe ^{4 +} = O) + H ⁺ + e → Intermediate II (ii)

Intermediate II + H ⁺ + e → Mb (Fe ^{3 +} ) + H ₂ O (iii)

The electrical signal can be measured by a variety of known methods such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), linear sweep voltammetry, LSV, and choronoampermetry. [0041] [36]

According to one specific embodiment of the present invention, the current signal was measured after adding hydrogen peroxide to the working electrode Mb / CeO ₂ / ITO under PBS (pH 7.0) and the linear regression equation was as follows:

I _p (μA) = 13.54 [H ₂ O ₂ mM] + 6.33, R ² = 0.9825. (Iv)

The sensitivity of Mb / CeO ₂ / ITO electrode to hydrogen peroxide was 5.4 μA. mM ^-1. cm ^-2 , and the detection limit of hydrogen peroxide is 0.6 μM, which allows detection of hydrogen peroxide at a lower concentration than in the case of using other sensing methods.

The method for detecting hydrogen peroxide of the present invention can be used not only for detecting the presence of hydrogen peroxide present in a sample but also for comparing the amount of hydrogen peroxide contained in the sample. That is, since the reaction with the sample can be observed in real time using the sensor of the present invention, the amount of hydrogen peroxide can be compared by comparing the electrochemical signals generated when the amounts of hydrogen peroxide contained in each sample are the same, Can be predicted.

In addition, since the sensor of the present invention has a very high specificity to hydrogen peroxide, it is not affected by other chemicals such as ascorbic acid, uric acid, sodium nitrite and sodium bicarbonate in the analytical sample, and thus has high selectivity for hydrogen peroxide . This characteristic is also related to the fast reaction rate of the sensor of the present invention, and the sensor of the present invention can reach a steady state current within 10 seconds by an electrical signal (current) generated by the presence of peroxide.

As used herein, the term " sample " includes, but is not limited to, a biological sample, a chemical sample or an environmental sample.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a sensor for detecting hydrogen peroxide using redox proteins, myoglobin and CeO ₂ .

(b) Since the sensor for detecting hydrogen peroxide of the present invention can generate a direct electrochemical signal of Mb on the CeO ₂ / ITO electrode, the hydrogen peroxide in the analytical sample can be accurately and precisely detected without being influenced by the intermediate compound have.

(c) Further, since the detection limit is much lower than that of the conventional sensor for detecting hydrogen peroxide, a very small amount of hydrogen peroxide contained in the sample can be detected, and the reaction time is also very short, enabling rapid sample analysis.

(d) According to the present invention, the sensor of the present invention can be produced in a simple and simple process and at a low cost. Since the excellent electrochemical catalysis by hydrogen peroxide contained in the sample itself avoids a complicated detection process, Is possible.

1A shows the electroadhesive electrodeposition of a cerium nanostructure film on the ITO surface as a cyclic voltammogram (CV): 0.01 M, (-) 0.1 M and (-) 0.2 M) . Scan speed (scan speed) 50 mVs ^-1 . CeO ₂ film SEM image on ITO electrode: (b) 0.01 M, (c) 0.1 M and (d) 0.2 M.
Fig. 2 shows SEM and AFM images of the ITO surface (a, d), the cerium film (b, e) on ITO, and myoglobin (c, f) immobilized on the cerium dioxide film on ITO.
Figure 3 shows a UV-Vis absorption spectrum showing XRD patterns of (a) ITO, (b) cerium dioxide on the ITO surface, and (c) CeO ₂ deposited films on the ITO surface.
Figure 4a shows the cyclic voltammogram at a scan rate of 50 mVs < ^-1 > under 10 mM PBS (pH 7.0). (i) non-deposited ITO and (ii) CeO ₂ deposited film on the ITO surface; Figure 4b shows the effect of a scan speed of 0.01 mVs on CeO ₂ / ITO surface mVs ^{-1 -1} to 0.1; FIG. 4C shows the CV of Mb / CeO ₂ / ITO in a wide potential difference range, and CeO ₂ And the redox properties of the Mb layer; 4d shows the effect of scan speed on Mb / CeO ₂ / ITO electrodes at 0.02 mVs ^-1 to 0.2 mVs ^-1 ; 4e shows plots of cathodic ( i _pc ) and anodic ( i _pa ) peak currents versus (scan speed) ^1/2 of the Mb / CeO ₂ / ITO electrodes; 4f shows plots of cathodic ( i _pc ) and anodic ( i _pa ) peak currents versus log (scan rate) ^1/2 of Mb / CeO ₂ / ITO electrodes.
Figure 5 shows a differential pulse voltammogram under 10 mM PBS (pH 7.0). _{(a) Mb / CeO 2 /} ITO (b) CeO 2 / ITO , and (c) Mb / ITO surface.
(c) and (d) show the linear swept voltamogram of CeO ₂ / ITO and Mb / ITO surface under 10 mM PBS (pH 7.0), respectively.
Figure 6a shows the function of the hydrogen peroxide for 5 μL hydrogen peroxide (100 mM) was added under 5 mL PBS (10 mM, pH 7.0) as the differential pulse voltammogram of Mb / CeO ₂ / ITO electrode. FIG. 6b shows the results of Mb / CeO ₂ / ITO electrode when 10 μL hydrogen peroxide (100 mM) was added to 5 mL of PBS (10 mM, pH 7.0) and then stirred constantly at -0.3 V potential under nitrogen purging (I / t) curves of FIG. The embedded graphs show the reaction times of (i) the calibration curve and (ii) the Mb / CeO ₂ / ITO electrode for the detection of hydrogen peroxide, respectively.
FIG. 7a shows the results of measurement of the concentration of uric acid (0.2 mM, AA), L-ascorbic acid (0.2 mM, AA), sodium nitrite (0.2 mM, NaNO ₂ ) and sodium bicarbonate 0.2 mM, NaHCO ₃ ) in the order of 5 μL, followed by constant stirring. 7B is a graph showing the relationship between the current (I / B) when 10 μL and 20 μL of uric acid, L-ascorbic acid, sodium nitrite, and sodium bicarbonate were sequentially added to 5 μL of hydrogen peroxide under 5 mL of PBS (pH 7.0) t) curves.
FIG. 8 is a schematic view illustrating a process of processing an Mb / CeO ₂ / ITO electrode for developing a hydrogen peroxide sensor of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Methods

1. Experimental material

Myoglobin (Mb) from the horse's heart was purchased from Sigma-Aldrich (USA) and used without further purification. Cerium (III) nitrate hexahydrate, Ce (NO ₃ ) ₃ .6H ₂ O) (purity 99%), Nafion and perfluorinated resin solution (20 wt%) in a mixture of aliphatic alcohol and water, Was purchased from Sigma-Aldrich. Hydrogen peroxide was purchased from the Counterpowder (Korea) and diluted in deionized water. For all electrochemical experiments, 10 mM PBS buffer solution (pH 7.0) was used. All solutions were prepared under purified deionized water (18 M? Cm) using a Milli-Q system (Millipore, USA) (28. 29).

2. Electrode cleaning

A 400 nm thick glass plate coated with ITO (indium tin oxide) (20 Ω / cm 2) was used. The transparent electrode was washed with Triton X-100 / water (1: 5, v / v), water and ethanol for at least 40 min. The ITO electrode was heated in a NH ₄ OH: H ₂ O ₂ : H ₂ O (1: 1: 5) solution at 80 ° C. for 40 minutes and then washed with water and then dried under a nitrogen gas (30). Electrical deposition of CeO ₂ (cerium dioxide) film and fixation of myoglobin (Mb) were performed on a room temperature electrochemical bath composed of 0.01, 0.1 and 0.2 M Ce (NO ₃ ) ₃ .6H ₂ O solution. The CeO ₂ film was electrochemically deposited using a CHI 660A electrochemical workstation. Electrochemical deposition was performed on a 3-electrode cell using ITO as a working electrode, and a platinum wire and an Ag / AgCl electrode were used as a counter electrode and a reference electrode, respectively. The deposition voltage was used in the range of -0.6 V to -1.8 V with respect to the reference voltage. After the deposition process, the coated film was washed with deionized water and then dried under nitrogen gas.

A 0.1 mg / mL myoglobin (Mb) suspension (20 μL) was dropped on the CeO ₂ / ITO electrode surface and water was evaporated for 6 hours to form a uniform film. Then, 10 μL of 0.1 wt% Nafion solution was added thereto, followed by drying at 4 ° C. Nitride films are commonly used in chemically modified electrodes and are used because of their specific ion-exchange, differentiation, chemical resistance and biocompatibility. The Mb / ITO electrode was made of CeO ₂ / ITO. The electrodes were fixed at 4 캜 for one day, then washed with deionized water and dried with nitrogen gas. The dried electrode was stored at 4 ° C before use.

3. Properties of processed film on ITO surface

The surface of the deposited or non-deposited film was analyzed by SEM measurement using a JEOL JSM S-4300 microscope (20 kV). An Atomic Force Microscope (AFM) image was obtained using a Nanoscope IV / Multimode (Digital Instrument, USA). All AFM images were recorded in tapping mode using a silicon cantilever with a resonance frequency (fo) of 250-300 kHz.

The prepared sample was recorded with a Cu Ka radiation (?) Of 1.54056 A using a Rigaku X-ray diffractometer at 0.02 / / sec per scanning rate 2 ?. The X-ray diffractometer was used at 40 kV and 150 mA. For the optical measurement, a JASCO-V-660 spectrophotometer was used. The size of the substrate is 20 × 20 mm ² . The non-deposited ITO substrate was used as a reference sample.

4. Electrochemical activity and hydrogen peroxide measurement

Cyclic voltammetry (CV) and linear sweep voltammetry (LSV) were performed using Mb / CeO ₂ / ITO substrate as working electrode, platinum as counter electrode and Ag / AgCl / KCl _sat Lt; / RTI > using a three-electrode system. A CHI 660A potentiostat with common electrochemical software was used in the experiments. Measurements of current (I / t) and differential pulse voltammetry (DPV) were performed on Mb / CeO ₂ / ITO electrodes using various hydrogen peroxide concentrations. The potential difference was measured to be -0.30 V and 10 μL of 100 mM hydrogen peroxide was continuously added under 5 mL of 10 mM PBS (pH 7.0) buffer. During the measurement of the current, convection was carried out with magnetic stirring at 600 rpm. The buffer solution added in the experiment was removed with high purity nitrogen.

Experiment result

One. ITO  Formation and optimization of metal oxide films on the surface

Porous CeO ₂ Film Electrical deposition while increasing the concentration of the cerium nitrate solution in the potential range from -0.6 V to -1.8 V against _{Ag / AgCl / KCl sat (0.01} M, 0.1 M and 0.2 M) rotation voltammetry using different working electrodes . As can be seen in FIG. 1A, reverse scan hysteresis means that the metal oxide film is deposited on the ITO surface. Hysteresis and current density were also increased with increasing concentration of cerium nitrate solution. In order to optimize the metal oxide for the development of the sensor with high sensitivity, we optimized the electrode performance for the metal oxide by loading the metal oxide at the deposition concentration showing higher current sensitivity after protein fixation. A biosensor containing different concentrations of CeO ₂ was fabricated and tested while the amount of myoglobin was maintained constant in the immobilized matrix. 0.1 M CeO ₂ film showed a higher current response. CeO ₂ The increase in concentration did not show high current intensity. 1B-1D illustrate the use of various CeO2 < RTI ID = 0.0 _> SEM images according to concentrations (0.01 M, 0.1 M and 0.2 M) are shown. The deposited films were observed at low concentrations like cerium nanoparticles and the particle size was increased with increasing concentration of cerium nitrate solution added to the porous structure.

2. Identification of protein fixation on the processed surface

Formation of the cerium oxide film on the machined surface (FIGS. 2B and 2E) and deposition of myoglobin (FIGS. 2C and 2F) are confirmed by SEM and AFM analysis. Both of these assays show that electro-deposition can form CeO ₂ films on ITO surfaces without any breaks. The ITO surface showed a rough surface as a whole as compared to the deposited film, and fragments of rough surface with a clear three-dimensional structure over the entire surface (FIGS. 2A and 2D). Nucleation of the oxides from the precursor solution occurs at the molecular level, and the film has a constant and dense microstructure. Also, when the protein and Nafion are immobilized on the structure of the rough surface, a protein molecule with a smoother surface is adsorbed (Figs. 2C and 2F); In addition, AFM analysis shows that molecules such as myoglobin (Mb) are well adsorbed on the CeO ₂ / ITO surface.

3. CeO ₂ / ITO  Electrode surface UV - vis  And X-rays Diffraction analysis

The ultraviolet spectra of the ITO non-deposited ITO showed an absorption peak at 550 nm, which is typical for the ITO surface, and an absorption edge of CeO ₂ / ITO of 360 nm (Figs. 3a and 3b). In previous studies, the absorption band of CeO ₂ / ITO was observed at 270400 nm wavelength and the charge transfer transition was observed from CeO ₂ O ^{2 -} (2p) to Ce ^{4 +} (4f) orbitals (33). Figure 3c also shows the nano-structured CeO2 < RTI ID = 0.0 _> The film was subjected to X-ray diffraction analysis (XRD). The XRD pattern results CeO ₂ The film is crystalline and has diffraction peaks corresponding to (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes, which is a pure cubic fluorite structure of it can be classified as CeO _2. The intensity and location of the peaks were in accordance with the literature (JCPDS card, No. 4-0593) (34). It is also shown that the ITO surface is decomposed into XRD peaks corresponding to (2 1 1), (2 2 2), (4 0 0), (4 1 1), (4 3 1) . The strength and location of the peaks corresponded to the JSPDS card file (JCPDS le: 01-088-2160).

4. CeO ₂ / ITO  And Mb / CeO ₂ / ITO  Electrochemical Properties of Electrode

The rotational voltage (CV) of the CeO ₂ / ITO and non-deposited ITO electrodes is shown in FIG. 4A.

No apparent redox peak was observed in the non-deposited ITO electrode (i), but apparent reversible redox peaks (Epa = 0.6 V and Epc = 0.3) were observed in the electrodeposited CeO ₂ -stable electrode (ii) V) appeared. These peaks were due to the formation of Ce ^{3 +} / Ce ^{4 +} couple. The results show that the surface of the ITO electrode has CeO ₂ , Which means excellent electrochemical properties of the film. FIG. 4B shows the rotational voltage current of CeO ₂ / ITO having different scan rates (scanning speed) under pH 7.0 PBS. Approximately symmetric redox peaks were observed and appeared at nearly the same height as the redox peak current at a scan rate of 0.01-0.1 mVs ^-1 . The redox peak current and the scan speed I _pc (μA) = (0.0071) (mVs ^-1 ) + 0.3597 (R ² = 0.9931) and I _pa (μA) = (-0.0062) (mVs ^-1 ) -0.5309 (R ² = 0.9952)]. This phenomenon can be attributed to CeO ₂ Electron transfer between the film and ITO is a surface-controlled electrochemical process. Figure 4c shows the direct electrochemical properties of Mb immobilized on the CeO ₂ / ITO electrode surface under 10 mM PBS. Two pairs of quasi-reversible reductive oxidation peaks were observed, indicating that CeO ₂ And Mb. The peaks at -0.38 and -0.2 V represent the Fe ³⁺ / Fe ²⁺ redox center of Mb. The form potential (E ^{0 '} ) of Mb was calculated from the midpoint of the reduction and oxidation peak potential (-290 mV (for Ag / AgCl)) and the potential difference between peak and peak ( E _p ) was 180 mV. These results confirm that Mb exhibits a quasi-reversible electrochemical reaction on CeO ₂ / ITO electrodes. Scanning speed analysis on Mb / CeO ₂ / ITO electrodes was performed in the range of 20-200 mVs ^-1 , under 10 mM PBS pH 7.0. As a result, the negative polarity and bipolar peak currents were nearly symmetric in the range of 0.01-0.2 mVs ^-1 and were linearly proportional to the square root of the scan speed. Figures 4d and 4e illustrate a typical divergence pattern. Regression equations are I _pc = 0.1449 僚^1/2 -0.528 (μA, Vs ^-1 , R ² = 0.982) and I _pa = -0.138 ν ^1/2 + 0.487 (μA, Vs ^-1 , R ² = 0.983) . E _p = f (log ν) showed two straight lines with a slope of -2.3RT / αn F and 2.3RT / (1α) n F as negative and positive peaks, respectively (FIGS. 4e and 4f). Therefore, the α value is calculated to be 0.533 from the linear slope according to the following equation (35).

(1)

(One)

The apparent heterogeneous electron transfer rate constant ( k _s ) was calculated using the following Laviron equation (35).

(2)

(2)

α is an electron transfer coefficient, n is the number of electrons, E _p is a dispersion of the reducing peak oxidation, and ν is the scan rate. The electron transfer rate constant ( k _s ) was calculated to be 1.57 s ^-1 .

According to the Ravi Long equation (36), the surface coverage (Γ) of the Mb immobilized on the surface of the CeO ₂ / ITO-modified electrode is 5.142 × 10 ¹¹ mol cm ² , And is involved in the electron transfer process. DPV and LSV were measured to investigate the electrochemical properties and constituents of Mb / CeO ₂ / ITO under 10 mM PBS. Figure 5a shows the DPV of Mb / CeO ₂ / ITO by potential sweeps in the range of -0.15 to 0.20 V (for Ag / AgCl), with CeO ₂ (0.10 V) and Mb (-0.075 V) A sharp reduction peak due to reduction appeared. 5B and 5C show that the DPVs of the CeO ₂ / ITO and Mb / ITO electrodes show peaks at -0.05 V and -0.125 V, respectively, and show a slight change in the peak potential distribution as compared with the hybrid structure. This phenomenon is expected to be manifested by the properties of the redox substance in a molecular environment that cause a change in the redox potential or by the surface fixation of the protein on the electrode surface. Thus, LSV shows the formation of apparent CeO ₂ / ITO, Mb / ITO and Mb / CeO ₂ / ITO electrode surfaces.

5. For hydrogen peroxide detection Mb / CeO ₂ / ITO  Analysis of electrodes

DPV and current (I / t) measurements were performed on Mb / CeO ₂ / ITO to investigate the electrocatalytic reaction to hydrogen peroxide.

The electrocatalytic reduction mechanism of hydrogen peroxide is as follows:

Mb (Fe ^{3 +} ) + H ₂ O ₂ → Intermediate I (Fe ^{4 +} = O) + H ₂ O (3)

Intermediate I (Fe ^{4 +} = O) + H ⁺ + e → Intermediate II (4)

Intermediate II + H ⁺ + e → Mb (Fe ^{3 +} ) + H ₂ O (5)

10 mM PBS under Mb / CeO After the addition of 100 mM hydrogen peroxide in 10 μL ₂ / ITO, was measured DPV signals (Fig. 6a). Whenever hydrogen peroxide was added, the Mb / CeO ₂ / ITO electrode showed excellent electrocatalytic reaction for detecting hydrogen peroxide. We selected a potential difference of -0.15 V which is lower than the reduction potential of Mb to fully reduce the test compound. Therefore, a chronoamperometric response was measured at a potential difference of -0.3 V. The biosensor under the blank buffer solution was tested with different hydrogen peroxide, and then a chronoamperogram was performed using nitrogen purging to avoid oxygen interference in order to obtain a calibration curve (FIG. 6B). The calibration graph (insert graph [i] in Figure 6b) is for hydrogen peroxide at a concentration of 0.2 mM to 5 mM (up to a dynamic range of 3 mM). The linear regression equation is:

I _p (μA) = 13.54 [H ₂ O ₂ mM] + 6.33, R ² = 0.9825.

The sensitivity of Mb / CeO ₂ / ITO electrode to hydrogen peroxide is 5.4 μA. mM ^-1. cm ^-2 and the detection limit of hydrogen peroxide is 0.6 μM. As shown in Table 1, the detection limit is lower than with other electrochemical methods (37-42).

- Fixed Matrix Detection substance Detection limit Sensitivity Reusability References One HRP / RANI / CeO ₂ / ITO H ₂ O ₂ 50 mM 159.6 nAmM ^-1 8 weeks 37 2
HRP / NanoCeO ₂ / ITO
H ₂ O ₂ 0.5 μM 8.44 x 10 ^-3 μA (μM cm ² ) ^-1 5 weeks 38 3 CeO ₂ / Au H ₂ O ₂ 1-30 μM 0.4806 nA μmol L ^-1 - 39 4 Lipase / nano-CeO ₂ / ITO Triglyceride 50-500 mg / dL 0.195 mA / mg dL cm < ² > 3 months 40 5 BSA / r-IgGs / nanoCeO ₂ / ITO Mycotoxin 0.5 ng dl ^-1 1.24 μA / ng dl ^-1 cm ² - 41 6
ssDNA / CeO ₂ / CHIT / GC
DNA 1.0 x 10 ^-11 molL ^-1
- - 42 7 Mb / CeO ₂ / ITO H ₂ O ₂ 0.6 μM 5.4 μA mM ^-1 cm ^-2 2 weeks Invention

The insertion graph [II] of FIG. 6B shows the reaction current in the steady state after adding hydrogen peroxide, and 95% of the steady state current appeared after about 10 seconds. The processed biosensor responded within 10 s and was judged to be suitable for in vivo diagnosis (38).

)의 효소 활성에 대한 산화금속 미세환경의 영향에 있어 정량적인 정보를 얻기 위해 라인웨버-버크식을 이용하여 효소-기질반응속도론에 반영하였다(39). A response plateau of the calibration curve appeared when the hydrogen peroxide concentration was > 3 mM, indicating the characteristics of the MichaelisMenten kinetic mechanism. Michaelis-Menten constant (

(39), using the Rhee Weber-Burk equation to obtain quantitative information on the effect of the oxidized metal microenvironment on the enzyme activity of the enzyme.

(6)

(6)

)는 3.15 mM이며, 이는 바이오센서가 과산화수소에 대하여 높은 친화도를 가짐을 나타낸다. I _ss is the steady-state current after substrate addition, C is the bulk concentration of the substrate, and I _max is the maximum current under substrate saturation. Apparent Michaelis-Menten constant (

) Is 3.15 mM, indicating that the biosensor has a high affinity for hydrogen peroxide.

Selectivity is also an important part of biosensor utilization. Ascorbic acid, uric acid, sodium nitrite and sodium bicarbonate were measured to evaluate the selectivity of the electrode at 2 mM. 5 [mu] L hydrogen peroxide (0.2 mM) was added continuously with the disturbance factors to perform an amperometric response of the biosensor (Fig. 7A). Indicating that the disturbance factor has a minimal effect on the hydrogen peroxide reaction. When 10 μL of the interfering substance and 5 μL of hydrogen peroxide (0.2 mM) were added together, a negligible reaction appeared, indicating that the biosensor had a high selectivity for hydrogen peroxide 7b). The stability of Mb / CeO ₂ / ITO was measured by monitoring the current response after cycling the deformed electrode over several cycles in the potential range of +0.3 to -0.25 V. In addition, the reproducibility of the biosensor was investigated in the linear range of hydrogen peroxide. After 1 week of storage at 4 ° C, the Mb / CeO ₂ / ITO electrode did not react with 10 mM hydrogen peroxide at pH 7.0 PBS. After two weeks, the sensor maintained 95% of the initial response to hydrogen peroxide. Thus, CeO ₂ and Nafion act as adhesives for film-forming and fixing Mb. As a result of 6 measurements using 3 mM hydrogen peroxide in 10 mM PBS (pH 7.0), the relative standard deviation was 2.3%, which means that the biosensor of the present invention is reproducible.

Argument

The present inventors synthesized a CeO ₂ film having a large surface area by fixing myoglobin (Mb) as a modified ITO electrode. Electrodeposited CeO ₂ provided electron transfer between Mb and the underlying IOT surface to electrochemically detect hydrogen peroxide. Electrochemical biosensors were able to measure hydrogen peroxide at a very low detection limit (0.6 μM). The high sensitivity sensor (5.4 μA.mu.- ¹ .mu.M) can be obtained by minimizing the background current level as an optimized process under a wide surface area, increased electrocatalytic activity of the CeO ₂ modified surface, conductivity due to metal oxide materials and various environmental conditions. cm < ^-2 & gt ^; ). Simple and simple processing, excellent electrochemical catalysis that shows low cost and high selectivity makes it possible to avoid complicated detection process and signal detection. The platform developed in the present invention has advantages in terms of simplicity, efficiency and speed for target measurement, which is essential for the development of a user-friendly sensor platform, and it is highly sensitive and highly specific And can be usefully used for detection.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

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Claims (9)

(a) a metal oxide bonded on a Conducting Oxide substrate; And
(b) a sensor for detecting hydrogen peroxide comprising myoblobin bound to the metal oxide.
The method of claim 1, wherein the conductive oxide is selected from the group consisting of phosphorous-tin oxide, phosphorous-zinc oxide, tin oxide, zinc oxide, copper-aluminum oxide, copper-gallium oxide, copper-scandium oxide, , Copper-yttrium oxide, and silver-phosphorus oxide.
The sensor according to claim 2, wherein the conductive oxide is phosphorus-tin oxide.
The sensor for detecting hydrogen peroxide according to claim 1, wherein the metal oxide is electrodeposited on the substrate of the conductive oxide.
The sensor for detecting hydrogen peroxide according to claim 1, wherein the myoglobin is indirectly bound to the metal oxide by a polymer solid electrolyte bonded to the surface of the metal oxide.
The sensor for detecting hydrogen peroxide according to claim 1, wherein the sensor further comprises a counter electrode and a reference electrode.
2. The sensor for detecting hydrogen peroxide according to claim 1, wherein the sensor has a detection limit of 0.6 [mu] M.
2. The sensor for detecting hydrogen peroxide according to claim 1, wherein the sensor reaches a steady state current within 10 seconds.
A hydrogen peroxide detection method comprising the steps of:
(a) preparing a sensor for detecting hydrogen peroxide according to any one of claims 1 to 8;
(b) contacting the sample to be analyzed with the sensor; And
(c) measuring an electrical signal generated in the step (b).

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