KR20080011481A - Hydrogen peroxide sensor using nano-metal particle and the method for producing said system - Google Patents
Hydrogen peroxide sensor using nano-metal particle and the method for producing said system Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
Description
도1. 폴리아닐린-Au-폴리프로필렌 필름 (PANI-Au-PP) 전극 위에 서양고추냉이 퍼옥시다제(HRP) 효소의 고정화 방법Figure 1. Method for Immobilizing Horseradish Peroxidase (HRP) Enzyme on Polyaniline-Au-Polypropylene Film (PANI-Au-PP) Electrode
도2. 전기화학적 방법에 의한 폴리아닐린(PANI)의 합성 메카니즘Figure 2. Synthesis Mechanism of Polyaniline (PANI) by Electrochemical Method
도3. 방사선법에 의해 제조된 나노골드입자의 FE-TEM 이미지Figure 3. FE-TEM image of nanogold particles prepared by radiotherapy
도4. Au-폴리프로필렌 필름전극에서 폴리아닐린(PANI)의 합성하는 동안의 순환전압전류법(CV)에 의한 순환전압전류곡선Figure 4. Cyclic Voltammetry of Cyclic Voltammetry (CV) During the Synthesis of Polyaniline (PANI) on Au-Polypropylene Film Electrode
도5. Au-폴리프로필렌 필름 전극위에 성장된 폴리아닐린(PANI)와 그 위에 로딩된 Au 나노입자의 FE-SEM 이미지와 SEM-EDX 데이터Figure 5. FE-SEM image and SEM-EDX data of polyaniline (PANI) grown on Au-polypropylene film electrode and Au nanoparticles loaded on it
도6. 폴리아닐린-Au-폴리프로필렌 필름 (PANI-Au-PP) 전극에 로딩된 Au나노입자에 고정화된 서양고추냉이 퍼옥시다제(HRP)의 FE-SEM이미지Figure 6. FE-SEM image of horseradish peroxidase (HRP) immobilized on Au nanoparticles loaded on polyaniline-Au-polypropylene film (PANI-Au-PP) electrode
도7. H2O2의 농도에 따른 서양고추냉이 퍼옥시다제(HRP) 효소전극의 크로노암페로메트리 응답Figure 7. Chronoamperometric Responses of Horseradish Peroxidase (HRP) Enzyme Electrode According to H 2 O 2 Concentration
본 발명은 나노금속입자를 이용하여 활성산소를 용이하게 실시간으로 측정할 수 있는 전극센서를 제작방법에 관한 것 이다.The present invention relates to a method for manufacturing an electrode sensor that can easily measure the active oxygen in real time using nano metal particles.
생물체는 환경스트레스 및 병원성 미생물의 침입 등에 의하여 다양한 종류의 활성 산소를 생성한다 특히 이들 활성산소는 여러 가지 생체반응의 시그널로서 작용 할 뿐 만 아니라 노화나 돌연사의 원인되기도 한다. 따라서 특정조건에 있는 생체가 스트레스를 받고 있는 상태인지를 확인하고 나아가 적절한 조치를 취하여 건강과 원활한 성장을 유도할 수 있게 된다.Organisms generate various kinds of free radicals by environmental stress and invasion of pathogenic microorganisms. Especially, these free radicals not only act as signals of various biological reactions but also cause aging and sudden death. Therefore, it is possible to check whether the living body under a certain condition is under stress and take appropriate measures to induce health and smooth growth.
활성 산소를 측정하는 방법은 여러 가지 알려져 왔으나 생체세포를 파괴하여 측정하거나 화학반응을 이용한 간접적인 측정방법이 주류를 이루고 있으나 정량적이지 못하며 실시간 측정에는 다소 무리가 있다.There have been many known methods for measuring free radicals, but indirect measurement methods using chemical reactions or destruction of living cells are the mainstream, but they are not quantitative and are somewhat unreasonable in real time measurement.
이를 개선하기 위하여 한국특허출원 제 10-200-62244호와 같이 전자자기공명장치를 이용한 방법이 개발되었으나 측정 비용이 높아 널리 사용되지 못하고 있다.In order to improve this, a method using an electronic magnetic resonance device, such as Korean Patent Application No. 10-200-62244 has been developed, but the measurement cost is not widely used.
한 편, 전극표면에 바이오물질, 특히 효소를 고정화하여, 이 효소와 상호 반응하는 화합물을 센싱하는 연구가 최근에 활발히 수행되어 오고 있다 (J.-L. He, Yu. Yang등 Sensors and Actuators B:Chemical (2006) 114 (1), 94-100, Z. Dai, J. Chen 등 Cancer Detection and Prevention (2005) 29(3), 233-240, A. Zhou 와 J. Muthuswamy Sensors and Actuators B: Chemical (2004) 101(1-2), 8-19). 그러나 상용의 봉전극 (Au, Pt, Carbon전극)의 경우, 구입가격이 최소 50만원선으로 매 우 고가일 뿐아니라, 전극 표면에 효소를 고정화하여 사용한 경우, 봉 전극을 재사용하기 (recycling) 위하여, 효소를 제거하여야만 한다. 이 경우 일반적으로 미세한 알루미나 파우더를 이용하여 전극표면 연마하게 되는데, 미세 알루미나 가격도 100g에 50만원선을 호가한다. 또한 봉전극표면을 연마하는 경우, 많은 시간이 소요 될 뿐만 아니라, 연마한 전극 표면이 거칠어서 측정 중에 자주 에러가 발생한다. 또한, 전극 표면에 링커(Linker)를 사용하여 효소를 고정화하여도, 효소가 고정화 되었는지를 분석할 수 없는 단점을 가지고 있다. 따라서, 봉전극의 경우 바이오센서로서의 상업화가 어려운 단점을 가지고 있다.On the other hand, researches on sensing biomaterials, especially enzymes, on the surface of the electrode to react with compounds that interact with the enzyme have been actively conducted (J.-L. He, Yu. Yang et al., Sensors and Actuators B). : Chemical (2006) 114 (1), 94-100, Z. Dai, J. Chen et al. Cancer Detection and Prevention (2005) 29 (3), 233-240, A. Zhou and J. Muthuswamy Sensors and Actuators B: Chemical (2004) 101 (1-2), 8-19). However, in case of commercial rod electrodes (Au, Pt, Carbon electrodes), the purchase price is very high, at least 500,000 won, and in order to recycle rod electrodes when enzymes are immobilized on the electrode surface. The enzyme must be removed. In this case, the surface of the electrode is generally polished using fine alumina powder. The price of fine alumina is also 500,000 won per 100g. In addition, when polishing the rod electrode surface, not only it takes much time, but also the surface of the polished electrode is rough, so that errors frequently occur during measurement. In addition, even if the enzyme is immobilized by using a linker (Linker) on the electrode surface, there is a disadvantage that can not be analyzed whether the enzyme is immobilized. Therefore, the rod electrode has a disadvantage in that it is difficult to commercialize as a biosensor.
이를 극복하기 위하여 최근 나노금입자 표면에 효소를 고정화시키고, 이 나노입자를 전극표면에 고정화하여 센싱하는 연구가 최근 보고 되었다 (S. Zhang, N. Wang 등 Sensors and Actuators B: Chemical (2005) 109(2), 367-374, Y.-C. Luo와 J.-S. Do Biosensors and Bioelectronics (2004) 20(1), 15-23). 이 경우 나노크기의 골드입자가 효소에 고정화하는 경우 효소의 삼차원적 구조가 유지되어 효소의 효율이 잘 보존되는 특성을 가지고 있으나 아직 활성 산소와 같이 반응시간이 빠른 인자를 측정하는 센서는 확립되어 있지 않다.In order to overcome this, a recent study has been reported on immobilizing an enzyme on the surface of a nano-gold particle and immobilizing the nanoparticle on an electrode surface (S. Zhang, N. Wang et al., Sensors and Actuators B: Chemical (2005) 109). (2), 367-374, Y.-C. Luo and J.-S. Do Biosensors and Bioelectronics (2004) 20 (1), 15-23). In this case, when nano-sized gold particles are immobilized on the enzyme, the three-dimensional structure of the enzyme is maintained and the efficiency of the enzyme is well preserved. However, a sensor for measuring a fast reaction time such as active oxygen is not established. not.
한편 방사선을 이용하여, Pd, Ag, Pt-Ru, Pd-Ag등의 나노입자을 제조하고, 이 나노입자를 이용하여, 연료전지 촉매, 수소화 반응 촉매, C-C 커플링(coupling) 반응 촉매 등에 이용연구를 수행하여 왔다 (S.-D. Oh, S. Lee등 Colloids Surf. A. (2006), 275 (1-3), 228-233, S.-D. Oh, K. R. Yoon 등 Journal of Non-Crystalline Solids (2006) 352, 355-360, M. I. Kim, H. O. Ham 등 Journal of Molecular Catalysis B: Enzymatic (2006) 39(1-4), 62-68). 그러나, 나노금입자를 제조하여 효소를 전극에 연결하는 링커(Linker)로의 사용에 관한 예는 아직 없다.On the other hand, nanoparticles such as Pd, Ag, Pt-Ru, and Pd-Ag are prepared using radiation, and the nanoparticles are used for fuel cell catalysts, hydrogenation catalysts, CC coupling catalysts, and the like. (S.-D. Oh, S. Lee et al. Colloids Surf. A. (2006), 275 (1-3), 228-233, S.-D. Oh, KR Yoon et al. Journal of Non- Crystalline Solids (2006) 352, 355-360, MI Kim, HO Ham et al. Journal of Molecular Catalysis B: Enzymatic (2006) 39 (1-4), 62-68). However, there is no example of the use of the nano gold particles as a linker for connecting the enzyme to the electrode.
본 발명은 상기와 같은 문제점을 해결하기 위하여 나노금속을 이용한 전극을 개발하여 활성산소를 저렴하게 실시간으로, 효과적으로 측정 할 수 있는 전극센서의 제작 방법을 제공하는데 그 목적이 있다.An object of the present invention is to provide a method for manufacturing an electrode sensor that can effectively measure the active oxygen in real time, by developing an electrode using a nano-metal to solve the above problems.
상기와 같은 목적을 달성하기 위하여 다음과 같이 구성되어졌다.In order to achieve the above object has been configured as follows.
(a) 방사선법에 의한 나노골드입자의 제조(a) Preparation of Nano Gold Particles by Radiation Method
수용액에 HAuCl4염 (100 ppm) 과 콜로이드 안정화제로 PVP, 그리고 라디칼 억제제로서 이소프로판올을 넣은 후, 질소 버블링 한 후 방사선을 조사하였다. 조사후, UV-Vis측정한 결과 520nm의 플라스몬 피크 (plasmon peak)가 나타나서 나노금입자가 형성되는 것을 확인 할 수 있었다. 또한, FE-TEM으로 분석 평가하였다.HAuCl 4 salt (100 ppm), PVP as a colloidal stabilizer, and isopropanol as a radical inhibitor were added to the aqueous solution, followed by irradiation with nitrogen after irradiation with nitrogen. After irradiation, UV-Vis measurement showed that the plasma peak of 520nm (plasmon peak) appeared to form the nano-gold particles. It was also analyzed and evaluated by FE-TEM.
(b) 주형법에 의한 평면전극의 제조(b) Preparation of Planar Electrode by Molding Method
먼저, 금 전극을 제조하기 위하여 주형틀을 제작하였다. 주형틀의 크기는 다양한 종류를 선정하였다. 이 주형틀을 이용하여 폴리프로필렌 필름 (PP film)에 금(Au)을 스퍼터링(sputtering)하여 Au-폴리프로필렌 필름 전극을 제조하였다. 순환전압전류법(CV)에 의한 폴리아닐린(PANI)의 합성 및 서양고추냉이 퍼옥시다제(HRP)의 고정화한다.First, a mold was manufactured to manufacture a gold electrode. Various types of molds were selected. Au-polypropylene film electrodes were prepared by sputtering gold (Au) on a polypropylene film (PP film) using this template. Synthesis of polyaniline (PANI) by cyclic voltammetry (CV) and fixation of horseradish peroxidase (HRP).
0.1M H2SO4 전해질 용액에 0.1M 아닐린(aniline)을 이용하여 Au-PP전극에 CV를 사용하여 폴리아닐린(PANI)을 성장시켰다 (이하 PANI-Au-PP 전극라고 명명함). Au colloid 용액 (100 ppm)에 PANI-Au-PP 전극을 6시간 침전 시켜 Au 나노입자를 로딩 시켰다. 로딩 된 정도를 FE-SEM 및 SEM-EDX를 통하여 확인하였다.Polyaniline (PANI) was grown by using CV on the Au-PP electrode using 0.1M aniline in 0.1MH 2 SO 4 electrolyte solution (hereinafter referred to as PANI-Au-PP electrode). The PANI-Au-PP electrode was precipitated in Au colloid solution (100 ppm) for 6 hours to load Au nanoparticles. The degree of loading was confirmed by FE-SEM and SEM-EDX.
Au 나노파티클-PANI-Au-PP 전극에 서양고추냉이 퍼옥시다제(HRP) (1mg/mL) 용액에 12시간 침전시켜 서양고추냉이 퍼옥시다제(HRP)을 로딩 하였다. 한편, 글루타릭 디알데하이드 (0.wt-％ to HRP)을 이용하여 PANI-Au-PP 전극표면에 HRP (2.0mg)을 12시간 반응시켜 공유결합 시켰다. 서양고추냉이 퍼옥시다제(HRP)의 고정화 여부를 FE-SEM으로 확인 하였다 (도 1 ).A horseradish peroxidase (HRP) was loaded by Au precipitated in a horseradish peroxidase (HRP) (1 mg / mL) solution for 12 hours on a nanoparticle-PANI-Au-PP electrode. Meanwhile, HRP (2.0 mg) was covalently bonded to the surface of PANI-Au-PP electrode for 12 hours using glutaric dialdehyde (0.wt-% to HRP). Wasabi peroxidase (HRP) was confirmed by the immobilization of FE-SEM (Fig. 1).
이하 첨부된 도면을 참조하여 본 발명의 구성을 상세하게 설명한다.Hereinafter, with reference to the accompanying drawings will be described in detail the configuration of the present invention.
도 1은 서양고추냉이 퍼옥시다제(HRP)효소 전극의 제조과정을 나타내고 있다. 서양고추냉이 퍼옥시다제(HRP)효소의 고정화는 Au나노입자를 통한 물리적 흡착과 글루타릭 알데히드 (gutaric dialdehyde)를 통한 공유결합을 이용하였다.Figure 1 shows the manufacturing process of horseradish peroxidase (HRP) enzyme electrode. The immobilization of horseradish peroxidase (HRP) enzyme was performed by physical adsorption through Au nanoparticles and covalent bonds through glutaric aldehyde (gutaric dialdehyde).
도 2는 (에우코에머랄딘 (leucoemeraldine)/에머랄딘 (emeradine)과 에머랄딘(emeraldine)/퍼니그라닐린 (pernigraniline)의 구조및 반응과정을 나타내고 있다.Figure 2 shows the structure and the reaction process of (leucoemeraldine / emeradine and emeraldine / emrandine / pernigraniline.
도 3은 방사선조사법으로 제조한 나노금콜로이드의 사진과 FE-TEM image을 나타내고 있다. 크기는 대략 5nm이하이고 분포가 매우 고른 금나노입자 임을 알 수 있었다. UV측정결과 문헌값과 유사하게 520 nm에서 흡수 피이크가 나타나서, 나노 입자가 성공적으로 제조됨을 확인 할 수 있었다.Figure 3 shows a photograph and FE-TEM image of the nano-gold colloid prepared by the irradiation method. The size was about 5nm or less and the distribution was very even gold nanoparticles. As a result of UV measurement, absorption peak appeared at 520 nm, similar to the literature value, and it was confirmed that nanoparticles were successfully manufactured.
도 4는 Au-폴리프로필렌 전극 표면에 폴리아닐린(PANI)가 성장해가는 순환전압전류곡선을 나타내고 있다. 2가지 형태의 환원 및 산화 피크가 나타나고 있다.4 shows a cyclic voltage current curve in which polyaniline (PANI) grows on an Au-polypropylene electrode surface. Two types of reduction and oxidation peaks are shown.
도 5는 Au-폴리프로필렌전극에서 성장한 전도성 폴리머인 폴리아닐린(PANI)의 FE-SEM image을 나타내고 있다. 폴리아닐린(PANI)는 로드형의 파이버로서 직경이 10nm정도 임을 확인 하였다. 이러한 폴리아닐린(PANI)에 물리적 방법으로 효소를 고정화하기 위하여, 도 3에 보여지는 Au나노입자를 로딩시킨 FE-SEM 데이터 및 SEM-EDX데이타를 도 5에 나타내고 있다. Au나노입자가 로딩되는 경우 성장한 폴리아닐린(PANI)의 형태학적인 변화가 보여졌다. 즉 폴리아닐린(PANI)표면의 결정성이 커짐을 알 수 있었다. FE-SEM 이미지에서 나타나듯이 폴리아닐린(PANI)표면에 나노금입자가 로딩된 것을 확인 할 수 있었다. 또한 SEM-EDX데이타에서 나타나듯이 나노금입자가 잘 로딩 된 사실을 알 수 있었다.5 shows an FE-SEM image of polyaniline (PANI), a conductive polymer grown on an Au-polypropylene electrode. Polyaniline (PANI) is a rod-shaped fiber was confirmed that the diameter of about 10nm. In order to immobilize the enzyme to the polyaniline (PANI) by physical methods, FE-SEM data and SEM-EDX data loaded with Au nanoparticles shown in FIG. 3 are shown in FIG. 5. Morphological changes of polyaniline (PANI) grown when Au nanoparticles were loaded were shown. In other words, it was found that the crystallinity of the polyaniline (PANI) surface is increased. As shown in the FE-SEM image, it was confirmed that nano gold particles were loaded on the polyaniline (PANI) surface. Also, as shown in SEM-EDX data, the nano gold particles were well loaded.
도 6은 금나노입자 표면에 서양고추냉이 퍼옥시다제(HRP)효소를 고정화한 전극의 FE-SEM 이미지를 나타내고 있다. 보는 바와 같이, 금나노입자 표면에 효소가 고정된 사실을 FE-SEM 이미지로 확연이 알 수 있었다.6 shows an FE-SEM image of an electrode immobilized with horseradish peroxidase (HRP) enzyme on the surface of gold nanoparticles. As can be seen, the fact that the enzyme is immobilized on the surface of the gold nanoparticles was confirmed by the FE-SEM image.
1. 도 7은 제조된 서양고추냉이 퍼옥시다제(HRP)효소 전극을 이용한 H2O2의 농도에 따른 chronoamperometric response로서 H2O2의 농도가 높아짐에 따라 전류 밀도가 증가하는 것을 볼 수 있다.1. FIG. 7 shows that the current density increases as the concentration of H 2 O 2 increases as a chronoamperometric response according to the concentration of H 2 O 2 using the prepared horseradish peroxidase (HRP) enzyme electrode. .
이하 실시예를 통해 본 발명에 의한 측정방법을 보다 상세히 설명한다. 하기 실시예는 본 발명을 설명하기 위한 예 일뿐 이에 의해 본 발명의 기술적 범위가 변경되거나 축소되는 것은 아니다.Hereinafter, the measuring method according to the present invention will be described in more detail. The following examples are only examples for describing the present invention, and thus the technical scope of the present invention is not changed or reduced.
실시예1.Example 1
서양고추냉이 퍼옥시다제(HRP)효소 전극을 이용하여, 과산화수소 (H2O2) 의 센싱 정도를 과산화 수소의 농도에 따라서 측정하여 보았다. 도. 7은 제조한 전극에 대한 과산화수소의 농도의 센싱 정도를 측정한 것이다.Using horseradish peroxidase (HRP) enzyme electrode, the degree of sensing of hydrogen peroxide (H 2 O 2 ) was measured in accordance with the concentration of hydrogen peroxide. Degree. 7 measures the degree of sensing of the concentration of hydrogen peroxide on the prepared electrode.
과산화수소의 농도가 증가함에 따라 전류밀도가 높아짐에 따라, 우리가 제조한 전극은 과산화수소를 센싱하는데 훌륭한 전극임을 확인 하였다As the concentration of hydrogen peroxide increased and the current density increased, we confirmed that the electrode we fabricated was an excellent electrode for sensing hydrogen peroxide.
본 발명에 따른 나노금속 전극센서는 활성산소를 아주 간편하게 적은 비용으로 실시간 측정을 가능하게 한다.The nanometal electrode sensor according to the present invention enables real-time measurement of active oxygen at a very simple cost.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101354975B1 (en) * | 2012-09-25 | 2014-01-24 | (주)바이오닉스 | Active oxygen analyzer |
| CN105016295A (en) * | 2015-06-11 | 2015-11-04 | 江苏科技大学 | Gold nanoparticle array structure produced by controlled heating and illumination synthesis and synthetic method thereof |
| KR20210122979A (en) * | 2020-04-02 | 2021-10-13 | 아주대학교산학협력단 | Biosensing chip to analyze cell-derived materials during cell culture, and method for quantitative analysis of cell activity using the same |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101354975B1 (en) * | 2012-09-25 | 2014-01-24 | (주)바이오닉스 | Active oxygen analyzer |
| CN105016295A (en) * | 2015-06-11 | 2015-11-04 | 江苏科技大学 | Gold nanoparticle array structure produced by controlled heating and illumination synthesis and synthetic method thereof |
| KR20210122979A (en) * | 2020-04-02 | 2021-10-13 | 아주대학교산학협력단 | Biosensing chip to analyze cell-derived materials during cell culture, and method for quantitative analysis of cell activity using the same |
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