압타머 성형 이중기능링커와 금나노입자의 응집 기반 식품 내 위해요소 검출 시스템 개발

학위논문(박사) -- 서울대학교대학원 : 농업생명과학대학 농생명공학부, 2021.8. 이준엽.The importance of developing analytical methods for rapid detection of harmful microorganisms and harmful substances in the food industry is becoming increasingly prominent as the industry develops. Currently, analysis of hazardous substances is performed through various methods. However, there remains a need for the development of simple, convenient, and rapid detection methods, because the food industry belongs to a relatively low value-added industry and many low-skilled workers are employed. Gold nanoparticle aggregation based colorimetric detection has been attracting attention due to their simplicity, rapidity, and accuracy. However, it also has a drawback in that it exhibits lower sensitivity compared to electrochemical or optical methods. To overcome these shortcomings, a colorimetric detection method based on a bifunctional linker that exhibits improved visual signals with higher sensitivity has been proposed. However, there were still disadvantages in that the research so far has been focused on the detection of microorganisms and the diversity of the bifunctional linker is insufficient. In this study, first, a bifunctional linker-based detection method was applied to detect a new target, Ara h 1, a peanut allergen. The target allergen was rapidly detected below the ED01 threshold, which is the minimum that can be recognized by allergy patients in cookies. By showing that it could detect less than 0.19 mg/mL within 45 minutes, including the extraction time, it was shown that the detection system based on the bifunctional linker can be applied to the detection of various hazards beyond microorganisms. Second, a novel bifunctional linker based on an aptamer, a DNA sequence that could selectively recognize a target, and a colorimetric detection method using this linker were devised. The bifunctional linker was designed by decorating the surface of silica nanoparticles with an aptamer, and on the contrary, for the gold nanoparticles, a DNA sequence complementary to the aptamer was decorated on the surface. In addition, dispersion stability and specific binding ability were imparted through additional surface molding of glutathione. Finally, a detection method for gluten, which induces celiac disease, was developed using a newly designed aptamer based bifunctional linker and gold nanoparticles whose surface was modified with a complementary DNA sequence. Through the development of this detection method, the concentration in the extract of gliadin, a constituent of gluten, was detected up to 0.5 ug/mL by naked eye without an analytical instrument. Although there are still many areas to be studied using the aptamer based bifunctional linker, this study is expected to pave the way for expanding the scope of application of rapid detection methods based on bifunctional linkers.식품산업에서 식품내 여러 위해요소의 신속한 검출의 중요성은 식품산업의 성장과 함께 커지고 있다. 현재 여러 분석법이 위해요소 검출에 사용되고 있지만, 식품산업은 저숙련 노동자가 많이 종사하고있고 부가가치가 상대적으로 낮은 산업에 속하기 때문에 더 쉽고, 더 간편하고, 더 저렴한 신속검출법의 개발이 여전히 필요하다. 금 나노입자 응집기반 비색검출법은 금 나노입자가 가시광선을 흡수하는 특성을 이용하기 때문에 특별한 분석장비 없이 눈으로도 검출 결과를 확인할 수 있다는 장점이 있다. 또한 금 나노입자는 저렴하게 제작이 가능하기 때문에 식품 내 위해요소 신속검출법 개발에 활용되기 적합하다. 하지만 금나노입자 응집 기반 비색검출법은 분석장비를 활용하는 전기적 또는 광학적 분석법보다 검출한도가 낮으며, 식품처럼 여러 물질이 혼합된 시료에서는 높은 선택성을 가지는 검출 시스템을 구축하는 것이 어렵다는 단점이 있다. 이러한 금나노입자를 활용한 비색검출법의 단점을 극복하기 위해, 금나노입자의 대규모 응집을 유도하여 신호를 증폭하는 동시에 선택적인 검출 대상 인지가 가능한 이중기능링커를 활용한 금나노입자 응집기반 신속 검출법에 대한 연구가 진행되고 있다. 하지만 아직 대부분의 연구가 미생물 검출에 한정되어 있으며 항체 기반 이중기능링커를 사용해야하기 때문에 한정된 확장성을 지니는 단점이 존재한다. 본 논문에서는 이러한 이중기능링커 활용의 어려움들을 극복하고자 하였다. 먼저, 금 나노입자의 응집반응으로 땅콩 알러젠인 Ara h1을 실제 식품인 쿠키에서 알러지 환자가 인지할 수 있는 최소한도인 ED01 기준치 이하로 신속히 검출하는 연구를 수행하였다. 추출시간을 포함하여 45분 이내에 0.19 mg/mL 이하의 땅콩 단백질을 검출할 수 있음을 보임으로서 이중기능링커 기반의 검출 시스템이 기존의 미생물뿐만 아니라 다른 위해물질 검출에도 적용 될 수 있음을 보였다. 두번째로 압타머 기반 이중기능링커와 금나노입자 표면 개질법을 고안하여 새로운 형태의 이중기능링커 기반 신속검출법을 개발하는 연구를 수행하였다. 항체와 같이 특정 단백질을 인지할 수 있는 기능이 있는 DNA 서열인 압타머(Aptamer)를 실리카 나노입자에 표면에 성형하고 이에 상보적인 DNA 서열을 금나노입자 표면에 성형함으로써 항체 없이도 타겟 물질 인지와 금나노입자 응집유도가 가능한 이중기능링커와 이를 활용한 응집 시스템을 고안하였다. 마지막으로, 압타머 기반 이중기능링커를 활용하여 셀리악 병의 원인 물질인 gliadin을 신속하게 검출하는 연구를 수행하였다. 새로 개발된 이중기능링커 기반 비색검출법을 활용하여 0.5 μg/mL 이하의 gliadin을 분석도구 없이 눈으로 검출할 수 있었다. 새로 개발된 압타머 기반 이중기능링커와 이를 활용한 신속검출법은 아직 연구되어야 할 부분이 많이 남아있지만 이를 활용하여 보다 다양한 식품 내 위해요소를 대상으로 이중기능링커 기반 신속검출법을 확장 적용할 수 있을 것이라 기대된다.Chapter I. Literature Review 1 I-1. Introduction 2 I-1-1. The necessity of developing a novel detection system for food safety screening 2 I-1-2. Material features of gold nanoparticles in detection method development 4 I-1-3. Methods for the synthesis of gold nanoparticles 5 I-1-4. Optical property of gold nanoparticles 7 I-1-5. Functionalization of gold nanoparticles 8 I-2. Gold nanoparticles based colorimetric detection systems 11 I-2-1. Principle of gold nanoparticles based colorimetric detection systems 11 I-2-1-1. Analysis based aggregation of gold nanoparticle 12 I-2-1-2. Analysis based anti-aggregation of gold nanoparticle 13 I-2-2. Application of gold nanoparticles detection systems in food safety 14 I-2-2-1. microorganisms 14 I-2-2-2. Microbial toxins 14 I-2-2-3. Chemical hazards 18 I-3. Motivation and Aims of the Dissertation 23 I-4. References 25 Chapter II. Visible on-site detection of Ara h 1 by the switchable-linker-mediated precipitation of gold nanoparticles 39 II-1. Introduction 40 II-2. Materials and Methods 43 II-2-1. Materials 43 II-2-2. Instrumentation 44 II-2-3. Preparation of gold nanoparticles (AuNP) 44 II-2-4. Preparation of streptavidin-coated gold nanoparticles (stAuNP) 45 II-2-5. Detection of Ara h 1 in standard solution 49 II-2.6. Preparation of model cookies for a detection experiment on a real matrix 49 II-2.7. Detection of Ara h 1 in cookie extract solution 50 II-2-8. Verification of the selectivity of the detection method 51 II-2.9. Statistical analysis 51 II-3. Results and Discussion 54 II-3-1. Overall detection procedures 54 II-3-2. Hypothesised mechanism for Ara h 1 detection 57 II-3-3. Detection of Ara h 1 in PBS using the proposed method 60 II-3-4. UV-Vis spectroscopic approach to Ara h 1 detection 66 II-3.5. Selectivity of the detection method 70 II-3.6. Detection of Ara h 1 in model cookies 73 II-4. Conclusions 79 II-5. References 80 Chapter III. Development of novel aptamer-based switchable linker and complementary DNA modified gold nanoparticle 85 III-1. Introduction 86 III-2. Materials and Methods 88 III-2-1. Materials 88 III-2-2. Instrumentation 88 III-2-3. Preparation of gold nanoparticles (AuNP) 88 III-2-4. Preparation of aptamer and complementary DNA (cDNA) modified gold nanoparticles 89 III-2-5. Removal of Nonspecific DNA Adsorption on surface of AuNP 90 III-2-6. Synthesis of nanoparticles functionalized with amino groups 90 III-2-7. Removal of Nonspecific DNA Adsorption on surface of SNP 91 III-3. Results and Discussion 92 III-3-1. Hypothesised structure of aptamer modified switchable linker and complementary nanoparticle 92 III-3-2. DNA modification on the surface of AuNP 95 III-3-3. Reducing nonspecific DNA adsorption on surface of AuNP 95 III-3-4. Selection of core nanoparticle material for switchable linker development 102 III-3-5. DNA modification on the surface of SNP 103 III-3-6. Determination of the aggregate-forming ability of the bifunctional linker 106 III-4. Conclusions 110 III-5. References 111 Chapter IV. Development of colorimetric detection method for gliaidn using aptamer based bi-functional linker 115 IV-1. Introduction 116 IV-2. Materials and Methods 118 IV-2-1. Materials 118 IV-2-2. Instrumentation 119 IV-2-3. Preparation of gold nanoparticles (AuNP) 119 IV-2-4. Preparation of aptamer and complementary DNA (cDNA) modified gold nanoparticles 120 IV-2-5. Removal of Nonspecific DNA Adsorption on surface of AuNP 121 IV-2-6. Synthesis of nanoparticles functionalized with amino groups 121 IV-2-7. Removal of Nonspecific DNA Adsorption on surface of SNP 122 IV-2-8. Extraction of gliadin 123 IV-2-9. Detection of gliadin in diluted extract solution 123 IV-2-10. Verification of the selectivity of the detection method 124 IV-2-11. Statistical analysis 124 IV-3. Results and Discussion 125 IV-3-1. Hypothesised mechanism for gliadin detection 125 IV-3-2. Detection of gliadin in extract solution using the proposed method 130 IV-3-3. UV-Vis spectroscopic approach to gliadin detection 135 IV-3-4. Selectivity of the detection method 139 IV-4. Conclusions 143 IV-4. References 144 국문 초록 150박

Rapid visible detection of Ara h1, major peanut allergen, using functionalized gold nanoparticle aggregation

학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부 식품생명공학전공, 2016. 8. 최영진.There has been a lot of research aimed to develop an easy, rapid and sensitive detection method for useful/harmful biomaterials and microorganism in food system. However, current techniques have their own problems such as complicate sample preparation of time-consuming steps, low sensitivity, and necessity of trained experts or sophisticated instruments. In the preceding studies, a rapid and simple detection method using gold nanoparticle (AuNPs) was developed. This method is carried out in two steps. In the first, the target is mixed with switchable linker (SLs) with various concentrations. After the first step, streptavidin coated AuNPs (stAuNPs) are added to the above solutions. Because of high affinity of biotin to streptavidin, biotins on the SLs connect with the streptavidin on the stAuNPs and form huge aggregates. Without the target, region of forming aggregates is naturally appeared by the relationship between the concentrations of the SLs and stAuNPs. However, when the targets are added, the SLs covered with targets have less ability to connect with stAuNPs than naked SLs. This resulted in a change of the quantitative relationship mentioned above, which led to shift of the aggregation region. Therefore, in this quantitative relationship, the number of streptavidin on stAuNPs is one of the key factors. In this study, streptavidin on stAuNPs was modified to improve this detection system. Firstly, the effect of the number of streptavidin on stAuNPs on the range exhibiting a visible color change (REVC) and reaction time was investigated. The less number of streptavidin on stAuNPs allowed REVC forming region to shift to lower concentrations of SLs and it took more time for making enough aggregation. However, by agitating during they are forming REVC, time for forming REVC was reduced. Finally, after optimized streptavidin modifying level on stAuNPs. Obtained particles are used to detect Ara h1, major peanut allergen. This detection system using modified stAuNPs could detect very low concentration of Ara h1 (10 nM), without sophisticated equipment. It was also tested with peanut extract and could be distinguished by naked eye in solution to 100-fold diluted extract. Thus, the result of this study could be used as valuable references for improving and applying this detection system to real food system.Ⅰ. INTRODUCTION 1 Ⅱ. MATERIALS AND METHODS 5 2.1. Materials 5 2.2. Instrumentation 5 2.3. Preparation of gold nanoparticles (AuNPs) 6 2.4. Preparation of streptavidin-coated gold nanoparticles (stAuNPs) 7 2.5. Detection of streptavidin in PBS buffer solution 9 2.6. Extraction of Ara h1 from peanut and preparation of standard Ara h1 solution 9 2.7. Detection of Ara h1 in standard solution and peanut extract 10 2.8. Selectivity of the colorimetric detection 12 Ⅲ. RESULTS AND DISCUSSION 12 3.1. Principle for REVC control by adjusting of the number of streptavidin on stAuNPs 12 3.2. Effect of the number of streptavidin of AuNPs surface on the detection system 16 3.2.1. Determination of streptavidin on stAuNPs in the detection system 16 3.2.2. Change of REVC forming region in the detection system 18 3.2.3. Effect of agitation on the detection system 25 3.2.4. Analysis of normalized absorbance ratio in the detection system 27 3.3. Application for detection of Ara h1 32 3.3.1 Detection of Ara h1 in standard solution 32 3.3.2 Detection of Aar h1 in peanut extract 34 3.3.3 Selectivity of the detection system 36 Ⅳ. CONCLUSION 38 Ⅴ. REFERANCE 40 Ⅵ. 국문 초록 43Maste

Global Politics of Technology and Knowledge Networks_The Cases of Linux and Wikipedia

이 글에서는 오픈소스형 기술지식 네트워크를 둘러싼 표준경쟁의 세계정치를 리눅스와 위키피디아의 사례를 중심으로 고찰했다. 특히 본 논문은 기존의 지식구조에 대한 도전을 관념, 이익, 제도의 차원에서 전개되는 표준경쟁으로 파악함으로써 그것이 가지는 세계정치적 의미를 다면적으로 살펴보았다. 우선, 리눅스가 세계화와 윈텔리즘의 지식구조에 대항하는 담론을 제공했다면, 위키피디아는 기존의 계몽주의 백과전서에 담겨 있는 지식구조에 대한 대항담론이자 지식생산에 배타적으로 참여하는 주류 지식인의 독점적 권위에 대한 도전이라고 할 수 있다. 둘째로 리눅스와 마이크로소프트의 경쟁은 사실상의 소프트웨어 시장에서의 기술표준 장악을 위한 경쟁이라고 할 수 있으며, 위키피디아에서 전형적으로 나타나는 탈근대적 분류체계의 부상은 근대적 지식 표준에 대한 도전을 의미한다. 셋째로 리눅스와 위키피디아를 둘러싸고 부상하는 탈 허브형 지식생산모델은 성당모델로 대변되는 기존의 제도표준에 도전하는 새로운 제도의 등장을 의미한다. 금후 대중협업을 통한 기술지식 생산방식이 비트로 구성된 소프트웨어와 인터넷 백과사전의 영역을 넘어 온·오프라인을 포괄하는 산업 패러다임으로 정착하고, 나아가 조직과 경제의 구조 및 운영방식에 관련된 변화를 의미하는 제도표준으로 정착하기 위해서는 담론과 시장, 그리고 조직의 차원에서 발견되는 한계와 가능성을 효과적으로 걸러내고 결합하는 방식을 발견하는 작업이 선행되어야 할 것이다.This paper is aimed at considering global political implications of the standard competition for open source style knowledge network, especially focused on cases of linux and Wikipedia. This paper tried to grasp various challenges to existing knowledge structure in terms of the standard competition from three dimensions of idea, interest and institution. First of all, as the linux project provided a discourse which opposes to the knowledge structure of globalization and Wintelism, so Wikipedia is a counter discourse upon existing knowledge structure which is being put in the enlightenment encyclopedia. and also Wikipedia is a challenge to monopolistic authority of the main stream intellectual which participates to knowledge production exclusively. Second, as the competition between Linux and Microsoft is a competition for the defacto standard in software market, so the rising of the post-modern classification system which appears typically in a case of Wikipedia means a challenge to modern knowledge standard. Third, the post-hub style knowledge production model which appears in cases of Linux and Wikipedia means the appearance of new institution challenging to existing institution standard such as the cathedral model. In near future, it is necessary to develop a proper method to combine or skip effectively limits and possibilities which are discovered in terms of discourse, market and organization that a mass collaboration model take root as a comprehensive industrial paradigm which exceeds the territory of software and internet encyclopedia composed of bits, and further as a institution standard which means a change related to the structure of organization and economy, operating system

The diffusion of mobile communications technical standard in East Asia-focusing on cases of Japan

이 글에서 필자는 기존 확산연구의 성과를 원용하여 첫째, 이동통신 기술표준에 있어 기술규격의 세대나 표준화의 국면(채택, 확산), 국가별 산업 발전단계, 기술 진보 등에 따라 다양한 확산패턴이 나타날 수 있다는 사실에 주목했다. 나아가 이전 세대에서의 학습이 다음 세대의 확산패턴에 큰 영향을 미치고 있다는 점, 동아시아 기술표준 후발국의 확산패턴에서 나타나는 일정한 경향성에 대해 고찰했다. 둘째, 기존 연구에서 확산의 결과에 대한 분석이 상대적으로 덜 중시되어 왔다고 한다면, 여기서는 그 결과나 확산과정에서의 편차와 그 원인 등에 대해서도 고찰했다. 3G에서 WCDMA를 채택한 일본이 유럽에서의 전례와는 달리 애초 예상되던 이익을 얻지 못하고 고립된 이유는 무엇인가에 대해 살펴보았다. 이러한 논의를 토대로 최근 진행되고 있는 4G 이동통신 표준을 둘러싼 국제적 경쟁과 확산 동향을 포함하여 이동통신 표준의 확산이 동아시아 지역주의 논의에서 갖는 시사점에 대해 살펴보았다.This article purports to analyze various patterns of diffusion in technical standard of mobile communications according to technology generations, phasis of standardization such as adoption or spread, stages of national mobile communications development, and technological advancements by using advanced studies on policy diffusion. Learning in previous generation has some decisive influences on the diffusion pattern of next generation, and also a certain trend in the technical standard diffusion patterns of developing East Asian countries can be found out. Furthermore, this article focuses on the deviation in the process of standard diffusion and its major causes and results. Especially this article examined why Japan was isolated in 3G mobile communications with no expected gains in spite of the adoption of dominant WCDMA standard, on the contrary to European precedents. On the basis of these arguments, this article tried to draw implications of the diffusion of mobile communications standard for East Asian regionalism including international competition and diffusion trend surrounding 4G mobile industry