코드화된 미세입자를 이용한 색기반의 동시다발적 현장 진단 플랫폼 개발

학위논문 (박사)-- 서울대학교 대학원 : 공과대학 전기·컴퓨터공학부, 2019. 2. Kwon, Sunghoon.In this dissertation, a multiplex colorimetric diagnosis platform using encoded microparticles is proposed. Multiple target biomolecules can be detected by an office scanner as a concept of point of care tests within low-resource settings. The encoded microparticles guarantee high multiplexing capacity up to millions. Detection using gold nanoparticles in platform was demonstrated with assay results according to the color change of the encoded microparticles. Realizing scanner-based multiplex assay, this platforms novelty lies in fabrication of the encoded particles with two materials and introduction of a signal enhancement step to the multiplex bead-based assay using deposition of gold for higher sensitivity. The encoded microparticles, in which the engraved codes indicate the types of target molecules, are prepared to capture target. The design of the particles including the size and the materials were determined, to analyze the assay results with images taken by scanners. Also, the high-throughput fabrication methods have been developed to guarantee that more than 1000 particles can be fabricated in less than 3 minutes. The encoded particles with a single code are coated by silica and chemically conjugated to one type of capture molecules. This pairing guarantees the code to indicate the type of target molecules in multiplexing assay. The encoded microparticles targeting various molecules are pooled and reacted to samples with target molecules. After capturing targets on the multiple types of encoded particles, the particles conjugated with targets react with detection molecules. The detection molecules include gold nanoparticles to change the levels of target molecules into color signals. If the signal is too weak, a signal enhancement step is introduced using gold deposition to the seed gold nanoparticles with targets. After the whole colorimetric assay, the reacted particles are imaged using an office scanner, from which the code and the assay results are analyzed using image processing. The size of the microparticles was considered according to the proper resolution of the scanners. To be applied to various situations, two types of particles have been developed and utilized. 900μm particles with 2.5 million kinds of character codes and 300μm particles with 70-256 kinds of binary codes are developed to be scanned with 1200 and 4800 dpi respectively. As a proof of concept to show a wide range of applications, proteins and genes are detected. Using 4-plex assay, multiple sclerosis autoimmune disease patients are classified from healthy people with p<0.0001 in an unpaired t-test. Using 3-plex assay, bacterial meningitis genes are detected within 1000 molecules. This scanner-based assay platform can expand the clinical impacts of the multiplex assay. This platform can be applied to various circumstances where high-resource settings have not been set. With operators and scanners, the platform can be applied to multiplex assay in high multiplexing capacity and high throughput.본 학위 논문에서는 동시다발적으로 단백질이나 유전물질을 색변화를 통해 진단할 수 있는 플랫폼을 개발하였다. 본 플랫폼은 최종적으로 오피스 스캐너로 분석할 수 있기에 고가의 장비 없이 환자와 보다 가까운 곳에서 활용될 수 있는 기술이다. 코드화된 미세입자를 통해 한 샘플에서 동시에 여러 가지 진단을 가능하게 하였으며, 금 나노입자를 통해 분석 결과가 색 변화로 나타나 스캐너로 검출할 수 있도록 하였다. 해당 기술을 구현하기 위하여 미세입자를 두 가지 물질로 구성되도록 제작하였고 빠르게 대용량으로 제작하는 기술 역시 개발하였다. 또한 색변화로 적은 양의 물질을 검출할 수 있도록 신호 증폭 기술을 입자 기반 진단 기술에 적용하였다. 본 플랫폼을 개발하기 위해서 우선 표적 생체물질을 잡을 수 있는 코드화된 미세입자를 제작하였다. 스캐너로도 분석에 충분한 이미지를 얻을 수 있도록 크기와 물질 등의 디자인이 고려되었다. 본 미세입자를 3분 이내에 1 000 개 이상 제작할 수 있는 방법 역시 개발하였다. 제작된 코드화된 미세입자는 표적 생체물질만을 붙잡을 수 있도록 실리카 코팅된 후 화학적으로 포획분자가 코드 별로 다르게 부착된다. 여러 물질을 표적하는 코드화된 미세입자들은 함께 섞인 뒤에 표적물질이 있는 샘플과 동시다발적으로 반응한다. 표적물질을 반응 시킨 후에는 표적물질의 존재를 색변화로 나타낼 수 있도록 미세입자에 부착된 표적물질에만 골드 나노파티클이 붙게 된다. 신호가 약한 경우 골드 나노파티클의 크기를 키우는 반응을 통해 신호를 증폭시켜 확인한다. 반응이 모두 끝난 이후 미세입자들은 스캐너로 관측이 되고, 이미지 처리를 통해 코드와 표적물질의 양에 따라 변화된 색변화를 분석한다. 코드는 표적 물질의 종류를 나타내고, 색변화는 표적물질의 존재 정도에 비례하여 나타난다. 미세입자의 크기는 스캐너의 해상도에 직접적으로 연관이 있으므로 1 200 dpi 에서 분석이 가능한 900μm 미세입자와 4 800 dpi 에서 분석이 가능한 300μm 미세입자를 개발하였다. 각 입자는 문자코드를 활용하여 250만 개 이상의 코드를 가질 수 있고, 2진법의 코드를 활용하여 70 에서 256 개의 코드를 가질 수 있도록 개발되었다. 본 플랫폼이 다양한 진단 상황에 적용될 수 있음을 보이기 위하여, 환자 샘플에서 자가면역질환 관련 항체를 검출하는 실험과 적은 양의 박테리아 뇌수막염 관련 유전체를 검출하는 실험을 진행하였다. 4 종류의 동시다발적 분석을 통해 자가면역 질환 환자와 건강한 사람을 비쌍체 t 검정에서 p<0.0001 로 구분할 수 있었으며, 3 종류의 동시다발적 분석을 통해 박테리아 뇌수막염 관련 유전체를 1 000 개까지 검출해낼 수 있었다. 본 플랫폼을 통해 동시다발적 진단 기술의 의료 혜택을 보다 널리 확장시킬 수 있을 것으로 기대된다. 본 플랫폼은 고가의 장비들이 구축되지 않은 환경에서도 스캐너만 있다면 구현될 수 있으며 많은 수의 표적 물질을 동시에 확인할 수 있고 병렬적으로 많은 샘플을 진단할 수 있도록 개발되었기 때문이다.Table of Contents ABSTRACT I MULTIPLEX COLORIMETRIC DIAGNOSIS FOR POINT OF CARE TEST USING ENCODED MICROPARTICLE I TABLE OF CONTENTS IV LIST OF TABLES VIII LIST OF FIGURES IX CHAPTER 1. INTRODUCTION １ 1.1. Multiplex point of care test ２ 1.1.1. Multiplex assay for diagnosis of patients ２ 1.1.2. Needs of multiplex point of care test near to patients ５ 1.2. Main Concept: Multiplex colorimetric assay platform with encoded microparticle ８ 1.2.1. Multiplex colorimetric assay platform with encoded microparticle ９ 1.2.2. Advantages of scanner as widely spread detecting device １０ 1.2.3. Core technology of platform １２ 1.3. Outline of dissertation １８ CHAPTER 2. BACKGROUND １９ 2.1. Process of multiplex assay ２０ 2.2. Previous multiplex point of care technology ２１ 2.2.1. Technology for multiplex point of care test technology ２２ 2.2.2. Positioning of previous technology for multiplex assay ２９ 2.3. Commercialized multiplex assay devices for point of care test ３１ 2.3.1. Pros and cons of conventional automated machines for multiplex point of care test ３８ 2.4. Previous research in the group ４３ CHAPTER 3. PLATFORM DEVELOPMENT ４６ 3.1. Preparing process of encoded microparticle conjugated with capture molecule ４７ 3.1.1. Particle design considerations for scanner-based detection ４９ 3.1.2. Fabrication process of dual-functional encoded particle ５２ 3.1.3. Strategy to fabricate dual-functional sequentially with fixing polymers at the same position. ５５ 3.1.4. High-throughput fabrication method ５７ 3.1.5. Process of chemically conjugating capture molecules on surface of encoded particles ６４ 3.2. Process of massively parallel multiplex colorimetric assay ６７ 3.3. Optimization of imaging process with office scanner ６８ 3.3.1. Optimization of imaging plate ６９ 3.3.2. Optimization of resolution of scanning ７０ 3.4. Data analyzing process ７１ 3.4.1. Particle detection and alignment process ７２ 3.4.2. Algorithm for decoding particles and analyzing results of colorimetric assay from scanned images ７４ CHAPTER 4. PLATFORM VALIDATION WITH APPLICATION: ANTIBODY FROM AUTOIMMUNE DISEASE AND GENE FROM BACTERIAL MENINGITIS ７８ 4.1. Validation for immunoassay with autoimmune disease samples ７９ 4.2. Validation for genotyping with bacterial meningitis target ８７ CHAPTER 5. CONCLUSION AND DISCUSSION ９１ 5.1. Summary of dissertation ９２ 5.2. Comparison with previous technology ９４ 5.3. Limit of platform ９７ 5.4. Future work ９９ BIBLIOGRAPHY １００ 국문 초록 １１０Docto

Microspinning: Local Surface Mixing via Rotation of Magnetic Microparticles for Efficient Small-Volume Bioassays

The need for high-throughput screening has led to the miniaturization of the reaction volume of the chamber in bioassays. As the reactor gets smaller, surface tension dominates the gravitational or inertial force, and mixing efficiency decreases in small-scale reactions. Because passive mixing by simple diffusion in tens of microliter-scale volumes takes a long time, active mixing is needed. Here, we report an efficient micromixing method using magnetically rotating microparticles with patterned magnetization induced by magnetic nanoparticle chains. Because the microparticles have magnetization patterning due to fabrication with magnetic nanoparticle chains, the microparticles can rotate along the external rotating magnetic field, causing micromixing. We validated the reaction efficiency by comparing this micromixing method with other mixing methods such as simple diffusion and the use of a rocking shaker at various working volumes. This method has the potential to be widely utilized in suspension assay technology as an efficient mixing strategy

발광 물질을 이용한 새로운 마이크로입자 코드화 기법 및 응용

학위논문 (석사)-- 서울대학교 대학원 : 전기·컴퓨터공학부, 2013. 2. 권성훈.바이오 의약 연구 분야에서 다중 분석 기술은 유전자 형질을 분석하고, 효과가 있는 약물을 검색하거나 병을 진단할 때 유용하게 사용되어왔다. 처리량이 많고, 사용자가 편리하게 사용할 수 있으며, 가격이 싼 다중 분석 방법들 중 하나로써, 코드화된 입자 방법이 있다. 코드화된 입자를 이용하면 다양한 분자들을 쉽게 다룰 수 있다. 분자들이 있는 액체 자체를 미세입자로 운반하고, 각 분자들을 확인하기 위해서는 새로운 마이크로입자가 필요하며, 이와 동시에 해당 입자를 코드화할 수 있는 기법이 개발되어야 한다. 본 논문에서는 2,2-dimethoxy-2-phenylacetophenone (DMPA) 광개시제의 발광 현상을 이용하여 미세입자에 적합한 새로운 코드화 방식을 소개하고, 이 방식이 다중 분석 기술에 응용될 수 있음을 보인다. 먼저, Perfluoropolyether로 된 마이크로캡슐과 디스크 형태의 마이크로입자를 미세유체학과 광미세유체 마스크리스 리소그래피를 이용하여 제작하였다. 다양한 패턴의 자외선을 조사하여 이 입자들에 코드를 생성하였다. 이러한 입자 코드화 방식은 다양한 패턴을 생성할 수 있어 많은 수의 코드를 생성할 수 있고, 오랜 시간 동안 코드가 유지되는 장점을 가진다. 코드의 세기는 DMPA의 농도와 UV 조사량에 의해 조절될 수 있다. 이 입자 코드화 방식은 실제 다중 분석 기술에 유용하게 쓰일 수 있고, 다단계 생화학 반응을 추적하는 데에도 사용될 수 있다.Multiplexed assay technologies have been used in biological and medical studies on gene profiling, drug screening, and clinical diagnostics. An encoded suspension array was developed as a high-throughput, convenient, and low-cost method. The encoded microparticles enabled easy handling of various molecules. A new encoding method needs to be developed to carry probe molecules in liquids and to identify them. This thesis presents a new encoding method using the photoluminescent material 2,2-dimethoxy-2-phenylacetophenone (DMPA) and demonstrates its applications. First, perfluoropolyether microcapsules and disk-shaped microparticles were generated using hillock microfluidic channels and an optofluidic maskless lithography setup to test the feasibility of the encoding method. Second, microparticle codes were created via diverse light patterns using the photoluminescence of DMPA. This new method has a high coding capacity and shows long-term durability. Furthermore, the code intensity can be controlled using DMPA concentration and ultraviolet light dose. As applications, the new multiplexed assay platform can be developed and code-changeable microparticles for multi-step assays can be fabricated.Abstract ii Contents vi List of Figures viii Chapter 1 Introduction 1 Chapter 2 Generation and Encoding of Microparticles Containing DMPA 3 2.1 Characteristics of DMPA Photoinitiator 4 2.2 Generation of Smart Microparticles Containing DMPA Photoinitiator 5 2.2.1 Sphere-shaped (core-shell) Microparticles Containing DMPA 5 2.2.2 Disk-shaped Microparticles Containing DMPA 17 2.2.3 Generation of Microparticle Codes 20 Chapter 3 Characterization of the Encoding Method Using DMPA Photoinitiator and its Applications 26 3.1 Characteristics of Microparticle Codes 26 3.1.1 Code Diversity 26 3.1.2 Code Durability 29 3.1.3 Code Intensity Controllability 32 3.1.4 Compatibility with Other Materials 35 3.2 Multiplexed Assay Platform Using Encoded Microcapsules 36 3.3 Code-changeable Microparticles for Multi-step Paricle-based Assays 38 3.3.1 Repeated Code Writing 39 3.3.2 Silica-coating of Code-changeable Microparticles 40 Chapter 4 Conclusion 42 Supplementary Information 45 Possible Mechanisms of Code Photoluminescence 45 Bibliography 48 Abstract in Korean 54Maste

환자맞춤형 치료를 위한 체외 항암제 스크리닝용 바이오칩

학위논문 (박사) -- 서울대학교 대학원 : 공과대학 전기·정보공학부, 2020. 8. 권성훈.정밀의학(Precision Medicine) 혹은 개인맞춤의학(Personalized Medicine)은 개개인의 최적화된 치료방법을 결정하는 것을 목표로 하는 의학의 패러다임이다. 특히, 임상종양학에서는 차세대염기서열분석(NGS), 전사체서열분석, 그리고 질량분석법들을 통한 환자의 분자 프로파일(molecular profile) 방법이 발전해오고 있으며, 이를 바탕으로 환자를 세분화하여 맞춤형 치료를 구현하려고 노력해오고 있다. 하지만, 여전히 현 수준에서 이해되지 못하는 수준의 종양 이질성(tumor heterogeneity)과 오랜 처방기록을 가진 환자군들의 항암제 획득내성(acquired resistance) 등의 원인으로 맞춤형 환자 처방은 쉽지 않은 경우가 많다. 이러한 경우 환자로부터 얻어진 암세포, 조직으로부터 얻어진 일차세포 혹은 체외 배양된 세포, 스페로이드, 장기유사체 등을 이용하여 고속다중약물스크리닝기술을 통한 맞춤형 항암제를 선별해내는 체외 약물진단 기술을 생각해낼 수 있는데, 이는 기존의 유전체 기반의 시도와 병행되어 개개의 환자들에게 더욱 적합한 치료방법을 찾는 것이 가능하게 한다. 하지만 이러한 목적의 고속다중약물스크리닝기술은 높은 활용가능성에도 불구하고, 광범위한 보급과 활용이 되기에는 제약점이 많았다. 기존의 고속다중약물스크리닝기술은 많은 양의 샘플이 소모되고, 값비싼 시약의 소모량도 적지 않았다. 게다가, 수천 가지 이상의 서로 다른 물질들을 탐색하기 위해 반드시 필요한 고가의 자동화된 액체 운반기(liquid handler) 등이 필요하였는데, 이러한 문제로 대형 제약사, 연구소 등을 제외하고는 도입이 쉽지가 않아 기술접근성이 제한되어 있었다. 본 연구에서는 반도체공정에서의 노광기술을 이용하여 개개의 식별할 수 있는 코드를 가지고 있는 코드화된 하이드로젤 기반의 광경화성폴리머 미세입자를 만들어, 이를 원하는 암세포에 약물 스크리닝을 해보고자 하는 다양한 약물라이브러리를 이용 각각의 코드화된 미세입자에 흡수시켜 약물-미세입자 라이브러리를 제작한다. 그후, 값비싼 어레이 제작용 스포터 혹은 디스펜서 장비없이 간단한 자기조립을 통해 대규모의 다양한 약물-하이드로젤 어레이를 제작할 수 있는 기술을 개발하였다. 또한, 소량의 세포들 만으로도 미세우물(microwell) 기반의 세포칩에 도포하는 방식을 개발하였으며, 이를통해 약물-하이드로젤 어레이와 미세우물기반의 세포칩의 결합으로 수백-수천의 다양한 어세이를 적은 수의 샘플만으로도 한번에 수행할 수 있는 고속다중약물스크리닝 기술을 수행할 수 있게 만들었다. 본 연구에서 제시한 소형화된 체외 항암제 스크리닝용 약물플랫폼은 다음과 같은 의의를 가진다. 적은 수의 환자세포 혹은 샘플의 양에 적용할 수 있는, 사용하기 손쉬운 기술로서, 기존의 값비싼 장비, 시약의 사용량을 획기적으로 줄일 수 있는 기술이다. 본 연구에서 제안된 기술을 통해 기존의 장비를 사용할 때 시약의 값이 비싸거나, 장비의 가격이 비싸서, 혹은 다루고자 하는 샘플의 양이 제한적이어서 기존에 접근하기 힘들었던 다양한 학술연구에 적용할 수 있으며, 병원에서의 임상연구 및 실제 환자맞춤형 치료에 사용 될 수 있는 접근성을 획기적으로 높일 수 있다. 특히, 비교적 중,소 규모의 연구환경에서도 다양한 희귀한 환자유래세포 혹은 환자유래오가노이드 등과 접목하여 사용된다면 본 플랫폼의 가능성을 더욱 극대화 할 수 있을 것으로 기대한다.Precision or Personalized Medicine is a medical paradigm aimed to determine optimal therapy for individual patient. In particular, clinical oncology has been using methods of molecular profiling for each patient through next-generation sequencing (NGS), mRNA-sequencing, and mass spectrometry, and has been trying to implement personalized treatment. However, personalized treatment based on molecular profiling to each patient is not always possible due to the high level of heterogeneity of tumor that is still not fully understood at the current level and acquired resistance of anti-cancer drug due to cumulative targeted therapy. In such cases, in vitro drug testing platform using primary cells obtained from patients, or patient-derived cells, spheroids, and organoids can make it possible to find a more appropriate treatment for each individual patient. However, though high-throughput drug screening technology for this purpose is of the utmost importance in saving lives, there were many limitations to its wide use in many hospitals. The existing high-throughput drug combination screening technology consumes a large number of samples and consumes a considerable amount of expensive reagents. In addition, expensive automated liquid handlers, which were essential for exploring thousands of different pipetting, were not easy to introduce except for large-sized pharmaceutical companies and research institutes, which limited access to technology. In this study, I construct a heterogeneous drug-loaded microparticle library by fabricating encoded photocurable polymer particle that has individually identifiable codes to track loaded drug. and I load various drug molecules, which I want to test to target cells, into each coded microparticle. Then, I developed to produce heterogeneous drug-laden microparticle arrays through simple self-assembly without the need for a microarray spotter or dispensing machine for generating microarray. I also have developed cell seeding method of seeding small-volume samples into the microwell-based cell chip. By utilizing the drug-laden microparticle hydrogel array and microwell-based cell chip technology, hundreds to thousands of different assays can be done at once with just a small number of samples and low cost. Through the implemented platform, the anti-cancer drug sequential combination screening was conducted on the triple-negative breast cooler (TNBC) cells, which are generally known to be difficult to treat due to lack of known drug target, and the results of screening were analyzed by establishing a library of drugs in the EGFR inhibitory type and drugs in the genotoxin type. In addition, another study was conducted to find optimal drug combinations using patient-derived cells derived from tumors in patients with non-small cell lung cancer that have obtained acquired resistance. Finally, as the growing need for three-dimensional culture, such as spheroid and organoid for having a similar response to in vivo drug testing, it was also developed that microwell-based cell chip that is capable of 3D culture with low-cost and small-volume of cells. The miniaturized in vitro anticancer drug screening platform presented in this study has the following significance. An easy-to-use technique that can be applied to a small number of patient cells or samples, which can dramatically reduce the use of conventional expensive equipment, reagents. The proposed technology in this study can be applied to a variety of academic studies previously inaccessible to high-throughput screening due to the high cost of reagents, the high price of equipment, or the limited amount of samples in conventional drug screening. and this platform can also dramatically increase access to clinical research in hospitals for personalized treatments. In particular, it is expected that the possibility of this platform will be further maximized if it is used in a relatively small and medium-sized research environment by the combined use of various rare samples such as patient-derived cells or patient-derived organoids.Chapter 1 Introduction １ 1.1 Motivation of this research ２ 1.2 Competing technologies and Previous works ８ 1.3 Main Concept: In vitro drug testing using miniaturized encoded drug-laden hydrogel array technology １５ Chapter 2 Platform Development of Drug Releasing Hydrogel Microarray ２０ 2.1 Encoded Drug-Laden Hydrogel & Library construction ２１ 2.2 Array generation of heterogenous drug-laden microparticles. ３４ 2.3 Cell Culturing on Cell Chip and bioassay ３６ Chapter 3 Sequential Drug Combination Screening Assy on TNBC ４０ 3.1 Background : Sequential Drug Combination as promising therapeutic option ４１ 3.2 Experimental design with sequential drug treatment assay ４３ 3.3 Technical Issue & its engineering solution ４４ 3.4 Assay Result ４９ Chapter 4 Drug Combination Assay on Patient-Derived Cells ５８ 4.1 Background : Simultaneous Combination Treatment using Patient-Derived Cells ５９ 4.2 Improvement of Platform for facilitating translational study ６２ 4.3 Study Design for small-volume drug combinatorial screening with NSCLC patient derived cell ６５ 4.4 Assay Result ６９ Chapter 5 Development of platform for 3D culture model ７２ 5.1 3D culturable platform ７３ 5.2 Development of 3D culture platform based Matrigel scaffold. ７８ 5.3 Advantage over conventional 3D culture-based drug testing platform. ８５ Chapter 6 Conclusion ８７ Bibliography ９０ Abstract in Korean ９７Docto

펄스 레이저를 이용한 고속 단분자 식별 및 분리 기법

최근 합성생물학 분야의 가장 큰 화두는 대량이다. DNA를 대량으로 빠른 시간에 읽어내는 차세대 염기서열 분석 기술의 급격한 성장은 다양한 종의 주요 유전회로 정보를 밝혔으며 이는 새로운 기능의 염기 서열을 쓰기 위한 유용한 단초를 제공해 주었다. 염기서열 쓰기 기술은 단백질 공학, 합성 유전체 연구 및 새로운 생물학적 회로의 구성뿐만 아니라 DNA 메모리와 같은 다양한 차세대 분야에서 응용이 가능하여 그 중요성이 더해지고 있다. 일반적으로 염기서열 쓰기 기술은 화학 합성 시 발생하는 오류로 인하여 충분한 길이의 염기 서열을 한번에 합성하는 것은 확률적으로 불가능하며 서열이 확인된 짧은 염기서열을 상향식으로 조립해 나가는 것이 합리적인 접근 방법이다. 따라서 저렴한 양질의 염기서열 재료를 대량으로 준비하는 것이 반드시 필요하다. 마이크로 어레이 DNA와 같은 최신의 합성 기술은 한 번에 수십만 종 이상의 짧은 염기서열을 합성할 수 있으나 오류가 섞여 있으며 원천적으로 혼합되어 있어 목적 염기서열로 조립하는데 사용하기 위해서는 적절한 대량의 식별 및 분리기법이 수반되어야 한다. 본 논문에서는 미세 입자 기술, 펄스 레이저 기술 및 위치추적 알고리즘 기술을 기반으로 차세대 염기서열 분석 장비를 통해 병렬적으로 분석된 수백만 개의 단분자 클론을 고속으로 분리하여 직접 사용하게 함으로써 마이크로어레이 칩 DNA의 효용성을 극대화 하는 기술을 소개한다 특정 탐침으로 표면이 기능화된 미세입자는 3차원 액상에 존재하는 혼합된 염기서열 집단으로부터 목적 염기서열만을 탐침과의 혼성화를 통해 분리한다. 코드화된 미세 입자는 혼성화 이후 펄스 레이저의 광압을 이용하여 물리적으로 분리하여 사용하도록 한다. 그러나 미세입자를 이용한 분리 기법은 동시에 여러 종류의 목적 염기서열을 3차원 반응상에서 분리할 수 있다는 장점에도 불구하고 목적 서열에 따라 개별적으로 미세입자를 준비해야 한다는 단점이 있다. 이러한 단점을 극복하기 위하여 DNA 마이크로 어레이의 탐침 영역을 펄스레이저 식각 기술을 응용하여 직접 분리하는 기술을 제시한다. DNA 마이크로 어레이는 한 번에 수십만 종 이상의 탐침영역을 표면에 기능화시킬 수 있으며 혼성화 반응을 통해 병렬적으로 혼합된 염기서열 집단을 분리할 수 있어 효율적이다. 다만 기판으로부터 물리적으로 분리하는 방법의 부재로 인하여 관찰의 용도로만 사용하여 왔다. 본 논문에서 제안한 펄스레이저 식각 기술은 탐침 및 탐침과 혼성화 분리된 목적 염기서열을 포함하는 기판영역 전체를 물리적으로 분리할 수 있도록 하여 쓰기의 재료로 이용 가능하게 한다. 한편 탐침 서열로 혼성화 하여 분리된 목적 염기서열들은 합성할 때 발생한 오류를 가지는 서열을 그대로 지니고 있다. 정확한 목적 염기서열만을 분리하기 위하여 전통적으로 클로닝 기법이 사용되는데 이는 근본적으로 무작위 추출 및 뒤따르는 개별 염기서열 분석을 기반으로 하고 있어 효율적이지 못하다. 본 논문에서 제안하는 Sniper cloning 기법은 펄스레이저 광압 분리 기술과 정교한 위치추적 알고리즘을 바탕으로 차세대 염기서열 분석장치를 이용하여 사전 검열된 막대한 양의 클론 미세입자들로부터 목적 염기서열을 고속 비 접촉 방식기술을 이용하여 선택적으로 분리하여 무작위 추출로 인해 발생하는 불필요한 노동력과 비용을 제거하였으며 매우 높은 순도의 전구 염기서열을 대량으로 생산한다. 본 논문에서는 염기서열 쓰기 기술이 가지는 잠재적 발전 가능성과 이를 뒷받침 하기 위해 개발된 다양한 기반기술에 대하여 소개하고자 한다.The key challenge of synthetic biology currently lies in the absence of cost effective high standard oligonucleotide precursor for constructing target long sequence. Microarray DNA is an ultra-rich source of oligonucleotides that generates millions of short oligonucleotide sequence in a single run. In spite of ensuring overwhelming advantages over conventional chemical oligonucleotide synthesis, the efficiency of the progress is dogged by high complexity and low quality of microarray DNA. In this thesis, I present various techniques including encoded-microparticle, DNA microarray and pulse laser sniper cloning, for the improvement of preparative tool for writing DNA. In the first part of this thesis, an important state-of-the-art element technologies for writing DNA are reviewed. Microarray DNA technology offers millions of short DNA in a cost effective single run that overcomes the problems related with conventional one-by-one column synthetic approach. Meanwhile, the downstream separation and the identification steps which normally consist a vector cloning and Sanger sequencing can also be replaced by high-throughput Next Generation Sequencing (NGS) platform. This chapter concludes in discussing the developmental possibility of a next generation writing technology by closely combining the elemental technologies into a preparative tool. The second part provides an overview to the fabrication method of various microparticles for complex pool separation. The microparticles which possess distinctive IDs and probe oligonucleotides on their surfaces plays a floating microfilter that selectively separates the target single strand DNA from complex pool according to the probe sequence. The fabrication of color barcoded microparticle and magnetochromatic sphere based on optofluidic technique is described followed by simple demonstration of DNA separation. Third part describes the pulse laser driven microstructure techniques. The focused nanosecond pulse laser exerts radiation pressure onto the microparticles containing hybridization selected DNA from mixed pool. Furthermore, the target microparticles can be physically separated without actual physical contact. More condensed energy of focused pulse laser ablates target substrate and therefore, generates a small explosion. The separated contents of an array of probe spots such as DNA microarray is also able to be individualized for utilization by directly ablation of target containing substrate. Chapter 4 presents the development of clone sniper method using parallel identification followed by high-throughput separation approach to construct ultra-high quality oligonucleotide library with low cost and high-throughput. This approach reduces the labor intensive conventional clonal separation and expensive Sanger derived identification. The custom made pulse laser retrieval system enables non-contact contamination high-throughput separation of perfect parts from sequencing plate with precise position data constructed by local mapping algorithm. The serial process consists of parallel synthesis parallel identification and high-throughput separation which not only increases the quality of contents, but also dramatically reduces the necessary resources, such ascost, labor and time. Chapter 5 provides a very compact summary of this research work, highlights the contributions made. Possibilities for future work to increase the significance of the approaches discussed.Contents Abstract ii Contents v List of Figures vii List of Tables xxxi Chapter 1 Introduction 1 1.1 Synthetic DNA 2 1.2 Separation of complex DNA pool 3 1.3 Identification of DNA 5 1.4 Deterministic clone targeting 7 Chapter 2 Encoded micropartle for pool separation 9 2.1 Color barcoded magnetic microparticle 12 2.1.1 Fabrication of color barcoded magnetic microparticle 14 2.1.2 Magnetic handling for multistep reaction 20 2.1.3 DNA separation 32 2.1.4 Summary 33 2.1.5 Materials and methods 35 2.2 Magnetochromatic microspheres 37 2.2.1 Fabrication of magnetochromatic microspheres 37 2.2.2 Optical response of magnetochromic microspheres 39 2.2.3 Summary 48 Chapter 3 Complex pool separation technique based on pulse laser 49 3.1 Radiation pressure driven microparticle separation 50 3.2 Ablation driven separation of microarray probe 57 3.3 Summary 60 Chapter 4 Identification followed by separation of single molecule 61 4.1 UPRandom separation and identification 62 4.2 URadiation pressure driven high-throughput separation system 67 4.3 Clone tracking algorithm 72 4.4 Results 79 4.5 Summary 88 Chapter 5 Conclusion and future work 89 Bibliography 92 Abstract in Korean 100Docto

고속 다중변수 세포기반 분석을 위한 코드화된 미세입자를 이용한 지역화된 바이러스 기반의 유전자 전달

학위논문 (박사)-- 서울대학교 대학원 : 전기·컴퓨터공학부, 2015. 8. 권성훈.In this dissertation, I develop an adenoviral vector-immobilised patch-type encoded microparticle for high-throughput, high-content cellular assays and name this encoded viral micropatch. This technology spatially confines the adenoviral gene delivery to only the cells under the micropatch by simply pipetting a heterogeneous mixture of the two-dimensional (2D) shape-coded viral micropatches on monolayer-cultured cells. Distinct clusters of transduced cells are then created in correspondence with the randomly positioned micropatches and the delivered gene into the cells within each cluster can be identified using the shape of the micropatch. For this purpose, shape-coded polymer microparticles are fabricated by photolithography, and highly localized gene delivery is achieved by specifically immobilizing adenoviral vectors on the microparticles. This unique feature allows high-throughput compound screening by virtue of multiplexing in a well of a standard microplate and creates no restriction for fluorescence-based assay formats with high-content imagers. To highlight the capabilities of this technology, I demonstrate a multiplex G-protein coupled receptor (GPCR) internalization assay that requires compound treatments followed by fluorescence-based target tracking at the sub-cellular level. First, I develop the maskless lithography system supporting an automated step-and-repeat operation for the fabrication of microparticles with various 2D graphical codes. Using this system, I explore new applications of the encoded microparticles and lithography technique such as anti-counterfeiting of drugs, parallel loading of small volume liquid for multiplexed bioassays, and conformal phosphor coating for white light-emitting diodes (LEDs). For the development of the encoded viral micropatch, various shape-coded microparticles are fabricated with carboxyl groups on the surfaces for specific immobilization of adenoviral vectors. The chemical functionalization is achieved by the incorporation of acrylic acid to photocurable polymer solution. Then, two adenoviral vector immobilization methods are developed with this shape-coded microparticle. The first method is to directly link the carboxyl groups on the microparticle and the primary amine groups on the surface proteins of adenoviral vectors using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) plus N-hydroxysulfosuccinimide (Sulfo-NHS) crosslinking reaction. The immobilization of adenoviral vectors in this approach is confirmed by an immunofluorescence test and a scanning electron microscope (SEM) observation. The second method utilizes an avidin-biotin interaction. In this approach, both the microparticles and adenoviral vectors are biotinylated using amine-activated and amine-reactive biotin reagents, respectively. Then, they are linked by the mediation of avidin. The immobilized adenoviral vectors are well observed using a SEM. The localized viral gene delivery of two types of the encoded viral micropatches is evaluated by transducing a human osteosarcoma cell line (U-2 OS) cultured in a standard 96-well microtiter plate. The first type of the encoded viral micropatch fabricated via EDC/Sulfo-NHS reaction shows low rate of the localization of gene delivery due to an escape of non-specifically bound adenoviral vectors. However, the second type of the encoded viral micropatch fabricated utilizing avidin-biotin interaction offers highly localized gene delivery. This is owing to the viral receptor-independent transduction of the biotinylated adenoviral vector, which is further supported by the transduction experiment of an adenovirus receptor-deficient cell line. Finally, I demonstrate a multiplexed GPCR internalization assay based on the localized gene delivery with the encoded viral micropatches. The development of high-throughput cell-based GPCR functional assays is very important for screening large compound libraries in the drug discovery process and ligand-induced receptor internalization assays have broad applicability to various GPCR subfamilies among several GPCR assay formats. However, high-content imaging is required for fluorescence-based intracellular measurement of receptor internalization. To address this issue, I fabricate three types of encoded viral micropatches with adenoviral vectors bearing green fluorescence protein (GFP)-tagged GPCR genes. Then, the responses of multiple GPCRs against one ligand treatment is acquired in one reaction site by achieving simultaneous expression of multiple GPCRs with the fabricated viral micropatches in a cell monolayer cultured in a well of a 96-well plate. High-content analysis of this micropatch-based multiplexed assay shows comparable results in the receptor internalization with the conventional singlet assay using free adenoviral vectors while reducing the number of pipetting actions.Abstract i Contents v List of Figures viii List of Tables xvii Chapter 1 Introduction 1 1.1 Cell-based Assays in Drug Discovery 4 1.2 Image-based High-content Screening 7 1.3 Cell Microarray for High-throughput Screening 10 1.4 Main Concept: Encoded Viral Micropatch 12 Chapter 2 Development of Encoded Viral Micropatch 15 2.1 Introduction 16 2.2 Fabrication of Encoded Microparticles 19 2.2.1 Maskless Lithography System 19 2.2.2 Shape-coded Microparticles for Encoded Viral Micropatch 32 2.3 Immobilization of Viral Vectors 37 2.3.1 Recombinant Adenoviral Vector 37 2.3.2 Direct Targeting of Viral Capsid via Carbodiimide Crosslinker (Type 1 Encoded Viral Micropatch) 39 2.3.3 Indirect Targeting of Biotin-tethered Viral Capsid via Avidin (Type 2 Encoded Viral Micropatch) 44 2.4 Conclusion 52 Chapter 3 Localized Viral Gene Delivery 53 3.1 Introduction 54 3.2 Localized Gene Delivery with Type 1 Encoded Viral Micropatch 57 3.3 Localized Gene Delivery with Type 2 Encoded Viral Micropatch 60 3.3.1 Evaluation of the Localized Gene Delivery 60 3.3.2 Consideration of the Localized Gene Delivery 65 3.3.3 Transduction of an Adenoviral Receptor-deficient Cell Line 67 3.3.4 Transduction Efficiency of the Encoded Viral Micropatch 69 3.4 Conclusion 74 Chapter 4 Multiplex GPCR Internalization Assay 75 4.1 G Protein-coupled Receptor (GPCR) 77 4.2 Materials for the Assay 79 4.2.1 GPCR Adenoviral Vectors 79 4.2.2 Ligands 79 4.2.3 Cell Culture 79 4.3 Conventional GPCR Internalization Assay 80 4.3.1 Assay Procedure 80 4.3.2 Assay Result 83 4.4 Multiplex GPCR Internalization Assay 85 4.4.1 Preparation of Encoded Viral Micropatches 85 4.4.2 Assay Procedure 85 4.4.3 Assay Result 87 4.5 Conclusion 91 Conclusion 92 Bibliography 94 국문 초록 103Docto

One-Step Generation of a Drug-Releasing Hydrogel Microarray-On-A-Chip for Large-Scale Sequential Drug Combination Screening

Large-scale screening of sequential drug combinations, wherein the dynamic rewiring of intracellular pathways leads to promising therapeutic effects and improvements in quality of life, is essential for personalized medicine to ensure realistic cost and time requirements and less sample consumption. However, the large-scale screening requires expensive and complicated liquid handling systems for automation and therefore lowers the accessibility to clinicians or biologists, limiting the full potential of sequential drug combinations in clinical applications and academic investigations. Here, a miniaturized platform for high-throughput combinatorial drug screening that is &quot;pipetting-free&quot; and scalable for the screening of sequential drug combinations is presented. The platform uses parallel and bottom-up formation of a heterogeneous drug-releasing hydrogel microarray by self-assembly of drug-laden hydrogel microparticles. This approach eliminates the need for liquid handling systems and time-consuming operation in high-throughput large-scale screening. In addition, the serial replacement of the drug-releasing microarray-on-a-chip facilitates different drug exchange in each and every microwell in a simple and highly parallel manner, supporting scalable implementation of multistep combinatorial screening. The proposed strategy can be applied to various forms of combinatorial drug screening with limited amounts of samples and resources, which will broaden the use of the large-scale screening for precision medicine

Synergism between particle-based multiplexing and microfluidics technologies may bring diagnostics closer to the patient

In the field of medical diagnostics there is a growing need for inexpensive, accurate, and quick high-throughput assays. On the one hand, recent progress in microfluidics technologies is expected to strongly support the development of miniaturized analytical devices, which will speed up (bio)analytical assays. On the other hand, a higher throughput can be obtained by the simultaneous screening of one sample for multiple targets (multiplexing) by means of encoded particle-based assays. Multiplexing at the macro level is now common in research labs and is expected to become part of clinical diagnostics. This review aims to debate on the “added value” we can expect from (bio)analysis with particles in microfluidic devices. Technologies to (a) decode, (b) analyze, and (c) manipulate the particles are described. Special emphasis is placed on the challenges of integrating currently existing detection platforms for encoded microparticles into microdevices and on promising microtechnologies that could be used to down-scale the detection units in order to obtain compact miniaturized particle-based multiplexing platforms