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Heterogeneous Chem-Bio Interfaces of Luminescent Materials for Accumulated Time-Gated Analyses

신호누적 시간 게이트 분석을 위한 발광소재의 화학-바이오 이종(異種)계면 연구

  • Yong Ho Cho (조용호, 대학원 화공생명공학과 화공생명공학전공)

초록/요약

ABSTRACT The objectives of this dissertation is to investigate interfacial phenomena occurring at heterogeneous interfaces of hybrid assemblies based on luminescent materials. We develop chem-bio hybrid interfaces capable of bio-recognition function by combining bioligands with organic semiconducting materials and observed photoluminescent changes after target recognition. Both fluorophors and phosphors can be used to form heterogeneous interfaces by hybridizing with DNA or peptide ligands. Particularly, the room temperature phosphor-based hybrid material can be used for time-gated analysis to avoid autofluorescence signals. In chapter 2, we successfully performed protein-cell recognition by combining an organic light-emitting diode (OLED) material and an aptamer with a three-dimensional DNA structure. Tris (8-hydroxyquinoline) aluminum (Alq3), one of the most widely used green OLED components used in displays, was chosen as an emitter. Epithelial cell adhesion molecule (EpCAM) was selected as a target because it is well-established in cancer studies. The generated micrometer-sized particles exhibited increased luminous intensity based on the specific recognition to proteins and cells on hybrid interfaces, proving that the aptamer maintained its three-dimensional structure while binding to its target. Interestingly, only human cells expressing EpCAMs were distinguishable, whereas mouse carcinoma cells were not. In chapter 3, we fabricated hybrid nanoparticles consisting of organic semiconducting material with peptide sequence to reflect the target protein interaction. A phosphorescent OLED material, platinum octaethylporphyrin (PtOEP) was self-assembled by reprecipitation with the A17 peptide selected as a probe ligand in order to recognize heat shock protein 70 (HSP70). The phosphorescence intensity of the PtOEP-A17 assembly was enhanced after treatment with HSP70. The specificity of the protein interaction was confirmed in both solution and solid states of the PtOEP-A17 assembly against to BSA and nucleolin. We figured out that the phosphorescence lifetime of PtOEP-A17 assembly after exposed to HSP70 increased significantly. In chapter 4, we introduced the time-gated analysis of room-temperature phosphorescence (RTP) for the in-situ analysis of the visible and spectral information of photons. Time-gated analysis is performed using a microscopic system consisting of a spectrometer. This is advantageous for in-situ analysis because it facilitates the real-time measurement of luminescence signal changes. An RTP material hybridized with a DNA aptamer that targets a specific protein enhances the intensity and lifetime of phosphorescence after selective recognition with the target protein. In addition, time-gated analysis allows for the millisecond-scale imaging of phosphorescence signals, excluding autofluorescence, and improves the signal-to-background ratio (SBR) through the accumulation of signals. We collect the time-gated images and spectra of RTP and autofluorescent materials and develop a method for obtaining phosphorescence signals by selectively excluding autofluorescence signals in simulated or real cell conditions. It is confirmed that the accumulated time-gated analysis can provide various information about luminescence signals for bioimaging and biosensing applications. In chapter 5, we developed a highly stable polymeric vesicle using a nanosilica-armor membrane to achieve a sustainable colorimetric/luminescent response. The silica armor can be grown directly as spherical nanoparticles on the surface of the diacetylene (DA) vesicle with liposomal structure. Once formed, the structural stability of the DA vesicles dramatically increased and remained so even in a dried powder form that could be stored for a period of approximately 6 months. Then, redispersed in water, the armored vesicles did not agglomerate because of the electric charge of the silica armor. After polymerization, the polydiacetylene (PDA) vesicles maintained an average of 87.4 % their sensing capabilities compared to unstored vesicles.

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초록/요약

국문 초록 본 학위논문은 발광물질을 기반으로 하는 융합체들의 이종 계면에서 발생하는 계면현상을 연구하는 것을 목표로 한다. 발광소재에 바이오리간드를 결합하여 바이오인식이 가능한 화학-바이오 융합계면을 형성하고 표적인식 후의 발광변화를 관찰하였다. 형광체와 인광체는 DNA 또는 펩타이드 리간드와 융합하여 이종계면을 형성한다. 특히, 상온인광 기반 융합소재는 자가 형광 신호를 회피하기 위한 시간 게이트 분석법에 사용할 수 있다. 2장에서는, 3차원 구조를 가지는 DNA 압타머와 유기발광다이오드(OLED) 소재를 융합하여 단백질 및 세포 인식을 성공적으로 수행했다. 발광체는 디스플레이에서 가장 널리 사용되는 녹색 OLED 물질 중 하나인 Alq3를 선택했다. 타겟물질은 암 연구에서 잘 알려져 있는 물질인 EpCAM을 선택했다. 생성된 마이크로미터 크기의 입자는 융합계면의 단백질과 세포에 대한 특이적 인식에 따라 형광이 증가하였으며 압타머가 타겟에 결합한 후에도 3차원 구조를 유지했음을 증명했다. 특히 쥐세포와 구분되어EpCAM을 발현하는 인간 세포만 구별이 가능했다. 3장에서는, 타겟 단백질 인식을 위해 펩타이드가 융합된 유기 반도체 나노입자를 제작했다. 인광 OLED 물질인 PtOEP는 HSP70단백질을 인식하기 위한A17 펩타이드와 융합하며 자기조립하였다. PtOEP-A17 조립체의 인광 강도는 HSP70타겟을 처리한 후 향상되었다. 단백질 인식 특이성은 용액 및 고체 상태에서 PtOEP-A17 조립체의 BSA 및 Nucleolin 단백질 처리로 확인했다. 동시에, HSP70단백질에 노출된 PtOEP-A17 조립체의 인광 수명이 크게 증가했음을 발견했다. 4장에서는, 광자의 가시광선 및 스펙트럼 정보의 in-situ 분석을 위한 상온 인광(RTP)의 타임 게이트 분석을 연구했다. 타임 게이트 분석은 분광계가 구축된 현미경 시스템을 사용하여 수행된다. 이는 발광 신호 변화의 실시간 측정이 용이하여 in-situ 분석에 장점이 있다. 특정 단백질을 타겟으로 하는 DNA 압타머와 결합된 RTP 물질은 단백질의 선택적 인식 후 인광의 강도와 수명이 향상되었다. 또한 타임 게이트 분석을 통해 자가형광을 제외한 인광 신호의 밀리초 단위의 이미징이 가능하며 신호의 축적을 통해 신호 대 배경비 (SBR)를 향상시켰다. RTP 및 자가형광 물질의 타임 게이트 이미지와 스펙트럼을 수집하고 인위 또는 실제 세포 조건에서 자가형광 신호를 선택적으로 제외하여 인광 신호를 얻는 방법을 개발했다. 축적된 타임 게이트 분석은 바이오 이미징 및 바이오센싱 응용을 위한 발광 신호에 대한 다양한 정보를 제공했다. 5장에서는, 지속 가능한 비색/발광 반응을 얻기 위해 나노실리카 보호층을 사용하여 매우 안정적인 고분자 소포체를 제작했다. 실리카 보호층은 리포좀 구조를 가지는 디아세틸렌(DA) 소포 표면에 구형 실리카 나노입자를 기반으로 제작한다. 막이 형성되면, DA 소포의 구조적 안정성이 급격히 증가하고 건조된 분말 형태에서도 약 6개월 동안 보관할 수 있게 유지된다. 이후 다시 물에 분산시키면 실리카 보호막층에 의해 소포체가 응집되지 않는다. 장기간 보존 후 고분자 중합한 폴리디아세틸렌(PDA) 소포체는 기존 소포체 대비 평균 87.4%의 타겟 검지능을 유지했다.

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목차

ABSTRACT i
국문 초록 iv
PREFACE vii
TABLE OF CONTENTS viii
LIST OF FIGURES xii
LIST OF TABLES xiv
Chapter 1. Introduction 1
1.1 Photoluminescence 2
1.2 Room-Temperature Phosphorescence (RTP) 6
1.3 Luminophor-Bio Hybrid Interfaces 7
1.4 Time-gated Microscopy 8
1.5 Objective 9
1.6 References 12
Chapter 2. Fluorophor-Aptamer Hybrid Interfaces for Recognition of Protein and Cell 15
2.1 Scope of Research 16
2.2 Experimental Section 19
2.2.1 Aptamer Preparation 19
2.2.2 Fabrication of CTAB-, ssDNA-, and aptamer-guided Alq3 rods 20
2.2.3 Preparation of Proteins and Interaction with aptamer-Alq3 rods 22
2.2.4 Preparation of human Human oral mucosal epithelial cells (OMECs) 23
2.2.5 Cell culture 24
2.2.6 Treatment of HSC-3 and CMT-93 cells with Alq3 crystals 25
2.2.7 Treatment of Alq3 crystals with GO 26
2.2.8 Characterization 27
2.3 Results and Discussion 28
2.3.1 Recognition of human EpCAM by aptamer-guided Alq3 microrods 28
2.3.2 Specific response of Alq3 microrods upon binding to different proteins 33
2.3.3 Cellular response of human EpCAM aptamer-Alq3 microrods in human epithelial cells 36
2.3.4 Signal amplification of the aptamer-Alq3 microrods quenched by graphene oxide 42
2.4 Summary 48
2.5 References 49
Chapter 3. Phosphor-Peptide Hybrid Interfaces for Recognition of Protein at Room Temperature 52
3.1 Scope of Research 53
3.2 Experimental Section 56
3.2.1 Fabrication of PtOEP-A17 assembly 56
3.2.2 Treatment conditions of proteins with PtOEP-A17 assembly 58
3.2.3 Characterization of PtOEP nanoparticles 59
3.2.4 Time-resolved phosphorescence measurements 60
3.3 Results and Discussion 61
3.3.1 Fabrication of PtOEP-A17 assembly with reprecipitation method. 61
3.3.2 Specific target recognition of PtOEP-A17. 64
3.3.3 Simultaneous enhancement in photoluminescence intensity and lifetime of PtOEP-A17 assembly 70
3.4 Summary 75
3.5 References 76
Chapter 4. Application of Room Temperature Phosphorescence Materials for Bioimaging 79
4.1 Scope of Research 80
4.2 Experimental Section 82
4.2.1 Installation of time-gated microscope system with spectrometer 82
4.2.2 Luminescent powder sampling for microscopy 83
4.2.3 Characterization of luminescence signal of IPA crystals under excitation with continuous-wave laser 84
4.2.4 Characterization of luminescence signal of IPA crystals under excitation with pulsed laser 85
4.2.5 Fabrication of IPA–aptamer hybrid assembly and selective recognition with the target protein 86
4.2.6 Cell culture 87
4.2.7 Characterization 88
4.3 Results and Discussion 89
4.3.1 Design of an accumulated time-gated RTP analysis for avoiding autofluorescence 89
4.3.2 Optical properties of isophthalic acid crystal with long-lived phosphorescence emission 93
4.3.3 In situ time-gated RTP images and spectrum of IPA crystal under pulsed laser irradiation 98
4.3.4 RTP enhancement for IPA–aptamer hybrid assembly with specific recognition of target protein 105
4.3.5 Accumulation of phosphorescence signals of IPA–aptamer hybrid assembly after treatment with target protein via time-gated microscopy 115
4.3.6 Obtaining spectral information using RTP and autofluorescence materials under gating time control 120
4.3.7 Differentiation between phosphorescence and autofluorescence using time-gated microscopy and application to actual cells 126
4.3.8 Determination of phosphorescence signal in cell imaging application using time-gated microscopy 135
4.4 Summary 141
4.5 References 143
Chapter 5. Silica-Armored Polymerizable Vesicles for Sustainable Sensor 149
5.1 Scope of Research 150
5.2 Experimental Section 153
5.2.1 Synthesis of TCDA vesicles 153
5.2.2 Formation of silica armor on TCDA vesicles 154
5.2.3 Confirmation of redispersibility 156
5.2.4 Reactivity tests by thermal and chemical stimuli 157
5.2.5 Size-selective response of TCDA vesicles in silica armor membranes 158
5.2.6 Characterization 159
5.3 Results and Discussion 160
5.3.1 Fabrication of silica armor membrane on the surface of DA vesicle 160
5.3.2 Optical properties of silica armored vesicles at different phases. 167
5.3.3 Sustainable responses of silica armored vesicles by powderization and redispersion process 171
5.3.4 Layer-by-layer method for repeated stacking of silica armor membrane 178
5.3.5 Size selective response of TCDA vesicles with multilayered silica armor membrane 182
5.4 Summary 188
5.5 References 189
APPENDICES 196
Appendix A. Other Research: Controlling Metal-Fluorophor Interfaces for Recognition of Membrane Cholesterol 196
A1. Scope of Research 196
A2. Experimental Section 200
A3. Results and Discussion 206
A4. Summary 233
A5. References 234
Appendix B. Papers in Publication/Submission 239
Appendix C. Patents 240

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