Development of optical immunosensing platform by employing retroreflective Janus particle as a signaling probe
- 주제(키워드) Retroreflection , immunoassay , biosensor , POCT , Cell imaging
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 윤현철
- 발행년도 2022
- 학위수여년월 2022. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000031392
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
This thesis aims to develop retroreflection-based optical immunosensing platforms using retroreflective Janus particles as a signaling probe. To construct the POCT biosensing application, retroreflective Janus particle (RJP) was fabricated and employed as a signaling probe. Employing retroreflection phenomena, sophisticated optical components, and a tailor-made filter system were not required to perform intuitive and simple immunosensing applications. In the presence thesis, we tried to facilitate a wash-free assay procedure, a simple signal processing protocol to develop a POCT immunosensor. Furthermore, to test the expandable usage of RJP, the RJPs were also applied to a cell migration monitoring application for live-cell imaging. The following three themes were covered in this thesis based on the RJP's inherent physical and biological properties. First, the retroreflection-based immunoassay platform was designed to perform the wash-free assay by introducing the intrinsic sedimentation characteristics of an RJP. Since the retroreflection ability of RJPs was generated by a half-coated metal layer, the RJP has a sedimentation property that could be used for passive particle modulation in the solution. The developed assay procedure introduces a forced contact and separation of particles toward the sensing surface, where the RJPs are eventually imaged and quantified by a retroreflective optical setup. Additionally, the signal processing protocol using time-lapse imaging techniques and logical operation was also implemented to show the applicability of noise signal exclusion procedure. As the retroreflection signals from non-specifically bound RJPs were registered as a dynamic object on the sensing surface, image calculation using logical operation “AND” could eliminate the variable signal counts from the sensing surface. One of the cardiac biomarkers, creatine kinase-MB fractions (CK-MB), was selected as a model target biomarker to evaluate the proposed immunoassay. Buffer- and human serum- spiked CK-MB was successfully quantified with high sensitivity. Second, to assess the applicability of the miniaturized retroreflective optical setup to integrate with the smart handset, the retroreflective optical gadget was designed and 3D-printed for inverted immunosensing. To address the integration of wash-free immunoassay using RJPs on the smart handset, signal registration property on smart handset-embedded camera and white LED flash was checked in advance of applying sedimentation-based assay protocol. To satisfy the distinctive signal registration from RJPs in the microfluidic chip, we designed the optical gadget for better light penetration yield and optical path in a miniaturized device. Furthermore, we also tried to develop an Android application package that could commence the time-lapse imaging and image calculation. The image calculation strategy was re-constructed as a model biomarker CK-MB was quantified with fluidic chips to lessen the burden of the processing capacity for the application package. Using this, a passive modulation of RJPs using sedimentation property was conducted to minimize the random error and enforce reproducibility. Third, to expand the application of RJPs for cell behavior sensing, RJPs were introduced to cells as a labeling probe that enables the live-cell monitoring while the cells are migrated. To avoid surplus complexity in experimental procedure in transwell migration assay, RJPs induced to be phagocyted to macrophage. Since conventional steps for migratory assays demand skillful, trained users, involve time-consuming processes, and cannot identify the progress of migration status until cell fixation and dye staining steps end, we designed to modify the migration assay to become a simplified method for analyzing migratory cell property. Thus, to omit the cell fixation and dye staining process RJPs were introduced to live macrophages. In this regard, we introduced a retroreflective Janus microparticle (RJP) as a new optical probe for analyzing the migratory macrophages using the principle of retroreflection. Based on the working principles of retroreflection and application of RJPs, a cell assay was designed and tested for applicability in migration assays. Based on these findings, we suggest a retroreflection-based immunosensing platform employing RJPs as optical probes, which could be applied to biosensors requiring point-of-care testing use.
more목차
Chapter I. General Introduction 1
1.1 Biosensor 2
1.2 Optical signaling probes 5
1.3 Limitation of the conventional optical biosensing system 8
1.4 Retroreflection-based optical probe 10
1.5 The proposed Solution and approach 25
1.6 Objectives of the thesis 26
1.7 References 29
Chapter II. Wash-free non-spectroscopic optical immunoassay by controlling retroreflective microparticle movement in a microfluidic chip 32
2.1 Introduction 34
2.2 Experimental section 40
2.2.1 Materials and apparatus 40
2.2.2 Modification of PMMA surface for microfluidic channel substrates 40
2.2.3 Modification of PMMA capturing surface 41
2.2.4 Modification of spatial-selective antibody conjugation on RJP 43
2.2.5 Preparation of antigen samples and specific protocol for inverted focusing assay for CK-MB detection 44
2.2.6 Imaging RJPs in microchannel and signaling protocol for optimized RJP counting 44
2.3 Results and Discussion 45
2.3.1 Whole process of inverted focusing method utilized retroreflective immuno -assay 45
2.3.2 Antibody modification on PMMA surface and RJP 49
2.3.3 Validation of sedimentation property of RJP in microchannel 53
2.3.4 Sedimentation-based inverted focusing assay procedure 57
2.3.5 Image processing-based retroreflection signaling strategy 60
2.3.6 Retroreflection-based inverted focusing CK-MB immunoassay 64
2.4 References 70
Chapter III. Practical design and operation of smartphone-integrated optical immunosensor using retroreflective microparticles 73
Abstract 74
3.1 Introduction 75
3.2 Experimental section 80
3.2.1 Materials and apparatus 80
3.2.2 Preparation of the biosensing surface and microfluidic chips 81
3.2.3 Fabrication of the retroreflective Janus microparticles 81
3.2.4 Antibody conjugation on retroreflective Janus particles 84
3.2.5 Wash-free immunosensing procedure using the sedimentation property of retroreflective Janus particles 85
3.2.6 The retroreflective optical gadget for smartphones 86
3.2.7 Procedure of retroreflection-based immunosensing on a smartphone using the inverted-focusing method 88
3.3 Results and Discussion 90
3.3.1 Introduction of the retroreflective Janus particles in the immunosensing procedure 90
3.3.2 Introducing morphological property of retroreflective Janus particles 93
3.3.3 Design of optical gadget and construction of the biosensing chip 94
3.3.4 Image processing package development of signal quantification strategy in PC and smartphone 102
3.3.5 Adaptation of retroreflection-based immunosensing on a smartphone 105
3.4 References 114
Chapter IV. A non-spectroscopic live-cell monitoring device by using retroreflective Janus microparticles 117
Abstract 118
4.1 Introduction 119
4.2 Experimental 123
4.2.1 Materials and apparatus 123
4.2.2 Preparation of retroreflective Janus microparticle 124
4.2.3 Cell culture 124
4.2.4 Cytotoxicity test 124
4.2.5 Preparation of Transwell insert for migration assay 125
4.2.6 Construction of retroreflection-based optical setup and retroreflection signal quantification 128
4.3 Results and discussions 129
4.3.1 Overall strategy of retroreflection-based migratory cell quantification and monitoring method 129
4.3.2 Validation of cytotoxicity and phagocytosis by macrophages at different RJP concentrations 132
4.3.3 Characterization of the RJP-phagocyted macrophage 135
4.3.4 Identification of phagocyted RJPs in macrophage for cell quantification 138
4.3.5 Retroreflection-based migratory macrophage quantification 141
4.4 Future works 145
4.5 References 147
Chapter V. Conclusions and perspectives 149
5.1 Conclusions 150
5.2 Perspectives 153
Summary in Korean 154
