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Development of optical immunosensing platform by employing retroreflective Janus particle as a signaling probe
- Author(s)
- 김가람
- Alternative Author(s)
- Ka Ram Kim
- Advisor
- 윤현철
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2022-02
- Language
- eng
- Keyword
- Cell imaging; POCT; Retroreflection; biosensor; immunoassay
- Alternative Abstract
- 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.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 4. Theses(Ph.D)
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