SHERP

Study on Multiplexed Immunoassay Platform Using Surface Enhanced Raman Spectroscopic Nanoprobes
표면 증강 라만 분광 나노프로브를 이용한 다중 면역분석에 관한 연구

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Authors
장혜진
Advisor
정대홍
Major
사범대학 과학교육과
Issue Date
2016
Publisher
서울대학교 대학원
Keywords
Surface-enhanced Raman scattering (SERS)immunoassayprostate specific antigen (PSA)SERS nanoprobecore-satelliteSERS hot spotnear-infrared probeuniform SERS activityquantitative analysisarea-scanningon-chip analysissensitivityreproducibility
Description
학위논문 (박사)-- 서울대학교 대학원 : 과학교육과 화학전공, 2016. 8. 정대홍.
Abstract
Recently, surface-enhanced Raman scattering (SERS)-based immunoassays (SIA) have drawn much attention as diagnostic tools with large multiplex capacity and high sensitivity. However, several challenges—such as a low reproducibility, a time-consuming readout process, and limited dynamic range—remain. In this study, we demonstrated a reliable and sensitive SIA platform for prostate specific antigen (PSA) detection. Reliability and sensitivity were achieved by two approaches: 1) well-established SERS probes, so-called SERS dots that have high sensitivity (single particle detection) and little particle-to-particle variation in SERS intensity
and 2) a whole area-scanning readout method for rapid and reliable chip analysis rather than point scanning. Therefore, this thesis is composed of three chapters, related to SERS nanoprobes for multiplexed and quantitative analysis, a sensitive and reliable readout method for 2-dimensional chip analysis, and immunoassay platform as a whole of former two, respectively. In chapter I, multiplexed and quantitative SERS nanoprobes for biological application were developed. An well-developed SERS nanoprobe, SERS dotTM, was anlayzed at single particle level for characterization of sensitivity, particle-toparticle distribution of signals, and photostability for its application to immunoassay. And also, an Ag shell-Au satellite (Ag-Au SS) nanostructure composed of an Ag shell and surrounding Au nanoparticles was described as a near-IR active SERS probe. It was a key strategy to create isotropic hot spots in developing a reproducible, homogeneous, and ultra-sensitive SERS probe. The heterometallic shell-satellite structure based SERS probe produced an intense and uniform SERS signals (SERS enhancement factor: ~1.4 × 106 with 11% relative standard deviation) with high detectability (100% under our measurement condition) by 785-nm photoexcitation. This signal enhancement was independent of the laser polarizations, which reflects the isotropic feature of the SERS activity of Ag-Au SS from the three-dimensional (3-D) distribution of SERS hot spots between the shell and the surrounding satellite particles. The Ag-Au SS nanostructure shows a great potential as a reproducible and quantifiable SERS probe for biological targets. In Chapter II, a whole area scanning readout method for rapid and reliable chip analysis was described. SERS techniques have been widely used for bioanalysis due to its high sensitivity and multiplex capacity. However, the point-scanning method using a micro-Raman system, which is the most common method in the literature, has a disadvantage of extremely long measurement time for on-chip immunoassay adopting a large chip area of approximately 1-mm scale and confocal beam point of ca. 1-μm size. Alternative methods such as sampled spot scan with high confocality and large-area scan method with enlarged field of view and low confocality have been utilized in order to minimize the measurement time practically. In this study, we analyzed the two methods in respect of signal-to-noise ratio and sampling-led signal fluctuations to obtain insights on a fast and reliable readout strategy. On this basis, we proposed a methodology for fast and reliable quantitative measurement of the whole chip area. The proposed method adopted a raster scan covering a full area of 100 μm × 100 μm region as a proof-of-concept experiment while accumulating signals in the CCD detector for single spectrum per frame. One single scan with 10 s over 100 μm × 100 μm area yielded much higher sensitivity compared to sampled spot scanning measurements and no signal fluctuations attributed to sampled spot scan. This readout method is able to serve as one of key technologies that will bring quantitative multiplexed detection and analysis into practice. In chapter III, a reliable and reproducible chip-based SERS immunoassay with high sensitivity and broad dynamic range was described. It was achieved by using the reproducible nanoprobes with single-particle sensitivity (SERS dot), and the reliable readout method from sub-millimeter area. As a feasibility test, PSA analysis was performed as a model system. Bio-functionality of SERS dot was demonstrated, and the number of antibody on the silica surface and captured antigens were evaluated by utilizing fluorescence-labeled antibody and antigen. Finally, PSA could be detected with high sensitivity (ca. 0.11 pg/mL, 3.4 fM LOD), with a wide dynamic range (0.001−1000 ng/mL). In addition, the developed platform was successfully adopted to detect PSA in the patient’s serum. Thus, the developed platform will facilitate development of reliable immunoassays with high sensitivity and a wide dynamic range.
Language
Korean
URI
http://hdl.handle.net/10371/129662
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College of Education (사범대학)Dept. of Science Education (과학교육과)Chemistry (화학전공)Theses (Ph.D. / Sc.D._화학전공)
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