Development of retroreflection-based optical biosensing platform using retroreflective Janus particle as a signaling probe
Development of retroreflection-based optical biosensing platform using retroreflective Janus particle as a signaling probe
- 주제(키워드) Biosensor , retroreflection , Janus particle , immunoassay , cardiac troponin I , Salmonella Typhimurium , retroreflective Janus particles , loop-mediated isothermal amplification , molecular diagnostic , optical biosensing system , Mercury ion sensing , optical biosensor , Hg2+-mediated thymine-thymine base pairing , stem-loop DNA probe
- 발행기관 아주대학교 일반대학원
- 지도교수 윤현철
- 발행년도 2019
- 학위수여년월 2019. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029080
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
We developed a nonspectroscopic optical biosensing platform by employing the principle of retroreflection. Retroreflection is the phenomenon of light rays striking a surface and being redirected back to the light source. Retroreflection is not related to the type of light source, but rather to the condition and structure of the reflecting surface and material. Since all conventional light sources can induce retroreflection, if retroreflection is introduced to optical biosensing, the optical system can be simplified and miniaturized. Based on these features, in order to overcome the limitations in conventional optical biosensors that require a sophisticated optics system, including a precisely aligned filter, mirror, and wavelength-tunable light source, we sought to implement a retroreflection-based optical biosensor that can be operated under a simplified optics configuration based on the general white light. To employ the retroreflection as a signaling principle, half-coated Janus particles that can induce interior retroreflection were developed and used as an optical signaling probe. First, to assess the applicability of the developed reflective Janus particles (RJPs) as an optical signaling probe, quantitative analysis for a disease biomarker was performed. As a model biomarker, cardiac troponin I (cTnI), which is a biomarker for the diagnosis of acute myocardial infarction, was selected. The RJPs were observed under a simple optics configuration and general light conditions such as a white light emitting diode (LED) and a complementary metal oxide semiconductor (CMOS) camera. Using this platform, the quantitative analysis of the cTnI ranging from 0 to 100 ng/mL was successfully performed with a low sensitivity and a good reproducibility. Based on these results, we confirmed that the RJPs can be used as a novel optical signaling probe. Second, to confirm the applicability of the RJPs for molecular diagnosis, the Salmonella Typhimurium (S. Typhimurium) was analyzed as a target molecule. To effectively amplify the S. Typhimurium, the loop-mediated isothermal amplification (LAMP) technique was employed. Due to the property of the LAMP technique where the amplified gene was generated dumbbell-shaped DNA structure containing a single-stranded loop region with two different sequences, the amplified gene could be measured in a manner similar to the sandwich-type immunoassay. Using the developed biosensing platform, a highly sensitive and selective quantification of S. Typhimurium (102 to 107 CFU) was successfully accomplished with the detection limit of 102 CFU. Based on the results, we proved that the developed RJPs could be applied for the molecular diagnostic field including pathogen. Third, to prove the possibility of RJPs utilization, the RJPs were applied to environmental monitoring. The water-soluble mercury ion was selected as the target model. To detect the mercury ion present in the water, Hg2+-mediated thymine-thymine (T-T) base-stabilization principle was integrated with a RJP-based optical sensing strategy. Using this system, various concentrations of Hg2+ (0 to 100 nM) could be quantitatively analyzed with the LOD of 0.027 nM. In addition, our results not only showed that the developed sensing system had a high selectivity, but also confirmed that it could be applied to a real sample. Based on these findings, we propose a retroreflection-based optical biosensing platform using RJPs as a novel optical signaling probe which can be adapted to various biosensing fields.
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CONTENTS
ABSTRACT·············································································································i
CONTENTS··········································································································iii
LIST OF FIGURES····························································································viii
LIST OF TABLES····························································································xv
Chapter I. General Introduction·························································1
1.1 Biosensor ·······················································································2
1.2 Optical signaling probe ·······································································5
1.2.1 Enzyme and chromogenic dyes ··························································5
1.2.2 Metal nanoparticles ·······································································6
1.2.3 Fluorophore ················································································7
1.2.4 Quantum dots (QDs) ······································································8
1.3 Limitation of the conventional optical biosensing system ·······························10
1.4 The proposed Solution and approach ······················································12
1.5 Objectives of the thesis ······································································16
1.6 References ····················································································19
Chapter II. Retroreflective Janus microparticle as a nonspectroscopic optical sensing probe················································································23
Abstract ···························································································24
2.1 Introduction ···················································································25
2.2 Experimental section
2.2.1 Materials and apparatus ·································································29
2.2.2 Synthesis of monodispersed silica microparticles ···································30
2.2.3 Fabrication of RJPs ······································································30
2.2.4 Characterization of RJPs using SEM and EDS ·······································33
2.2.5 Construction of an optical analysis system for retroreflection evaluation ·········33
2.2.6 Analysis of the effect of the aluminum thickness of RJP toward its retroreflection
and dispersion properties ································································34
2.2.7 Spatio-selective antibody conjugation on RJPs ······································35
2.2.8 Fluorescence microscopic tests of RJPs ···············································38
2.2.9 Design and fabrication of the immunosensing chip allowing easy signal
counting ··················································································38
2.2.10 Preparation of the immunosensing surface and RJP probe for cTnI
immunoassay ··········································································40
2.2.11 Retroreflective immunosensing of cTnI ·············································40
2.2.12 Retroreflective Immunoanalysis on an Imaging Setup ·····························42
2.3 Results and Discussion
2.3.1 Design and fabrication of retroreflective Janus particles ····························44
2.3.2 Quantitative analysis of the retroreflective property of RJPs ·······················49
2.3.3 Qualitative evaluation of the retroreflective property of RJPs ······················54
2.3.4 Characterization of optical signal from RJPs under the microscopic imaging
system ····················································································56
2.3.5 Spatio-selective biofunctionalization of RJP ·········································60
2.3.6 Retroreflection-based cTnI immunosensing ··········································62
2.4 References·····················································································66
Chapter III. A Salmonella Typhimurium sensing platform based on the loop-mediated isothermal amplification using retroreflection Janus particle as a nonspectroscopic signaling probe·······················································70
Abstract ···························································································71
3.1 Introduction ···················································································72
3.2 Experimental section
3.2.1 Materials and apparatus ·································································76
3.2.2 Design of primers for the LAMP assay ················································76
3.2.3 DNA extraction ···········································································77
3.2.4 LAMP assay ··············································································77
3.2.5 Preparation of the detection DNA-conjugated RJP and the sensing surface ······78
3.2.6 Measurement process of the amplified LAMP product ······························79
3.3 Results and Discussion
3.3.1 Signaling principle of the developed Salmonella detection system ················81
3.3.2 Confirmation of the DNA amplification antibody of the designed LAMP primer·····················································································82
3.3.3 Verification of the functionalization of the RJP surface ·····························85
3.3.4 Verification of the functionalization of the sensing surface ·························87
3.3.5 Cross-reactivity test between capture-DNA of the sensing surface and detection-
DNA of the RJPs ········································································89
3.3.6 Confirmation of the developed optical sensing principle ····························93
3.3.7 Calibration study of the developed Salmonella detection platform using a non-
purified LAMP product ··································································95
3.4 References ····················································································98
Chapter IV. Water-soluble mercury ion sensing based on the thymine-Hg2+-thymine base pair using retroreflective Janus particle as an optical signaling probe························································································103
Abstract ·························································································104
4.1 Introduction ·················································································105
4.2 Experimental
4.2.1 Materials and apparatus ································································110
4.2.2 Preparation of streptavidin-RJP ·······················································111
4.2.3 Preparation of sensing surface ························································113
4.2.4 Measurement process of mercury ion concentration ·······························115
4.2.5 Selectivity test and verification of practical possibility using real samples ······115
4.3 Results and discussions
4.3.1 Signaling principle of mercury ion detection using Hg2+-mediated T-T base
pairing and retroreflection ····························································117
4.3.2 Confirmation of functionalization of RJP surface ··································118
4.3.3 Confirmation of functionalization of sensing surface ······························120
4.3.4 Calibration study using developed mercury ion detection platform ··············122
4.3.5 Verifying the selectivity of the developed mercury ion sensing system for the
mercury ion ············································································127
4.3.6 Recovery test of mercury ion in real water samples ································130
4.4 References ····················································································132
Chapter V. Conclusions and perspectives············································137
5.1 Conclusions ··················································································138
5.2 Perspectives ··················································································139
Summary in Korean······································································141
