An optical biosensor for the detection of pathogenic Salmonella Typhimurium based on a stem-loop DNA probe and retro-reflective signaling
An optical biosensor for the detection of pathogenic Salmonella Typhimurium based on a stem-loop DNA probe and retro-reflective signaling
- 주제(키워드) Salmonella , Molecular diagnostic , Retroreflective Janus particles , Optical biosensor , Stem-loop DNA
- 발행기관 아주대학교 일반대학원
- 지도교수 윤현철
- 발행년도 2019
- 학위수여년월 2019. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000029072
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The detection of foodborne pathogenic microorganisms is an essential issue in molecular diagnostics. Owing to their ability to detect and measure low analyte concentrations, fluorescence-based assays have been widely used in molecular diagnostics. However, conventional fluorescence-based assays require sophisticated optics systems, such as a specific light source and light filter. To overcome these limitations, in the present study, we developed an optical sensing system using a retroreflective Janus microparticle (RJP) as a signaling probe. Compared to fluorescent dyes, the advantage of RJPs is that they do not require complicated optic systems, as they can be observed using visible light without a filter. To confirm that RJPs can be used as a probe for molecular diagnostics, in the present study, Salmonella was detected using a biotinylated stem-loop DNA probe to capture the target gene DNA and a streptavidin-conjugated RJP (SA-RJP) as the detection molecule. When the target gene DNA was present at the sensing surface where the stem-loop DNA probe was immobilized, the biotinylated stem-loop DNA probe was stretched, exposing biotin, which can react with SA-RJP. Since the amount of exposed biotin increased according to the concentration of the applied target gene DNA, the number of RJPs observed on the sensing surface increased with the concentration of the target gene DNA. Consequently, the concentration of Salmonella could be quantitated by counting the number of observed RJPs. Using this system, Salmonella at concentrations ranging from 0 to 100 nM could be effectively analyzed with high sensitivity and selectivity, with the limit of detection of 2.48 pM.
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Contents
Abstract
Contents
List of Figures
List of Tables
An optical biosensor for the detection of pathogenic Salmonella Typhimurium based on a stem-loop DNA probe and retro-reflective signaling
1. Introduction
1.1 Salmonella as a foodborne pathogen 1
1.2 Salmonella diagnostic system 1
1.3 Stem-loop type DNA probe-based Salmonella detection system using fluorescence 2
1.4 Utilization of retroreflective Janus particle (RJP) as a signaling probe 4
1.5 Aim of thesis 12
2. Materials and methods
2.1 Reagents and apparatus 13
2.2 Preparation of the sensing surface and signaling probe
2.2.1 Immobilization of stem-loop DNA probe on sensing surface 14
2.2.2 Conjugation of streptavidin on RJP 18
2.3 Target gene analysis using developed system
2.3.1 Quantitative assay for detecting target gene of Salmonella 20
2.3.2 Selectivity test with mismatched target genes 21
3. Results and discussion
3.1 Confirmation of bio-conjugation on sensing surface and RJP 23
3.2 Optimization of stem-loop DNA probe concentration on the sensing surface 28
3.3 Salmonella quantitative assay and calibration curve 32
3.4 Verification of the selectivity in developed system for detecting Salmonella 37
4. Conclusions 42
5. References 43
