Microfluidic kit for macrophage activation monitoring based on chromogenic and electrochemical dual-sensing
- 주제(키워드) Microfluidic , Macrophage activation , dual-sensing , chromogenic , electrochemical
- 발행기관 아주대학교
- 지도교수 윤현철
- 발행년도 2021
- 학위수여년월 2021. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000030541
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
In this study, we developed a macrophage activation monitoring kit based on electrochemical and optical methods for evaluating the activation of macrophages. Macrophages are immune cells, and upon immune activation through external stimuli, they develop pseudopodia-, become irregular in shape, and increase secretion of H2O2. Therefore, using these immune cell properties, we developed a macrophage activation monitoring kit to perform activation testing by measuring macrophage-derived H2O2 in a culture medium. The developed macrophage activation monitoring kit consists of a cell chip and an analysis module, which is designed to be integrated into a cell chip. The developed cell chip contains an analysis section and a cell culture chamber, with a microvalve between them, to allow H2O2 analysis without destroying the cells. In addition, two different analytical methods were introduced to increase sensitivity and efficiency compared to using a single analytical method. For this, the analysis module based on the enzyme-mediated catalytic reaction was developed, and a paper-based analytical device was used for color development reactions, and a carbon paste electrode was used for the electrochemical method. To verify the H2O2 analysis capability of the cell chip, we cultured RAW264.7 cells, a murine macrophage cell line, and activated them using phorbol 12-myristate 13-acetate. As a result, we confirmed that RAW264.7 cells stably adhered and grew on the surface of the cell culture chamber. Furthermore, their activation was confirmed based on cathodic current values, both of which were proportional to RAW264.7 cell concentrations.
more목차
1. Introduction 1
1.1 Immunogenicity test 1
1.2 Macrophages as immune cells 1
1.3 immune activation test using macrophages 2
1.4 Hydrogen peroxide detection 3
1.5 Aim of thesis 4
2 Materials and methods 5
2.1 Materials and apparatus 5
2.2 Cell chip manufacture for macrophage activation analysis 6
2.2.1 Cell chip manufacture 6
2.2.2 RAW264.7 cell culture 7
2.2.3 Cell viability and activation test 8
2.3 Colorimetric assay 9
2.3.1 Fabrication of PAD for H2O2 analysis 9
2.3.2 PAD and TMB immobilization 9
2.3.3 PAD integrated with cellulose powder 10
2.3.4 Manipulation of smartphone gadget 11
2.3.5 Assay of buffer spiked analysis 11
2.4 Electrochemical analysis 12
2.4.1 Carbon paste electrode preparation 12
2.4.2 TEOS sol-gel preparation 12
2.4.3 Synthesis of Gold nanurchine (AuNU) 13
2.4.4 Preparation of AuNU/HRP-modified CPE 13
2.4.5 Cyclic voltammetry and chronoamperometry 14
3 Results and discussion 15
3.1 Strategy for macrophage activation monitoring 15
3.1.1 Macrophage activation monitoring kit configuration 15
3.1.2 Macrophage-derived H2O2 measurement 18
3.2 Monitoring RAW264.7 cell activation using the kit 21
3.2.1 RAW264.7 cell viability test 21
3.2.2 Inducing macrophage activation using PMA 24
3.3 Colorimetric assay for analysis of H2O2 27
3.3.1 Main principle of analysis for H2O2 by colorimetric assay 27
3.3.2 Configuration of PAD 27
3.3.3 Illustration of smartphone-based device for PAD image acquisition 30
3.3.4 Optimization of PAD pattern size 32
3.3.5 Difference in color intensity depending on H2O2 concentration 35
3.4 Electrochemical analysis of H2O2 38
3.4.1 Principle of analysis the H2O2 using the electrochemical method 38
3.4.2 Characterization of AuNU structure 41
3.4.3 Electrochemical properties of AuNU/HRP-modified CPE 43
3.4.4 Determination of levels of H2O2 derived from RAW264.7 cell 46
4 Conclusions 49
5 References 50
