Title Development of Microdevice-based Immunoassay Systems for Point-of-Need Testing Author(s) 西山, 慶音 Citation 北海道大学. 博士(工学) 甲第14471号 Issue Date 2021-03-25 DOI 10.14943/doctoral.k14471 Doc URL http://hdl.handle.net/2115/81280 Type theses (doctoral) File Information NISHIYAMA_Keine.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP Development of Microdevice-based Immunoassay Systems for Point-of-Need Testing 現場即時検査のための マイクロデバイスを用いたイムノアッセイシステムの開発 Keine Nishiyama Graduate School of Chemical Sciences Engineering Hokkaido University Title Development of Microdevice-based Immunoassay Systems for Point-of-Need Testing Author Keine Nishiyama Degree Doctor of Philosophy (Engineering) Supervisor Professor Manabu Tokeshi CONTENTS CHAPTER 1 General Introduction .................................................................................................................. 1 1.1 Point-of-Need Testing ......................................................................................................................... 2 1.2 Immunoassays ..................................................................................................................................... 3 1.2.1 Heterogeneous Immunoassay ..................................................................................................... 5 1.2.2 Homogeneous Immunoassay ...................................................................................................... 6 1.3 Microdevices for analytical chemistry .............................................................................................. 8 1.3.1 The “μTAS” Concept .................................................................................................................. 8 1.3.2 Microdevices for Immunoassays ................................................................................................ 9 1.3.2.1 Immunoassay systems for easy-to-use detection ....................................................................... 9 1.3.2.2 Highly sensitive immunoassay systems .................................................................................... 12 1.4 Objectives of the Thesis .................................................................................................................... 14 1.5 References .......................................................................................................................................... 17 CHAPTER 2 Ultrasensitive Detection of Disease Biomarkers Using an Immuno-wall Device with Enzymatic Amplification ................................................................................................................................. 29 2.1 Introduction ....................................................................................................................................... 30 2.2 Experimental ..................................................................................................................................... 31 2.2.1 Materials and reagents .............................................................................................................. 31 2.2.2 Fabrication of immuno-wall device ......................................................................................... 32 i 2.2.3 Assay procedure ........................................................................................................................ 34 2.2.4 Fluorescence microscope .......................................................................................................... 35 2.3 Results and Discussion ...................................................................................................................... 36 2.3.1 Biomarker detection with fluorescence-labeled antibody ..................................................... 36 2.3.2 Selection of fluorogenic substrates for enzymatic amplification ........................................... 37 2.3.3 Biomarker detection with the enzyme-labeled antibody, alkaline phosphatase-labeled antibody ..................................................................................................................................................... 42 2.4 Conclusions ........................................................................................................................................ 46 2.5 References .......................................................................................................................................... 47 CHAPTER 3 Simple Approach for Fluorescence Signal Amplification Utilizing a Poly(vinyl alcohol)- based Polymer Structure in a Microchannel ................................................................................................. 53 3.1 Introduction ....................................................................................................................................... 54 3.2 Experimental ..................................................................................................................................... 55 3.2.1 Materials ..................................................................................................................................... 55 3.2.2 AWP-wall in a microchannel .................................................................................................... 56 3.2.3 Evaluation of AWP-wall in a microchannel as a fluorescence signal amplifier .................. 56 3.2.4 Evaluation of transfer of fluorescent molecules to AWP in a microplate ............................ 57 3.2.5 Fluorescence spectrum of DDAO ............................................................................................. 57 3.2.6 Prediction of octanol-water partition coefficient .................................................................... 58 3.3 Results and discussion ...................................................................................................................... 58 ii 3.4. Conclusions ........................................................................................................................................ 67 3.5 References .......................................................................................................................................... 69 CHAPTER 4 Non-competitive Fluorescence Polarization Immunoassay Based on a Fab Fragment for Protein Quantification ..................................................................................................................................... 73 4.1 Introduction ....................................................................................................................................... 74 4.2 Experimental ..................................................................................................................................... 76 4.2.1 Chemicals ................................................................................................................................... 76 4.2.2 Labeling procedure ................................................................................................................... 76 4.2.3 Assay procedure ........................................................................................................................ 77 4.2.4 Fluorescence analysis of human serum ................................................................................... 77 4.2.5 Portable FP analyzer ................................................................................................................. 78 4.2.6 Microfluidic device .................................................................................................................... 78 4.3 Theoretical fluorescence polarization value ................................................................................... 79 4.4 Results and Discussion ...................................................................................................................... 80 4.4.1 Development of non-competitive FPIA based on a Fab fragment ........................................ 80 4.4.2 Application: diagnosis of inflammation based on CRP level in serum ................................ 84 4.5 Conclusions ........................................................................................................................................ 89 4.6 References .......................................................................................................................................... 90 CHAPTER 5 Application of non-competitive fluorescence polarization immunoassay for antigen and antibody detection of H5 avian influenza virus ............................................................................................. 95 iii 5.1 Introduction ....................................................................................................................................... 96 5.2 Experimental ..................................................................................................................................... 98 5.2.1 Materials ..................................................................................................................................... 98 5.2.2 Preparation of purified H5N3 AIV .......................................................................................... 98 5.2.3 Preparation of F-Fab ................................................................................................................. 99 5.2.4 Preparation of Fluorescein-labeled antigen