체내에 미량으로 존재하는 바이오마커 측정을 위한 면역 센싱 바이오칩
Immunosensing biochip for marker proteins at minute concentrations in human bodily fluids
- 발행기관 아주대학교
- 발행년도 2011
- 학위수여년월 2011. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000011874
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
A fluoro-microbead counting platform was developed to perform an optical immunoassay of various markers in real human samples. The fluoro-microbead guiding chip (FMGC) has four immunoreaction regions on a silicon oxide substrate, with five gold patterns imprinted on each region for multiple simultaneous assays. The FMGC assay clearly distinguished immunospecific binding from nonspecific binding by comparing optical signals from inside and outside of the patterns. To detect specific biomarkers, a sandwich immunoassay was performed using antibody-tagged fluoro-microbeads. The antigen-specific capture antibody was immobilized to the FMGC surface by reaction with 3-3'-dithiobis-propionic acid N-hydroxysuccinimide ester to create a self-assembling antigen-sensing monolayer (DTSP SAM) on the chip. A sample was applied to the antigen-sensing monolayer and allowed to react. To generate a binding signal, detection antibody-linked fluoro-microbead preparation step was added. The concentration of antigen such as cardiac troponin I (cTnI) and cartilage oligomeric matrix protein (COMP) in real human samples was determined by counting the number of biospecifically bound fluoro-microbeads on the corresponding five patterns on the FMGC. The optical signal showed a linear dependence with antigen concentrations in human samples such as blood and synovial fluid (SF). Optical detection and quantification of binding by fluorescence microscopy gave results that correlated well with results from a commercial ELISA for biomarkers in human plasma. Based on these findings, we propose that the FMGC-based immunoassay system may be adapted to detect and quantify a variety of clinically important targets in human samples.
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ABSTRACT i
CONTENTS ii
LIST OF FIGURES vi
LIST OF TABLES vi
Chapter I. General Introduction 1
1.1 Biosensor 2
1.2 Substrate materials for biosensors 6
1.3 The biological components 9
1.4 Immobilization of biological elements 17
1.5 Transducers 21
1.6 Review of fluorescence-based detection method 24
1.7 Objectives of the dissertation 27
Chapter II. A Fluoro-Microbead Guiding Chip for Simple and Quantifiable Immunoassay of Cardiac Troponin I (cTnI) 29
Abstract 30
2.1Introduction 31
2.2 Experimental section
2.2.1 Chemicals and apparatus 35
2.2.2 Antibody conjugation to fluoro-microbeads 36
2.2.3 Fabrication of the fluoro-microbead guiding chip (FMGC) 36
2.2.4 Fabrication of sensor surface 40
2.2.5 Optical immunosensing with the sandwich immunoassay and fluoro-microbead counting 42
2.2.6 Application to human plasma 43
2.3 Results and Discussion
2.3.1 Preparation of fluoro-microbead conjugate 44
2.3.2 Selection of reactive surface for the cTnI immunoassay 47
2.3.3 Fluorescence-based optical analysis of sandwich immunoassays 50
2.3.4 Cross-reactivity test with related troponins 53
2.3.5 Analysis of cTnI in human plasma 55
Chapter III. Chip-based Cartilage Oligomeric Matrix Protein (COMP) Detection in Serum and Synovial Fluid for Osteoarthritis Diagnosis 59
Abstract 60
3.1 Introduction 61
3.2 Experimental section
3.2.1 Chemicals and apparatus 64
3.2.2 COMP detection Antibody-tagged Fluoro-micro beads 65
3.2.3 Fabrication of a COMP-sensing interface 65
3.2.4 Detection of COMP on the FMGC with a fluorescence immunoassay 66
3.2.5 Application to real human samples 68
3.2.5.1 Human serum samples 68
3.2.5.2 Human synovial fluid sample 68
3.3 Results and Discussion
3.3.1 Optical analysis of the FMGC-based immunoassays 70
3.3.2 Analysis of COMP in human serum 73
3.3.3 Analysis of COMP in human synovial fluid 78
Chapter IV. Microfluidics Chip-Based Cartilage Oligomeric Protein (COMP) Detection in Serum and Synovial Fluid for Diagnosis of Osteoarthritis 82
Abstract 83
4.1 Introduction 84
4.2 Design
4.2.1 Design of microfluidics 87
4.2.2 Immunosensing strategy based on the sandwich immunoassay 88
4.3 Experimental
4.3.1 Materials and apparatus 90
4.3.2 Fabrication 91
4.3.3 Fabrication of a COMP-sensing Interface 93
4.3.4 Detection of COMP on the MFGC with a Sandwich Immunoassay 93
4.4 Results and discussions
4.4.1 Flow characterization 96
4.4.2 Solvent stability 97
4.4.3 Fluorescence-based optical analysis of sandwich immunoassays 101
Chapter V. Conclusions 103
Reference 106
LIST OF FIGURES
Fig. 1-1 Schematic diagram showing the main components of a biosensor 3
Fig. 1-2 The process for chip manufacturing by photolithography and wet etching 8
Fig. 1-3 Structures of different immunoglobulin classes 12
Fig. 1-4 Important attributes of an antibody. Detailed structure of immunoglobulin G (IgG) 13
Fig. 1-5 Schematic diagram of sandwich immunoassay 16
Fig. 1-6 Immobilization of the biological element 20
Fig. 1-7 Schematic diagram of the FMGC-based sandwich immunoassay 28
Fig. 2-1 (A) Design of the fluoro-microbead guiding chip (FMGC). (B) Photograph of the FMG 32
Fig. 2-2 Schematic illustration of the conjugation reaction between the fluoro-microbead and cTnI detection antibody 38
Fig. 2-3 Fabrication process for the fluoro-micobead guiding chip (FMGC) 39
Fig. 2-4 Schematic diagram of the sandwich immunoassay 41
Fig. 2-5 Fluorescence images of fluoro-microbead conjugates adsorbed on the glass surface 45
(A) before PEG coating
(B) after
Fig. 2-6 Photographs of microbead conjugates binding on different biosensing surfaces 49
(A) Dend-BA-modified chip surfaces
(B) Cys-GA-modified chip surfaces
(C) DTSP-modified chip surfaces
Fig. 2-7 Fluorescence images from FMGCs after sandwich immunoassay of cTnI at varying concentrations 52
(A) antigen/antibody binding system (System 1)
(B) avidin/biotin affinity binding (System 2)
Fig. 2-8 Cross-reactivity with nonspecific antigens (cTnT, cTnC and sTnI) in the FMGC-based
immunoassay 54
Fig. 2-9 Detection of cTnI in diluted plasma samples using a sandwich immunoassay on the FMGC. Calibration curves for fluorescence as a function of cTnI concentration in plasma diluted 2-, 5- and 10-fold 57
Fig. 2-10 Correlation between cTnI levels measured in spiked real plasma samples using the developed sandwich immunoassay (System 1 and System 2) and measurements using a conventional ELISA. Inset: a FMGC fluorescence image for 340 pM cTnI sample 58
Fig. 3-1 Schematic diagram of the FMGC-based sandwich immunoassay for COMP. Procedure for sensing layer formation, the fluorescence-signaling principle, and a magnified view of the registered image are presented 67
Fig. 3-2 (A) Photographs of fluoro-microbead conjugates binding at different COMP concentrations in buffer. (B) Calibration curves for fluorescence as a function of COMP concentration in standard samples in buffer. Each data point represents the average and standard deviation of independent triplicate assays 72
Fig. 3-3 Quantification of COMP concentration based on fluorescence signals from the spiked COMP concentration in 50-fold and 100-fold dilutions of human serum samples. Each data point represents the average and standard deviation of independent triplicate assays 75
Fig. 3-4 A COMP calibration curve in serum, and blind test results for unknown serum samples from patients. The red line corresponds to the calibration curve using the standard serum sample, and various patterns show the statistical means of FMGC-based immunoassay data for 11 unknown serum samples. The inset shows the correlation between COMP levels measured with the FMGC-based immunoassay and those with a commercial ELISA kit 76
Fig. 3-5 Detection of COMP in diluted synovial fluid samples using an FMGC-based sandwich immunoassay 80
(A) FMGC fluorescence images for synovial fluids diluted 1/500-1/3000.
(B) Calibration curves as a function of COMP concentration in SF samples diluted 500-, 1000-, 2000- and 3000-fold.
Fig. 3-6 (A) Photographs of FMGC-based assay results and data for the analysis of COMP at different concentrations in SF. (B) The FMGC-signaling results recorded for real SF samples as a function of COMP concentration. Inset: the correlation of COMP levels in SF measured with the FMGC-based immunoassay and those with a commercial ELISA kit？ 81
Fig. 4-1 Design of microfluidic guiding chip (MFGC) 89
Fig. 4-2 Photograph of the MFGC 92
Fig. 4-3 Schematic representation of MFGC-based sandwich immunoassay 95
Fig. 4-4 Characterization of mixing in basic type of microchannel using colored solution 97
Fig. 4-5 Characterization of dilution in microchannel using fluorescence material 99
Fig. 4-6 Photographs of microchannel and fluoro-microbead conjugates with different solvent (A) DTSP solution in DMSO, (B) DTSSP solution in DDW 100
Fig. 4-7 Fluorescence images from MFGCs after sandwich immunoassay of COMP in SF 102
LIST OF TABLES
Table 1-1 Biosensor market by sectors and commercial glucose sensors 4
Table 1-2 Classification of biosensor by biocomponent, transducer and immobilization method 5
Table 1-3 Various optical geometries used for biosensors 26
Table 2-1 Changes in the z-potential and sizes of fluorescence microbead conjugates using different blocking processes 46
Table 3-1 COMP concentrations were measured in patient samples using both the FMGC-based sandwich immunoassay and a commercially available ELISA. Difference (%) refers to the difference in COMP levels as measured by the two assays 77
