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Development and characterization of aptamers for the use in surface plasmon resonance sensors for the detection of glycated blood proteins
Table of Contents
Abstract
Acknowledgements
Table of Contents
List of Tables
List of Figures
List of Abbreviations
1 Introduction
1.1 Background and Significance
1.2 Research Objectives
1.3 Thesis Chapters
2 Theory and Applications
2.1 Glycated Proteins as Biomarkers
2.2 Aptamer Theory
2.3 SELEX Theory
2.4 SPR Overview and Theory
2.5 Other SPR Applications and Configurations
2.6 SPR Opto-Chemical Junction Methods
2.7 SPR Instrumentation: BIAcore® 3000
3 Aptamer Development
3.1 Magnetic Bead Based SELEX
3.2 PCR Overview
3.3 Real Time PCR
3.4 Transformation and Cloning
3.5 SELEX Materials and Methods
3.6 SELEX Results and Discussion
3.7 Sequencing Methods and Results
3.8 Aptamer Potential Structure and Binding Sites
3.9 Redesign of Initial Oligonucleotide Pool Primers
3.10 Chapter Summary
4 Aptamer Characterization
4.1 Equilibrium Dissociation Constants
4.2 Langmuir Binding Model
4.3 SPR Sensing Surface Regeneration
4.4 Characterization Materials and Methods
4.5 Characterization Results and Discussion
4.6 Sensor Applications
4.7 Chapter Summary
5 Future Work and Conclusions
5.1 Testing and Use of the New Oligonucleotide Pool
5.2 Primer-Free SELEX and Stem-Loop Characterization
5.3 Counter-Selection Methods
5.4 SAM Development to Prevent Non-Specific Binding
5.5 Conclusions
References
A SELEX Protocol
A.1 Coupling Target Protein to Magnetic Beads (MB)
A.2 Binding and Amplification
A.3 Cloning and Sequencing
B Additional SELEX Results
B.1 DNA Melting Temperature Curves
C Identified Aptamer Sequences
C.1 Potential Glycated Hemoglobin Aptamers
C.2 Potential Hemoglobin Aptamers
C.3 Potential Fibrinogen Aptamers
D Aptamer Structure and Binding Sites
D.1 Potential Aptamer Binding Sites - Hemoglobin
D.2 Potential Aptamer Binding Sites – Glycated Hemoglobin
D.3 Potential Aptamer Binding Sites – Fibrinogen
D.4 Aptamer Structures – Hemoglobin
D.5 Aptamer Structures – Glycated Hemoglobin
D.6 Aptamer Structures – Fibrinogen
E Redesign of Initial Oligonucleotide Pool and Primers
E.1 Primer Design Rules
E.2 Analysis of Original Primers
E.3 Analysis of New Primers
F Derivation of the Langmuir Model
F.1 Langmuir Binding Model
G Experimental Data and Global Fits
G.1 Aptamer HA1
G.2 Aptamer GHA1
G.3 Aptamer HA2
G.4 Aptamer GHA2
H DynabeadTM Coupling Protocol
H.1 Protocol Bài viết liên quan
