Microarray Technology for Kinetic Analysis of Vesicle Bound Receptor-Ligand Interactions

A proof-of-concept for a novel microarray used to study protein-ligand interaction in real-time using label-free detection is presented. Many of todays commercially available instruments lack the ability to immobilize membrane proteins. At the same time, the pharmaceutical industry develops drugs directed towards membrane-bound receptors. The need to study drug-target kinetics and to be able to screen for new medical substances is high. To study the biomolecular interactions in real-time, imaging surface plasmon resonance (iSPR) is used. A patterned sensor surface with hydrophobic barriers assisting in the piezodispensing of NeutrAvidin with complex-bound biotin-ssDNA is created. Histidine-tagged proteins are immobilized at the vesicle surface using divalent nitrilotriacetic acid. The concept of the vesicle immobilization, the protein-binding to vesicles and the protein-ligand interaction is initially studied using a Biacore instrument…


1 Introduction
1.1 Background
1.2 Aims of the Master’s Thesis
1.3 Key Ideas for the Solution of the Problem
2 Theory
2.1 Protein Kinetics – A Way to Characterize Protein-Ligand Interaction
2.1.1 General Protein Kinetics
2.1.2 Studied Proteins
2.2 Vesicles
2.2.1 Tagging the Vesicles with ssDNA
2.2.2 Nitrilotriacetic Acid (NTA)
2.3 Creating the Biochip
2.3.1 Self Assembled Monolayers (SAMs)
2.3.2 Microcontact Printing
2.3.3 Piezodispensing
2.3.4 Dextran
2.3.5 The Neutravidin-Biotin-ssDNA Complex
2.4 Null Ellipsometry
2.4.1 The Polarization of Light
2.4.2 Optical Component Setup
2.5 Surface Plasmon Resonance (SPR)
2.5.1 Imaging SPR
3 Experimental Details
3.1 Surfaces
3.2 Preparation of PDMS Master and µCP Stamp
3.3 Preparation of SAMs
3.3.1 µCP and Back-filling
3.4 Dextran
3.4.1 Dextran Coupling to the SAM
3.4.2 Biotin Coupling to Dextran
xi3.5 NA-Biotin-ssDNA
3.6 Piezodispensing
3.7 Vesicle Preparation
3.7.1 DNA-tagging of the Vesicles
3.8 Surface Characterization
3.9 SPR
4 Results & Discussion
4.1 Non-imaging Measurements using Biacore
4.1.1 Adsorbing ssDNA-tagged Vesicles to the Sensor Surface
4.1.2 Detection of Receptor-Ligand Interaction
4.1.3 Non-Specific Binding of Hexadecanolthiol and EG3
4.2 Qualitative Surface Characterization
4.2.1 µCP Performance
4.2.2 Wetting Microscopy
4.3 Measurements Using Imaging SPR
4.3.1 Bovine Serum Albumin Minimizes NSB of Vesicles
4.3.2 Vesicle-Vesicle Interaction
4.3.3 Loading Vesicles with Receptors On-line
4.3.4 Formation of a Ternary Complex
5 Summary & Outlook
A DNA-sequences

Author: Brian, Björn

Source: Linkoping University

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