surface plasma resonance (SPR)

Introduction

Identifies:

• specificity of binding between 2 molecules
• concentration: how much of a molecule is present
• binding kinetics: rates of association & dissociation
• binding affinity

Advantages

• molecules > 100 daltons
• no need to label with fluorescent or radioactive tags
• molecules may be studies in their native state

Methods

• the sensor chip consists of a glass surface, covered with a thin layer of gold
• in the most widely used sensor chip, the gold surface is modified with a carboxymethylated dextran layer
• the dextran hydrogel layer forms a hydrophilic environment for attached biomolecules, preserving them in a non- denatured state
• other derivatized surfaces enable other immobilization chemistries
• a microfluidics system allows analyte to pass over the sensor surface in a continuous, pulse-free, controlled flow maintaining constant analyte concentrations at the sensor chip surface
• up to 4 channel may be run in parallel (allows for subtraction of blank)
• SPR detects changes in mass in the aqueous layer close to the sensor chip surface by measuring changes in refractive index
• when molecules in the test solution bind to a target molecule, the mass increases; when they dissociate, the mass decreases
• this (8) allows continuous, real-time monitoring of the association & dissociation of molecules to the target
• software analyzes & presents the data