Biophysical Characterisation

Introduction to Biophysical Characterisation

using Dual Polarisation Interferometry

Dual Polarisation Interferometry (DPI) is a highly versatile, powerful analytical technique for

biophysical characterisation of proteins and other biomolecules. It extends the typical dynamic

measurements of conventional biosensors by including an additional quantitative, sub molecular,

conformational measurement.

DPI delivers a unique perspective on biochemistry, linking conformational changes to biochemical activity at a resolution normally associated with ‘big physics’.

DPI is an interferometric technique to derive dynamic information concerning the thickness,

density and mass of the molecular layer. As well as mass-based affinity and kinetic

determinations possible with other label-free optical and acoustic biosensor technologies, DPI

additionally provides real-time data on the orientation of the surface immobilised layer and

any conformational changes involved upon binding.

Two polarisations of light are passed through the sensor which consists of an upper ‘sensing’ layer and an embedded ‘reference’ layer. The output from each of the layers then combine producing two interference patterns which are detected by a camera. As molecules bind (or change shape) on the surface of the ‘sensing’ layer, they are probed by the evanescent field from the ‘sensing’ surface. This, in turn, changes both interference patterns. As material binds, the interference pattern shifts one way.

As material is removed from the surface, the interference pattern returns.

The AnaLight® Resolver software automatically analyses both sets of data and outputs the real time changes in the thickness, density and mass of the molecular layer. All of the calculations are based on classical optics theory and have been independently validated by, amongst others, the National Physics Laboratory (NPL) in the UK.