Interrogation of Biomolecular Interactions Utilizing Backscattering Interferometry

Abstract

Backscattering interferometry (BSI) is a relatively new technique used to study molecular interactions in a label-free format and is proving to be a versatile biosensing technique. BSI quantifies minute changes in refractive index created by a binding event to interrogate molecular interactions with zeptomole sensitivity and seven decades dynamic range for determining dissociation constants, from low picomolar to micromolar. BSI requires little a priori knowledge of the binding pair, allowing this technique to perform novel binding affinity determinations on uncharacterized molecules. BSI is unique in that it can be used to study binding interactions in both heterogeneous (surface-immobilized) and homogeneous (free-solution) configurations. In the heterogeneous format, both monovalent and polyvalent glycan – lectin interactions were studied and the relative binding affinities quantified. BSI has also shown exceptional sensitivity in the homogeneous format, detecting just tens of thousands of binding events and monitoring interactions with picomolar affinities, without the use of any type of label or immobilization strategy. A variety of binding events were studied in the homogenous and label-free format; these include antibody – antigen, membrane associated receptor – ligand interactions, and aptamer – small molecule binding. Several of the interactions studied were on systems that would have been difficult or impossible to measure with other biosensing techniques. These assays involved determining the binding affinity of several small molecule ligands to membrane bound proteins expressed in membrane vesicles and a heteroaryl dihydropyrimidine binding to encapsidated aptamers. BSI has also been shown to be compatible with complex matrices allowing biomolecular interactions to be performed in cell media and serum. Capitalizing on this unique characteristic, it was shown that BSI can potentially be used in a clinical setting as a reactive serum detector. Overall, BSI represents a powerful biosensing tool that is broadly applicable and has the potential to shift the paradigm for assays that require the quantification of affinity, determination of labeling perturbation, or screening for binding.