Abstract
Analytical technologies based on binding assays have evolved substantially since their inception nearly 60 years ago, but our conceptual understanding of molecular recognition has not kept pace. Contemporary technologies, such as single-molecule and digital measurements, have challenged, or even rendered obsolete, core concepts behind conventional binding assay design. Here, we explore the fundamental principles underlying molecular recognition systems, which we consider in terms of signals generated through concentration-dependent shifts in equilibrium. We challenge certain orthodoxies related to binding-based detection assays, including the primary importance of a low dissociation constant (K(D)) and the extent to which this parameter constrains dynamic range and limit of detection. Lastly, we identify key principles for designing binding assays that are optimally suited for a given detection application.