Modulation of the diffusion coefficient of a hapten-modified redox species as a basis for an amplified electrochemical affinity assay

An affinity assay was developed that is based on the modulation of the diffusion coefficient of an electroactive label upon complementary recognition leading to an increase of the molecular weight. Using an electroanalytical technique, which is correlated to the diffusion coefficient of the redox species, e.g., cyclic voltammetry, the decrease of the diffusion coefficient can be monitored as a decrease of the diffusion-limited current. Signal amplification was achieved by redox recycling using a microelectrode that is positioned in close distance to a conducting surface. The amplification rate can be adapted by varying the distance between the microelectrode and the conducting surface. As a model system for molecular recognition, the biotin–streptavidin system has been chosen using a ferrocene-labeled biotin derivative as electroactive species. The generality of the approach was proved by extension of the basic assay to related sandwich assays and by miniaturization using a wall-free droplet cell.