Online affinity micro free-flow electrophoresis for the continuous monitoring of insulin via a competitive immunoassay

Abstract

An online, micro free-flow electrophoresis (μFFE) assay was developed for the real-time measurement of insulin in a continuous, competitive immunoassay. Fluorescently labeled insulin and anti-insulin monoclonal antibody were mixed with the sample stream online and flowed through an incubation capillary into the μFFE device. Under an electric field, the bound antibody : insulin-FITC complex was separated from free insulin-FITC. When unlabeled insulin was introduced, labeled and unlabeled insulin competed for binding sites on the antibody, and the separated peaks of the bound complex and free insulin-FITC responded in real-time. Temporal resolution of 30 seconds was achieved following injections of unlabeled insulin, with a limit of detection of 63 nM and a linear response over the tested range of 50 nM to 1 μM insulin. The development of this assay supports the eventual integration of an organ-on-a-chip system capable of measuring direct cellular efflux in tandem with the affinity μFFE detection system. To approximate such an online cellular response, glucose-stimulated insulin secretion was collected offline from human islets and injected into the affinity μFFE instrument. A measurable change in insulin secretion was observed between samples exposed to low and high glucose, demonstrating sufficient limit of detection, linearity, the compatibility of the instrument with a complex biological matrix.