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, glucosestimulated 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.