Design of an islet-on-a-chip device to dynamically measure insulin secretion from individual islets

Abstract

Glucose-stimulated insulin secretion (GSIS) from pancreatic islets shows a biphasic pattern. Investigating the temporal dynamics and patterns of GSIS could give insight into the mechanisms underlying islet beta-cell function and dysfunction; however, these dynamics are lost when using bulk (i.e., multiple islet) assays for insulin secretion. Here, we present a novel microfluidic device to detect insulin secretion from individual islets using on-chip miniaturization of a fluorescence anisotropy immunoassay (FAIA). Insulin is co-secreted with c-peptide after ∼5-10 min of glucose stimulation. Here, we designed a 10-residue synthetic c-peptide tagged with fluorophore based on antibody binding and response of the FAIA. This included selection of the sequence, tagging site (N- versus C-terminal), and c-peptide antibody. Subsequently, a continuous-flow method was applied to monitor reaction kinetics to determine the equilibrium time of the assay. Using the maximum time required to reach equilibrium, we designed an islet-on-a-chip that individually mixes the effluent of four islets within ∼100 s of residence time. This assay is downstream of the islet, which leaves an ideal optical window to simultaneously image islet responses (e.g., Ca2+ activity). Ultimately, we used this device to measure secretion from individual mouse pancreatic islets in response to 11- and 20-mM glucose. The secretion detected on-chip shows the characteristic biphasic pattern and was further validated with pharmacological activators (e.g., tolbutamide) and inhibitors (e.g., diazoxide) of insulin secretion. Finally, we will explore the dynamics of GSIS relative to islet Ca2+ activity and thus demonstrate how this method can be coupled to simultaneously imaging of other parameters of islet beta-cell response.