Parallel Separations on Microfluidic Chips for High Throughput Monitoring of Insulin Secretion from Single Islets of Langerhans.

[摘要] Microfluidic devices for the simultaneous characterization of insulin release from four and 15 isolated pancreatic islets were developed.Quantification of released insulin from islet samples was performed using parallel immunoassays coupled to capillary electrophoresis with fluorescence detection.Assays for insulin were completed in a serial fashion on each channel every 6 – 10 s, giving fast temporal resolution used for investigations into insulin secretion dynamics.Assay limits of detection were between 0.5 – 10 nM insulin.Individual islets were housed on the chips while perfusion streams carrying glucose or other secretagogues were used to stimulate insulin release.Secreted insulin was then mixed with fluorescently-labeled insulin and anti-insulin antibody in reaction channels for a competitive immunoassay.Portions of the continuously flowing reaction streams were injected onto separation channels where bound fluorescent insulin:antibody and free fluorescent insulin complexes were separated electrophoretically and detected via fluorescence.Relative amounts of these products were used to determine the amount of released insulin.The 15-islet microchip was used to investigate possible roles of leptin signaling on insulin secretion.Through a collaborative effort, islets from mice lacking leptin receptors only in the pancreas were compared to control islets through insulin release studies.Specifically, the effects of leptin, glibenclamide, glucagon-like peptide-1, and palmitic acid on glucose-stimulated insulin secretion were investigated.It was observed that leptin produces an inhibitory effect on insulin release and that lack of leptin signaling in islets enhances insulin release stimulated with glucose.Modifications were made to the 15-islet chip to ensure more uniform sampling of insulin from islets.The new islet sampling method was used to characterize oscillatory insulin release under various conditions.It was found that when treated appropriately, islets from individual mice displayed similar insulin secretion and Ca2+ flux oscillation frequencies.These frequencies were shown to be different from mouse to mouse, complementing previous studies.Additionally, the effects of free fatty acid-induced liptoxicity on pulsatile insulin release were investigated.Results from these experiments demonstrate the usefulness of single islet data not previously available at this level of throughput.

[发布机构] University of Michigan

[效力级别] Chemistry

[关键词] Microfluidics;Chemistry;Science;Chemistry