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Pulsatile insulin from pancreatic islets is crucial for glucose homeostasis, but the mechanism behind coordinated pulsatility is still under investigation. One hypothesis suggests that cholinergic stimulation of islets by pancreatic ganglia resets these endocrine units, producing synchronization. Previously, it was shown that intracellular Ca 2+ oscillations within islets can be entrained by pulses of a cholinergic agonist, carbachol (CCh). Although these proxy measurements of Ca 2+ provided insight into the synchronization mechanism, measurement of insulin output would be more direct evidence. To this end, a fluorescence anisotropy competitive immunoassay for online insulin detection from single and grouped islets in a microfluidic system was developed using a piezoelectric pressure-driven fluid delivery system and a squaraine rotaxane fluorophore, SeTau-647, as the fluorescent label for insulin. Due to SeTau-647 having a longer lifetime and higher brightness compared to the previously used Cy5 fluorophore, a 45% increase in the anisotropy range was observed with enhanced signal-to-noise ratio (S/N) of the measurements. This new system was tested by measuring glucose-stimulated insulin secretion from single and groups of murine and human islets. Distinct islet entrainment of groups of murine islets by pulses of CCh was also observed, providing further evidence for the hypothesis that pulsatile output from the ganglia can synchronize islet behavior. We expect that this relatively straightforward, homogeneous assay can be widely used for examining not only insulin secretion but other secreted factors from different tissues.

analytical chemistry

Profiling Glucose-Stimulated and M3 Receptor-Activated Insulin Secretion Dynamics from Islets of Langerhans Using an Extended-Lifetime Fluorescence Dye