Monitoring hormone release from islets of Langerhans via rapid electrophoresis-based immunoassays on a microfluidic chip

A microfluidic device utilizing an immunoassay for monitoring the chemical environment around living cells is described. The immunoassay is implemented using capillary zone electrophoresis with laser-induced fluorescence detection, allowing for both highly sensitive and rapid assays to be performed. Each immunoassay is performed within 6 s with limits of detections as low as 0.8 nM, allowing for the study of cellular secretion kinetics. Use of the glass microdevice permits a high degree of automation and integration within the system.; Real-time detection of glucose-stimulated insulin secretion from single islets of Langerhans is demonstrated. Islets are housed in a microchamber on the device while perfusing biological media by a pressure driven system. Perfusate containing secreted insulin is sampled via electroosmotic flow and mixed on-line with fluorescein isothiocyanate-labeled insulin (FITC-insulin) and anti-insulin immunoglobulin (Ab). The reaction stream is then sampled and injected onto an electrophoresis channel via flow-gated injection for the separation of FITC-insulin:Ab complex and free FITC-insulin.; To accommodate the large volumes of data generated by the microdevice, a computer program was developed for high-throughput analysis of electropherograms. The program, designated "Cutter", utilizes batch processing for determining chromatographic figures of merit such as peak centroid times, heights, areas, etc. Incorporated within the program is a novel method for automated peak deconvolution using an Empirically Transformed Gaussian model.; Various genetic mutations of islets were evaluated for glucose-stimulated insulin secretion including null mutations of insulin receptor, insulin growth factor receptor, and liver X receptor beta. All mutants exhibited impaired insulin response to elevated glucose concentrations. The mutant phenotypes are similar to characteristics displayed by diabetics; the results provide further evidence of the importance of the receptors in normal regulation of insulin secretion and glucose homeostasis.; Modifications to the immunoassay are described for monitoring glucose-mediated glucagon secretion from groups of islets. In order to minimize sample dilution, an improved electrical scheme was developed to increase the on-chip mixing ratio of sampled perfusate to immunoreagents. Detection limits of 1 nM for glucagon were obtained. Islets were quantitatively monitored for changes in glucagon secretion as the glucose concentration was decreased from 15 to 1 mM.