A method was developed that allowed simultaneous monitoring of the acute secretory dynamics of insulin and islet amyloid polypeptide (IAPP) from islets of Langerhans using a microfluidic system with two-color detection. A flow-switching feature changes in the perfusion media within 5-s allowing rapid exchange of the glucose concentrations delivered to groups of islets. The perfusate was continuously by electroosmotic flow and mixed online with Cy5-labeled insulin, fluorescein isothiocyanate (FITC)-labeled IAPP, anti-insulin, and anti-IAPP antibodies in an 8.15-cm reaction channel maintained at 37 °C. The immunoassay mixture was injected for 0.3-s onto a 1.5-cm separation channel at 11.75-s intervals and immunoassay reagents detected using 488- and 635-nm lasers with two independent photomultiplier tubes for detection of the FITC and Cy5 signal. RSD of the bound-to-free immunoassay ratios ranged from 2–7% with LODs of 20 nM for insulin and 1 nM for IAPP. Simultaneous secretion profiles of the two peptides were monitored from groups of 4–10 islets during multiple step changes in glucose concentration. Insulin and IAPP were secreted in an approximately 10:1 ratio and displayed similar responses to step changes from 3 mM to 11 mM or 20 mM glucose. The ability to monitor the secretory dynamics of multiple peptides from islets of Langerhans in a highly automated fashion is expected to be a useful tool for investigating hormonal regulation of glucose homeostasis.

The capillary electrophoresis (CE) conditions for a competitive immunoassay of glucagon were optimized for highest sensitivity of the immunoassay and resolution of the electrophoretic peaks using a Box-Behnken design. Injection time, voltage ramp time, and separation voltage were varied between three levels and two responses, bound-to-free (B/F) ratio of the immunoassay peaks and resolution between the peaks, were measured. Analysis of variance was applied to fit a predictive model, and a desirability function was used to simultaneously optimize both responses. A 10 sec injection, 1.6 min ramp time, and a 22 kV separation voltage were the conditions found when high B/F was given more emphasis than high resolution. To test the model, calibration curves of a glucagon immunoassay were measured at the optimum and least optimum CE conditions. Optimal conditions increased the sensitivity of the immunoassay by 388% compared to the least optimum conditions while maintaining adequate resolution.

A microfluidic device was developed to produce temporal concentration gradients of multiple analytes. Four on-chip pumps delivered pulses of three analytes and buffer to a 14 cm channel where the pulses were mixed to homogeneity. The final concentration of each analyte was dependent on the temporal density of the pulses from each pump. The concentration of each analyte was varied by changing the number of pump cycles from each reservoir while maintaining the total number of pump cycles per unit time to ensure a constant total flow rate in the device. To gauge the independent nature of each pump, sinusoidal waves of fluorescein concentration were produced from each pump with independent frequencies and amplitudes. The resulting fluorescence intensity was compared to a theoretical summation of the waves and the experimental data matched the theoretical waves within 1%, indicating that the pumps were operating independently and outputting the correct frequency and amplitude. The device was used to demonstrate the role of ATP-sensitive K+ channels in glucose-stimulated increases in intracellular [Ca2+] in islets of Langerhans. Perfusion of single islets of Langerhans with combinations of glucose, diazoxide, and K+ resulted in intracellular Ca2+ patterns similar to what has been observed using conventional perfusion devices. The system will be useful in other studies with islets of Langerhans, as well as other assays that require the modulation of multiple analytes in time.