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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 with 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 adenosine triphosphate-sensitive K channels in glucose-stimulated increases in intracellular [Ca] in islets of Langerhans. Perfusion of single islets of Langerhans with combinations of glucose, diazoxide, and K resulted in intracellular Ca 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. [ABSTRACT FROM AUTHOR]

A microfluidic system was developed to produce sinusoidal waveforms of glucose to entrain oscillations of intracellular [Ca2+] in islets of Langerhans. The work described is an improvement to a previously reported device where two pneumatic pumps delivered pulses of glucose and buffer to a mixing channel. The mixing channel acted as a low pass filter to attenuate these pulses to produce the desired final concentration. Improvements to the current device included increasing the average pumping frequency from 0.83 to 3.33 Hz by modifying the on-chip valves to minimize adhesion between the PDMS and glass within the valve. The cutoff frequency of the device was increased from 0.026 to 0.061 Hz for sinusoidal fluorescein waves by shortening the length of the mixing channel to 3.3 cm. The value of the cutoff frequency was chosen between the average pumping frequency and the low frequency (∼0.0056 Hz) glucose waves that were needed to entrain the islets of Langerhans. In this way, the pulses from the pumps were attenuated greatly but the low-frequency glucose waves were not. With the use of this microfluidic system, a total flow rate of 1.5 ± 0.1 μL min-1 was generated and used to deliver sinusoidal waves of glucose concentration with a median value of 11 mM and amplitude of 1 mM to a chamber that contained an islet of Langerhans loaded with the Ca2+-sensitive fluorophore, indo-1. Entrainment of the islets was demonstrated by pacing the rhythm of intracellular [Ca2+] oscillations to oscillatory glucose levels produced by the device. The system should be applicable to a wide range of cell types to aid understanding cellular responses to dynamically changing stimuli. [ABSTRACT FROM AUTHOR]

A microfluidic perfusion system was developed for automated delivery of stimulant waveforms to cells within the device. The 3-layer glass/polymer device contained two pneumatic pumps, a 12 cm mixing channel, and a 0.2 μL cell chamber. By altering the flow rate ratio of the pumps, a series of output concentrations could be produced while a constant 1.43 ± 0.07 μL/min flow rate was maintained. The output concentrations could be changed in time producing step gradients and other waveforms, such as sine and triangle waves, at different amplitudes and frequencies. Waveforms were analyzed by comparing the amplitude of output waveforms to the amplitude of theoretical waveforms. Below a frequency of 0.0098 Hz, the output waveforms had less than 20% difference than input waveforms. To reduce backflow of solutions into the pumps, the operational sequence of the valving program was modified, as well as differential etching of the valve seat depths. These modifications reduced backflow to the point that it was not detected. Gradients in glucose levels were applied in this work to stimulate single islets of Langerhans. Glucose gradients between 3 and 20 mM brought clear and intense oscillations of intracellular [Ca2+] indicating the system will be useful in future studies of cellular physiology. [ABSTRACT FROM AUTHOR]