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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 microL 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 microL/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 [Ca(2+)] indicating the system will be useful in future studies of cellular physiology.

The IR-mediated polymerase chain reaction (IR-PCR) in microdevices is an established technique for rapid amplification of nucleic acids. In this report, we have expanded the applicability of the IR-PCR to quantitative determination of starting copy number by integrating fluorescence detection during the amplification process. Placing the microfluidic device between an IR long-pass filter and a hot mirror reduced the background to a level that enabled fluorescence measurements to be made throughout the thermal cycling process. The average fluorescence intensity during the extension step showed the expected trend of an exponential increase followed by a plateau phase in successive cycles. PUC19 templates at different starting copy numbers were amplified, and the threshold cycle showed an increase for decreasing amounts of starting DNA. The amplification efficiency was 80%, and the gel separation indicated no detectable nonspecific product. A melting curve was generated using IR heating, and this indicated a melting temperature of 85 °C for the 304 bp amplicon, which compared well to the melting temperature obtained using a conventional PCR system. This methodology will be applicable in other types of IR-mediated amplification systems, such as isothermal amplification, and in highly integrated systems that combine pre- and post-PCR processes.

A microfluidic system was developed to produce sinusoidal waveforms of glucose to entrain oscillations of intracellular [Ca(2+)] 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 (approximately 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 microL 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 Ca(2+)-sensitive fluorophore, indo-1. Entrainment of the islets was demonstrated by pacing the rhythm of intracellular [Ca(2+)] 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.