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 Hz 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 Hz 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 islets of Langerhans. In this way, the pulses from the pumps were attenuated greatly, but the low frequency glucose waves were not. Using 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 in understanding cellular responses to dynamically changing stimuli.

There are numerous detection methods available for methods are being put to use for detection on these miniaturized systems, with the analyte of interest driving the choice of detection method. In this article, we summarize microfluidic 2 years. More focus is given to unconventional approaches to detection routes and novel strategies for performing high-sensitivity detection.Microfluidic devices are becoming a common fixture in many laboratories. Besides the wellknown advantages of reduced sample volumes and decreased analysis times when compared with macro-sized components, these devices also offer significant advantages in other ways. For instance, the ability to couple multiple channels together with minimal dead volume allows for easy handling of low mass samples. Also, the ability to easily and accurately control fluid flow has prompted researchers in other areas, such as biology or biochemistry, to use these devices.Regardless of the nature of the analysis, the end result of the reactions, separations and other processes that occur on these miniaturized devices must be detected. In this article, we aim to provide a review of detection methods for use with microfluidic devices. Due to the evergrowing popularity of microfluidic devices, we have limited the timeframe of this review to papers published after 2007. We refer the readers to previous reviews on detection schemes used in microfluidic systems for earlier time-frames and more specific applications [1-3]. We have not attempted a comprehensive review of the literature, but have selectively chosen a sample of the articles that we believe present unique approaches to the three most common detection methods (optical, electrochemical and mass spectrometric). Optical detection methodsThe most predominant detection method in microfluidic analyses by far has been with optical means. Within this broad class, fluorescence-based detection can be considered routine. The popularity of this technique is mostlikely due to the simplicity with which microfluidic devices can be coupled to fluorescence excitation and detection schemes, as well as their ability to detect from low volume samples. There have been multiple examples of advances in detection using fluorescence-based methods as applied to microfluidic devices. Recent advances have included fluorescence lifetime-imaging (FLIM), high-

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.