Articles Selective Ion Extraction: a Separation Method for Microfluidic Devices Matthew B. Kerby, Michael Spaid, Spencer Wu, J. Wallace Parce, and Ring-Ling Chien* Caliper Technologies Corporation, 605 Fairchild Drive, Mountain View, California 94043 A separation concept, selective ion extraction (SIE), is proposed on the basis of the combination of hydrodynamic and electrokinetic flow controls in microfluidic devices. Using a control system with multiple pressure and voltage sources, the hydrodynamic flow and electric field in any section of the microfluidic network can be set to desired values. Mixtures of compounds sent into a T-junction on a chip can be completely separated into different channels on the basis of their electrophoretic mobilities. A simple velocity balance model proved useful for predicting the voltage and pressure settings needed for separation. SIE provides a highly efficient separation with minimal ad- ditional dispersion. It is an ideal technique for high- throughput screening systems and demonstrates the power of lab-on-a-chip systems. Miniaturization of chemical analysis systems and the “lab-on-a chip” concept has generated a great deal of interest in the scientific community in the past decade. 1-5 Many chemical and biochemical applications were developed and demonstrated using this new technology. 6-8 One area of interest and commercial application is high-throughput screening (HTS) using microfluidic devices. 9,10 Miniaturization reduces the amount of biological and chemical reagents used per assay and provides high quality data with high throughput. The ability to program and control fluid transport has always been the most promising feature for lab-on-a-chip devices. Elec- trokinetic forces have the advantages of direct control, fast response, simplicity, and allowing analytes to be selectively moved through a complex network of channels, which permits the implementation of a wide variety of chemical and biochemical analyses. While electrokinetic material transport systems provide numerous benefits in the microscale movement, mixing and aliquoting of fluids, pressure-driven flow avoids detrimental effects of electric fields, such as biased sampling and disruption of enzyme reactions. We previously described a universal multiport system capable of controlling pressures and voltage on multiple wells in a lab-on-a-chip microfluidic device. 11 Precise flow control from each individual channel can be achieved, assuming that the hydrodynamic resistance of the network is known. In this report, we describe a separation technique that uses the combination of hydrodynamic and electrokinetic flow control to perform enzy- matic assays for high-throughput screening. There are several different approaches toward assay miniatur- ization. One approach is based on the concept of a continuous flow assay. 10 Small plugs of the preincubated substrate, enzyme, and inhibitor solution from a library of compounds are sipped onto the chip through the capillary from the microtiter plate. Buffer is sipped between each sample as a spacer. To prevent sample biasing caused by electrokinetic injection, pressure-driven flow is commonly used to transport these plugs through a network of interconnecting channels to a waste well, usually located at the end of the fluidic network, where a negative pressure (vacuum) is applied. When all of the flow on a chip is driven by a single pressure source, the hydrodynamic flow distribution or the dilution ratio in the channel network is predetermined by the fixed hydrodynamic resistances of the channels. For some assays, an electric field is also applied in part of the channel to provide separation on the basis of differences in the electrophoretic mobility of the substrate and product. An electrophoretic mobility difference between the substrate and product molecules generates a finite difference of velocity in the separation channel. This velocity change is recorded as a fluorescent intensity peak shift at the detector. Nevertheless, both substrate and product flow downstream together to the waste well. Since all reagents flow into a single waste well, the detector is usually located near the end of the separation channel in which the electric field is applied to maximize the separation power. 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