Huanchun Cui 1 Prashanta Dutta 2 Cornelius F. Ivory 1 1 School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, USA 2 School of Mechanical and Materials Engineering, Washington State University, Pullman, WA, USA Received August 23, 2006 Revised September 28, 2006 Accepted September 28, 2006 Research Article Isotachophoresis of proteins in a networked microfluidic chip: Experiment and 2-D simulation This paper reports both the experimental application and 2-D simulation of ITP of proteins in a networked microfluidic chip. Experiments demonstrate that a mixture of three fluo- rescent proteins can be concentrated and stacked into adjacent zones of pure protein under a constant voltage of 100 V over a 2 cm long microchannel. Measurements of the iso- tachophoretic velocity of the moving zones demonstrates that, during ITP under a constant voltage, the zone velocity decreases as more of the channel is occupied by the terminating electrolyte. A 2-D ITP model based on the Nernst–Planck equations illustrates the stacking and separation features of ITP using simulations of three virtual proteins. The self-shar- pening behavior of ITP zones dispersed by a T-junction is clearly demonstrated both by experiment and by simulation. Comparison of 2-D simulations of ITP and zone electro- phoresis (ZE) confirms that ZE lacks the ability to resharpen protein zones after they pass through a T-junction. Keywords: 2-D simulation / ITP / Microfluidic chip / Proteins / T-junction DOI 10.1002/elps.200600525 1138 Electrophoresis 2007, 28, 1138–1145 1 Introduction ITP is a well-known electrophoretic technique used in the separation of a variety of ionic compounds, ranging from small molecules like metal ions to large molecules like pro- teins. ITP is also a powerful sample preconcentration tech- nique which is useful in the analysis of low abundance spe- cies. ITP has been successfully coupled with a number of analytical techniques, such as zone electrophoresis (ZE) [1, 2], IEF [3], LC [4], MS [5], Raman spectroscopy [6], and NMR spectroscopy [7, 8]. Analytical and preparative ITP of proteins has been extensively explored in gels and capillary tubes since the 1970s [9]. In an effort to replace conventional benchtop elec- trophoresis systems, microchip-based electrophoresis has received rapidly growing interest during the last decade be- cause it has the potential to provide higher throughput, lower sample consumption, and lower fabrication costs. Although integration of ZE and IEF on microchips for protein separa- tion shows a peak capacity comparable to 2-D PAGE [10, 11], protein detection is challenging due to the low sample mass loadings in a microfluidic chip. The best way to increase the loading capacity of a microfluidic chip is to preconcentrate the sample. Sample preconcentration is very important when analyzing biological samples which may have a large dy- namic range of protein concentrations extending from the millimolar down to the femtomolar [12]. ITP is a simple and effective preconcentration and separation method which can be easily integrated on a chip prior to other on-chip opera- tions, especially ZE. As is the case with two other electrophoretic techniques, ZE and IEF, great progress has been made on the miniatur- ization of ITP. However, most of the published works on miniaturized ITP were focused on the separation of small organic molecules [13, 14] and metal ions [15, 16] which were primarily used in the food and beverage industry and for water analysis. ITP preconcentration and separation of pro- teins on chips has so far received relatively little interest. In this work, we demonstrate ITP of proteins in a PDMS channel with T-junctions which we consider the key ele- ments for the integration of unit operations, e.g., sample loading. As briefly discussed in our previous work [17], dis- persion of protein zones as they pass by a T-junction during electrophoresis is due to the deformation of electric field lines as current passes by the open channel. However, dis- Correspondence: Dr. Cornelius F. Ivory, School of Chemical Engi- neering and Bioengineering, Washington State University, Pull- man, WA 99164-2710, USA E-mail: cfivory@wsu.edu Fax: 1509-335-4806 Abbreviations: APC, allophycocyanin; EACA, e-amino-n-caproic acid; GFP, green fluorescent protein; KRF, Kohlrausch regulating function; LE, leading electrolyte; MC, methylcellulose; PE, r-phy- coerythrin; TE, terminating electrolyte; ZE, zone electrophoresis  2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com