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Stanford University

Stanford Microfluidics Laboratory

High-Sensitivity Isotachophoretic Stacking CE Devices

Principal Investigators: J.G. Santiago, Byoungsok Jung, and Tarun Khurana


Isotachophoresis (ITP) stacking is a well-known, well-established sample pre-concentration technique implemented using a leading electrolyte (LE) with relatively high mobility ions and a trailing electrolyte (TE) with low mobility ions. The ion mobilities of the TE and LE are respectively lower and higher than those of the sample ions. Upon application of up to 3 kV/cm electric fields, the disparate mobilities of LE and TE ions cause sample ions to focus within a narrow, self-sharpening zone between them and then migrate at the same velocity (hence “isotacho”).


We fill one well with slow trailing electrolyte (T) mixed with samples (S1,S2), and the other well with fast leading electrolyte (L). When we apply an electric field, ions electromigrate through a microchannel according to their electrophoretic mobilities.



Sample ions overspeed the slow trailing electrolyte, but cannot overspeed the fast leading electrolyte; consequently, they focus at the interface.


Sample continues to accumulate. If sample concentration approaches the concentration of the leading electrolyte, samples self-segregate into discrete zones.

We have designed and characterized a novel on-chip ITP/CE method that can be implemented with available off-the-shelf standard chip systems using off-the-shelf voltage control systems and buffer chemistries. We have demonstrated concentration enhancement of more than two million-fold in a fast (< 120 s), single step electromigration assay (Jung et al., 2006). We also demonstrated injection, preconcentration, separation of 100 attomolar (60 parts per quadrillion) fluorophores (Alexa Fluor 488 and bodipy) using the ICP/CE protocol combined with a high sensitivity, laser induced confocal fluorescence microscopy detection system.


On-chip ITP assay showing regions of TE and LE, and sample ions (Alexa Fluor 488) compressed into 5 um zone.


In the past three years, we have placed extensive effort into developing on-chip ITP as a robust pre-concentration assay with novel functionality. We have demonstrated on-chip ITP for small organic ions, amino acids, dsDNA, ssDNA, and proteins (e.g., casein, angiotensin-II, and green fluorescent protein). We have developed robust ITP protocols and have demonstrated these in a variety of substrates including fused silica, borosilicate, free-standing capillaries (using standard interconnects), acrylic, and hybrid acrylic/glass channels. We have also demonstrated stable ITP waves in the presence of large pressure disturbances and tortuous geometries.


Isotachophoretically focused green fluorescent protein (GFP). The protein band is held stationary with pressure-driven flow; when the pressure is removed, the band tightens and electromigrates through the channel.

See related publications here