We are developing optimized and robust isotachophoresis (ITP) processes for on-chip sample preconcentration (i.e., sample stacking). These methods lower the limits of detection to new levels. ITP is easily coupled with microchip-based capillary electrophoresis (CE) to achieve robust and efficienty stacking.
ITP uses various ion mobilities to create zones of relatively purified sample ions in a microchannel. These “concentration shock waves” effect significant increases in sample concentration. The process can be performed as follows. First, two solutions are created: a leading electrolyte (LE) with relatively high mobility ions and a trailing electrolyte (TE) with low mobility ions. The sample can be dissolved in, say, the TE electrolyte solution. The ion mobilities of the LE and TE are respectively lower and higher than those of sample ions
where m is ion mobility (velocity per unit electric field) and T, S, and L refers respectively to trailing, sample, and leading ions. The process is summarized in the schematic below.
L-: Leading electrolyte
T-: Trailing electrolyte
S-: Sample ion
Counter ions not shown
Figure 1. Schematic of a ITP sample stacking process. The schematic shows sample ions as circles with a negative charge and denotes regions of trailing and leading ions with a T and L, respectively. Counterions (e.g., positive sodium ions) (not shown) maintain electroneutrality in each zone. In the trailing ion region, sample ions overspeed trailing ions and race ahead. Sample ions cannot race ahead of leading electrolyte ions, L, and so segregate and focus into a narrow region of purified sample (and counter ion).
After preconcentration, we can easily terminate ITP-mode stacking and initiate CE separation on-chip.
Below are several example visualizations of on-chip ITP stacking.
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Figure 2. Experimental visualization of ITP front forming and in electrophoretically migrating downstream. ITP front is formed at the intersection of two 50 by 20 um (width by depth) glass microchannels. The ITP concentration shock wave is clearly visible and focuses to a width of less than 5 um.
Figure 3. Image of ITP-focused band showing TE, LE, and sample ion shock wave. (a) (b)
Figure 4. Signal enhancement via ITP stacking on-chip. (a) shows the early stage of sample preconcentration showing near linear increase of peak sample intensity (traces separated every 2 sec). (b) shows stacking ratio in both early and late-stage ITP stacking. At later stages, ITP reaches a nearly steady flow state.
We have optimized ITP stacking by using on-chip flow control, minimizing dispersion, and leveraging the high electric fields made possible by high surface-to-volume ratios of microfabricated channels. ITP lowers achievable limits of detection (LOD) of electrophoretic detections and increases resolution by providing self-sharpening concentration field. We are able to concentrate ionic samples by more than a factor of 106 in less than one minute. We can also inject, concentration, and detect initial sample concentrations of less than 100 fM concentrations in less than 50 seconds. We have also applied our method successfully to the analysis of various analytes including water soluble organic ions and single and double stranded DNA. Below is a brief comparison of our stacking performance and that of 34 other studies.
Figure 5. Review of on-chip and capillary-based sample stacking methods. Using our optimized on-chip ITP methods, we are able to concentration samples by more than one million fold in less than 100 sec.
Simulation of ITP-based stacking
We are currently developing numerical models for generalized, non-linear electrokinetic processes including FASS and ITP based stacking.
The model is a reduced-order, low computational cost electrokinetic flow model for the prediction and design of electrophoretic separations. The model adopts a depth-averaged approach that accurately captures convective-dispersion processes (Lin et al. 2005), and includes important physical effects such as electrical body force and fully nonlinear multi-species electromigration. We can simulate arbitrary electrolyte and sample configurations (and therefore different stacking and separation processes) by varying initial conditions. The corresponding numerical simulation is based on a finite volume approach using a monotonic upstream-centered construction (MUSCL), and can capture the complex evolution of sharp, narrow-banded peaks and high-stacking ratios.
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Figure 6. Simulation of ITP front of three samples. These data were generated by a numerical non-linear electrokinetics model we have developed in our group.
Figure 7. Detail of ion concentrations from ITP simulation above at the near steady state condition.
Miscellaneous ITP data
Figure 8. Early-stage focusing of a 100 fM sample (on-chip fluorescence measurement)
Figure 9 Concentration profile of ITP shock front showing sharp spike at the interface between trailing and leading electrolytes.
Lin, H., B.D. Storey and J.G. Santiago, “A depth-averaged electrokinetic flow model for thin microchannels,” submitted to Proceedings of the Royal Society A, 2005.
Storey, B.D., B.S. Tilley, H. Lin, and J.G. Santiago, "Electrokinetic Instabilities in Thin Microchannels," Physics of Fluids, Vol. 16, No. 6, p.1922-1935, 2004.
“...of the incalculable trillions of billions of millions of imperceptible molecules contained by cohesion of molecular
affinity in a single pinhead.”
Ulysses, Episode 17: Ithaca, by James Joyce