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Stanford University

Stanford Microfluidics Laboratory

Optimization of On-Chip Field Amplified Sample Stacking

Sensitivity to low analyte concentrations is a crucial challenge towards the development of robust miniaturized bioanalytical devices. Field amplified sample stacking (FASS) [1] is a promising method of achieving increased sensitivity for on-chip assays in a scheme that is easily integrated with electrophoretic separation techniques. In addition, experimental studies of sample stacking provide data applicable to a more general understanding of electrokinetic phenomena such as electromigration dispersion and flow instabilities.

Field Amplifies Sample Stacking (FASS) Concept
FASS uses gradients in electrolyte conductivity to subject sample ions to non-uniform electric fields.  As shown in the schematic below, sample ions are dissolved in a relatively low conductivity electrolyte which has a high electrical resistance in series with the rest of the flow.  This high resistance results in large electric fields within the sample and, therefore, large local electrophoretic velocities.  Sample ions stack as they move from high field, high velocity region to the low field, low velocity regions. 

Figure1. Schematic of field amplified sample stacking showing sample ions (in yellow) stacking as they exit low conductivity and enter high conductivity regions of the channel. 

Project Description
Efficiency of sample stacking is strongly affected by internal pressure induced dispersive fluxes [2]. In presence of electroosmotic flow (EOF) gradients in buffer concentration, required for stacking, lead to gradients in electric field and hence in electroosmotic velocity. An additional secondary effect is that the electroosmotic mobility (e.g., the zeta potential) is also a function of local concentration and chemistry. This mismatch in EOF velocity near the wall generates an internal pressure consistent with the mass continuity constraint on the system. The internal pressure causes dispersion of the stacked analyte bands limiting the concentration enhancement.

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Figure 2. Micro PIV velocity measurements in a microchannel.  Velocity measurements obtained at a fixed location in the channel while the conductivity interface sweeps by.  The measurements clearly show the adverse pressure gradient upstream and, after the interface sweeps by, the favorable pressure measurements downstream

We are developing experimental, analytical, and numerical simulation techniques to probe the dynamics of FASS. Unlike most prior studies of FASS, which employed point-wise detection system, we use quantitative, full-field CCD imaging and eipfluorescence microscopy to measure high resolution temporal and spatial concentration fields. We have developed a robust single buffer-buffer interface experiment to study FASS [3, 5].

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Figure 3. FASS of fluorescein dye at a single buffer-buffer interface. Evidence of favorable pressure gradient due to mis-matched EOF can be seen in the stacked fluorescein region.

Recently, we have designed, fabricated, and characterized a novel FASS CE chip design that uses a photoinitiated porous polymer structure to facilitate sample injection and flow control for high gradient FASS. We have demonstrated an electropherogram signal increase by a factor of 1100 in an electrophoretic separation of fluorescein and bodipy dye [4]. In addition, we are developing novel stacking schemes for on-chip applications (see movie below).

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Figure 4. mFASS followed by electrokinetic sample injection at a cross intersection


1.) Mikkers, F.E.P., Everaerts, F.M., and Verheggen, Th.P.E. M., "Concentration Distributions in Free Zone Electrophoresis," Journal of Chromatography, Vol. 169, 1-10, 1979.

2.) Burgi, D.S., and Chien, R.L., "Optimization in Sample Stacking for High Performance Capillary Electrophoresis," Analytical Chemistry, Vol. 63, 2042-2047, 1991.

3.) Bharadwaj R. and J.G. Santiago, "On-chip Field Amplified Sample Stacking Under Suppressed Electrosomotic Flow Conditions," Proceedings of the 7th International Conference on Miniaturized Chemical and BioChemical Analysis Systems, Squaw Valley, CA, 2003.

Jung, B., R. Bharadwaj, and J.G. Santiago, "Thousand-Fold Signal Increase using Field Amplified Sample Stacking for On-Chip Electrophoresis," Vol. 24, No. 21, Electrophoresis, 2003.

Bharadwaj, R., and Santiago, J.G., "Dynamics of Field Amplified Sample Stacking," Proceedings of the International Mechanical Engineering Congress and Exposition, New York, 2001

6.)Devasenathipathy, Shankar; Bharadwaj, Rajiv; Santiago, Juan G." Investigation of field amplified sample stacking with particle image velocimetry" American Society of Mechanical Engineers, Fluids Engineering Division (Publication) FED; 2002; v.258, p.401-405 Conference: 2002 ASME International Mechanical Engineering Congress and Exposition; Nov 17-22 2002; New Orleans, LA, United States