High sensitivity electrokinetic assays based on propagation and interaction of ion concentration shock waves
Abstract/Contents
- Abstract
- The advent of microfluidics has ushered a renewed interest in electrokinetic transport phenomenon for controlling, mixing, concentrating, and separating a wide variety of charged species. This dissertation focuses on two electrokinetic techniques, capillary zone electrophoresis (CZE) and isotachophoresis (ITP), each of which enables detection of charged analytes based on their differential migration velocities under applied electric fields. CZE is a linear electrokinetic technique wherein analytes separate into distinct zones under a uniform electric field and while diffusing over time. Whereas ITP leverages nonlinear electrokinetic transport to generate propagating ion-concentration shock waves which counter diffusion of analytes and enable analyte focusing and separation. This dissertation deals with theoretical and experimental studies to explore new regimes of shock wave propagation and interaction in electrokinetics which enable higher performance of microchip ITP and CZE. We begin by presenting a quasi one-dimensional (1-D) model based on averaging of the 3-D equations along the local cross-sectional area, and an associated numerical scheme to simulate nonlinear electrokinetic processes in channels with non-uniform cross-sectional area. Our approach uses techniques of lubrication theory to approximate electrokinetic flows in channels with arbitrary variations in cross-section; and we include chemical equilibrium calculations for weak electrolytes, Taylor-Aris type dispersion due of non-uniform bulk flow, and the effects of ionic strength on species mobility and on acid-base equilibrium constants. To solve the quasi 1-D governing equations, we provide a finite volume scheme with limited numerical dissipation, coupled with an adaptive grid refinement algorithm to improve accuracy. Simulations of nonlinear electrokinetic problems, including ITP and electromigration-dispersion effects in CZE show that our approach yields fast, stable and high resolution solutions using an order of magnitude less grid points compared to the existing dissipative schemes. We have also validated our simulations with a wide range of data from ITP and CZE experiments. We then use our model and simulations to design and optimize two methods for increasing detection sensitivity of ITP. The first method employs axial variations in channel cross-sectional area to elongate analyte zones in ITP and thereby increase sensitivity. We show that using strongly convergent channels can result in large increase in sensitivity and simultaneous reduction in assay time, compared to uniform cross-section channels. Using our model, we develop simple analytical relations for dependence of zone length and assay time on geometric parameters of strongly convergent channels. We have validated our theoretical predictions with detailed experiments by varying channel geometry and analyte concentrations, and demonstrated indirect fluorescence detection with a sensitivity of 100 nM. In the second method for increasing sensitivity of ITP, we use bidirectional ITP to create a concentration cascade of leading electrolyte (LE) in ITP. In bidirectional ITP, we set up simultaneous shock waves between anions and cations such that these waves approach each other and interact. This shock interaction causes a sudden decrease in the LE zone concentration in the region ahead of the focused anions and a corresponding decrease in analyte zone concentrations. This readjustment of analyte zone concentrations is accompanied by a corresponding increase in their zone lengths, in accordance to conservation laws. We have developed an analytical scaling relation for the gain in analyte zone length due to shock interaction, verified it with detailed simulations, and validated it with experimental visualizations of bidirectional ITP zones. Lastly, we present a method to enhance resolution of detection by coupling ITP and CZE via shock interaction in bidirectional ITP. In this method, we use anionic ITP to focus anionic sample species prior to shock interaction. The interaction of the counter-propagating anionic and cationic ITP shocks then changes the local pH (and ionic strength) of the focused analyte zones and the trailing anion zone. Under this new condition, the analytes no longer focus and begin to separate electrophoretically. We illustrate the technique with numerical simulations, validate theoretical predictions with experimental visualization of bidirectional ITP zones. We then show the effectiveness of the technique by coupling ITP preconcentration and high resolution separation of a 1 kbp DNA ladder via shock interaction in bidirectional ITP. We also demonstrate applicability of this coupled ITP-CZE method for rapid, sequence-specific detection of multiple DNA fragments. For these experiments, we leverage the high preconcentration ability of ITP to accelerate slow, second-order DNA hybridization kinetics, and the high resolving power of CZE to separate and identify reaction products.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2013 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Bahga, Supreet Singh |
|---|---|
| Associated with | Stanford University, Department of Mechanical Engineering |
| Primary advisor | Santiago, Juan G |
| Thesis advisor | Santiago, Juan G |
| Thesis advisor | Lele, Sanjiva K. (Sanjiva Keshava), 1958- |
| Thesis advisor | Mani, Ali, (Professor of mechanical engineering) |
| Advisor | Lele, Sanjiva K. (Sanjiva Keshava), 1958- |
| Advisor | Mani, Ali, (Professor of mechanical engineering) |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Supreet Singh Bahga. |
|---|---|
| Note | Submitted to the Department of Mechanical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2013. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2013 by Supreet Singh Bahga
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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