Sample Zone Dynamics in Peak Mode Isotachophoresis

Khurana, Tarun K.; Santiago, Juan G.

doi:10.1021/ac800792g

Cited by 97 publications

(185 citation statements)

References 34 publications

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“…Here we account for a semi-infinite injection mode, wherein both target and reporter molecules are mixed homogenously within the TE buffer and continuously focus into the ITP zone. 24 Experiment results are in good agreement with our numerical model ( Figure 2B; see the Supporting Information for a detailed description of the numerical simulations).…”

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confidence: 85%

Integration of On-Chip Isotachophoresis and Functionalized Hydrogels for Enhanced-Sensitivity Nucleic Acid Detection

Garcia-Schwarz

Santiago

2012

Anal. Chem.

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We introduce an on-chip electrokinetic assay to perform high-sensitivity nucleic acid (NA) detection. This assay integrates electrokinetic sample focusing using isotachophoresis (ITP) with a background signal-removal strategy that employs photopatterened, DNA-functionalized hydrogels. In this multistage assay, ITP first enhances hybridization kinetics between target NAs and end-labeled complementary reporters. After enhanced hybridization, migration through a DNA-functionalized hydrogel region removes excess reporters through affinity interactions. We demonstrate our assay on microRNAs, an important class of lowabundance biomarkers. The assay exhibits 4 orders of magnitude dynamic range, near 1 pM detection limits starting from less than 100 fg of microRNA, and high selectivity for mature microRNA sequences, all within a 10 min run time. This new microfluidic framework provides a unique quantitative assay for NA detection.

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confidence: 85%

Integration of On-Chip Isotachophoresis and Functionalized Hydrogels for Enhanced-Sensitivity Nucleic Acid Detection

Garcia-Schwarz

Santiago

2012

Anal. Chem.

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“…The ITP interface width, δ, can be measured as a function of time from an ITP experiment focusing a fluorescent species. See Khurana et al (23) for general formulation of the analyte accumulation rate parameters V ITP , c LE , c TE well and ion mobilities. We numerically solve this set of non-linear ordinary differential equations (ODEs) for the volume averaged species concentrations to describe nucleic acid hybridization kinetics under ITP focusing.…”

Section: Resultsmentioning

confidence: 99%

Rapid hybridization of nucleic acids using isotachophoresis

Bercovici

Han²,

Liao³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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150

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We use isotachophoresis (ITP) to control and increase the rate of nucleic acid hybridization reactions in free solution. We present a new physical model, validation experiments, and demonstrations of this assay. We studied the coupled physicochemical processes of preconcentration, mixing, and chemical reaction kinetics under ITP. Our experimentally validated model enables a closed form solution for ITP-aided reaction kinetics, and reveals a new characteristic time scale which correctly predicts order 10,000-fold speed-up of chemical reaction rate for order 100 pM reactants, and greater enhancement at lower concentrations. At 500 pM concentration, we measured a reaction time which is 14,000-fold lower than that predicted for standard second-order hybridization. The model and method are generally applicable to acceleration of reactions involving nucleic acids, and may be applicable to a wide range of reactions involving ionic reactants.hybridization kinetics | DNA | RNA | molecular beacons | electrophoresis N ucleic acid hybridization is ubiquitous in molecular biology, biotechnology, and biophysics, and has been instrumental in the development of numerous important techniques including genetic profiling (1, 2), pathogen identification (3, 4) sequencing reactions (5), and single-nucleotide polymorphism typing (6). In nucleic acid hybridization, two single-stranded nucleic acid molecules with complementary sequences bind and form more stable double-stranded molecules. Diffusion, transport, and reaction rates limit hybridization of nucleic acids at low concentrations (7). While diffusion and transport limitations can be effectively overcome using mixing and flow control methods (8-10), reaction rates still limit assay times and sensitivity (7,11).Nucleic acid amplification techniques (e.g., polymerase chain reaction, PCR) are often used as an initial step to improve sensitivity and accelerate hybridization. However, amplifications such as PCR can suffer from as much as 10,000-fold amplification bias (1, 4), require significant sample preparation (12), can be difficult to reproduce quantitatively across laboratories (13), and require a well-controlled environment (12). Amplification-free hybridization avoids amplification-associated sequence bias and may be particularly important for applications beyond the conventional laboratory settings.Temperature, monovalent salt concentration, and divalent cation concentration (in particular, magnesium ion) are most commonly used to control the rate of hybridization. The hybridization of short oligonucleotides in the presence of 50 mM MgCl 2 has been reported as fivefold faster than at 1 mM MgCl 2 (14). Similarly, higher salt concentration and higher temperatures can accelerate reactions (14-16). However, these approaches reduce the energy of binding events and strongly affect specificity. Adjustment of these hybridization parameters is therefore often a trade-off between acceleration and specificity (12, 17). Other methods such as volume exclusion by inert polymers (e.g., dext...

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“…For example, electrophoretic separations at high ionic strengths have been suggested to minimize peak distortion due to electromigration dispersion [14], and high ionic strength separations have also been used to reduce wall adsorption of cationic proteins [15] and increased analyte selectivity [16]. Other studies have reported on the adverse effects of high ionic strength, which may lead to zone reversal in CZE and ITP [17,18], and decreased focusing rate in ITP [19].…”

Section: General Aspectsmentioning

confidence: 99%

“…a ''semi-infinite sample'' configuration), the sample is mixed in the TE well (reservoir) and continuously overspeeds the TE ions to focus at the TE-LE interface. The TE and sample ions displace the LE ions and, in bulk liquid regions formerly occupied by LE ions, adjust to concentration levels dictated by the LE [19]. The sample concentration, C S,te , in the new ''regulated'' TE zone can be calculated from flux balance at the interface of TE well and regulated TE zone.…”

Section: Sample Preconcentration In Peak Mode Itpmentioning

confidence: 99%

“…The sample concentration, C S,te , in the new ''regulated'' TE zone can be calculated from flux balance at the interface of TE well and regulated TE zone. C S,te is related to the initial sample concentration C S,te well as [19] The amount of sample accumulated in peak mode, N s , depends primarily on the ratio of the effective mobility of the analyte in the TE zone to that of the TE anion in the TE zone, the initial sample concentration, and the time elapsed between injection and detection. The rate of accumulation with respect to the distance traveled by the interface (neglecting EOF) is given by [19].…”

Section: Sample Preconcentration In Peak Mode Itpmentioning

confidence: 99%

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Ionic strength effects on electrophoretic focusing and separations

2010

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We present a numerical and experimental study of the effects of ionic strength on electrophoretic focusing and separations. We review the development of ionic strength models for electrophoretic mobility and chemical activity and highlight their differences in the context of electrophoretic separation and focusing simulations. We couple a fast numerical solver for electrophoretic transport with the Onsager-Fuoss model for actual ionic mobility and the extended Debye-Huckle theory for correction of ionic activity. Model predictions for fluorescein mobility as a function of ionic strength and pH compare well with data from CZE experiments. Simulation predictions of preconcentration factors in peak mode ITP also compare well with the published experimental data. We performed ITP experiments to study the effect of ionic strength on the simultaneous focusing and separation. Our comparisons of the latter data with simulation results at 10 and 250 mM ionic strength show the model is able to capture the observed qualitative differences in ITP analyte zone shape and order. Finally, we present simulations of CZE experiments where changes in the ionic strength result in significant change in selectivity and order of analyte peaks. Our simulations of ionic strength effects in capillary electrophoresis compare well with the published experimental data.

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Sample Zone Dynamics in Peak Mode Isotachophoresis

Cited by 97 publications

References 34 publications

Integration of On-Chip Isotachophoresis and Functionalized Hydrogels for Enhanced-Sensitivity Nucleic Acid Detection

Integration of On-Chip Isotachophoresis and Functionalized Hydrogels for Enhanced-Sensitivity Nucleic Acid Detection

Rapid hybridization of nucleic acids using isotachophoresis

Ionic strength effects on electrophoretic focusing and separations

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