Sample dispersion in isotachophoresis

Garcia-Schwarz, Giancarlo; Bercovici, Moran; Marshall, Lewis A.; Santiago, Juan G.

doi:10.1017/jfm.2011.139

Cited by 57 publications

(116 citation statements)

References 31 publications

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“…For the numerical model, we regard the ITP interface width as a time-varying quantity δðtÞ and assume colocated Gaussian concentration profiles for all focused species. As described by Garcia-Schwarz et al (31), this assumption is reasonable when the mobilities of focused species are significantly greater and less than the those of TE and LE, respectively, and when concentration of focused species is significantly lower than those of both TE and LE, as we consider here. The species concentrations c i are assumed unsteady and one-dimensional, so the conservation equations in the frame of reference of the moving ITP zone are…”

Section: Resultsmentioning

confidence: 69%

“…The characteristic ITP interface width δ is determined by a balance between electromigration and diffusion. For constant current, ITP theory predicts constant δ; however, in practice, several factors may cause its increase in time (30,31). For the numerical model, we regard the ITP interface width as a time-varying quantity δðtÞ and assume colocated Gaussian concentration profiles for all focused species.…”

Section: Resultsmentioning

confidence: 99%

“…ITP interface width determines volume-averaged concentrations and therefore hybridization rate. ITP width can vary as ITP interface migrates through the channel due to changes in the dispersion dynamics (31).…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Rapid hybridization of nucleic acids using isotachophoresis

Bercovici

Han²,

Liao³

et al. 2012

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

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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Section: Resultsmentioning

confidence: 69%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Rapid hybridization of nucleic acids using isotachophoresis

Bercovici

Han²,

Liao³

et al. 2012

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

150

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show abstract

“…The distribution of sample ions is determined by the self-sharpening interface between neighboring zones (here the TE and LE) and the value of the sample effective mobility relative to these zones. 14 Multiple sample ions focus within the same narrow ITP interface region as largely overlapping peaks. The interface and peak widths, as well as the associated preconcentration factor, scale inversely with the applied current (see experiments in Figure 2b).…”

Section: Physics Of Itpmentioning

confidence: 99%

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Garcia-Schwarz

Rogacs

Bahga

et al. 2012

JoVE

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Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration 1,2 and efficient separation and extraction based on ionic mobility. 3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation 4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood. 9-12ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al. 13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids. Video LinkThe video component of this article can be found at https://www.jove.com/video/3890/ Protocol Physics of ITPITP forms a sharp moving boundary between ions of like charge. The technique can be per...

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“…In CZE, pH and ionic strength in the separation channel are uniform and determined directly by the background buffer chemistry, which can be quantified ex situ. Furthermore, CZE is easily compatible with systems with unsuppressed EOF, as CZE avoids non-uniform electric fields and non-uniform electroosmotic mobilities which can give rise to significant analyte zone dispersion [27,28].…”

Section: Introductionmentioning

confidence: 99%

Electrophoretic mobility measurements of fluorescent dyes using on‐chip capillary electrophoresis

et al. 2011

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We present an experimental study of the effect of pH, ionic strength, and concentrations of the electroosmotic flow (EOF)-suppressing polymer polyvinylpyrrolidone (PVP) on the electrophoretic mobilities of commonly used fluorescent dyes (fluorescein, Rhodamine 6G, and Alexa Fluor 488). We performed on-chip capillary zone electrophoresis experiments to directly quantify the effective electrophoretic mobility. We use Rhodamine B as a fluorescent neutral marker (to quantify EOF) and CCD detection. We also report relevant acid dissociation constants and analyte diffusivities based on our absolute estimate (as per Nernst-Einstein diffusion). We perform well-controlled experiments in a pH range of 3-11 and ionic strengths ranging from 30 to 90 mM. We account for the influence of ionic strength on the electrophoretic transport of sample analytes through the Onsager and Fuoss theory extended for finite radii ions to obtain the absolute mobility of the fluorophores. Lastly, we briefly explore the effect of PVP on adsorption-desorption dynamics of all three analytes, with particular attention to cationic R6G.

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Sample dispersion in isotachophoresis

Cited by 57 publications

References 31 publications

Rapid hybridization of nucleic acids using isotachophoresis

Rapid hybridization of nucleic acids using isotachophoresis

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrophoretic mobility measurements of fluorescent dyes using on‐chip capillary electrophoresis

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