Suppression of electroosmotic flow and its application to determination of electrophoretic mobilities in a poly(vinylpyrrolidone)-coated capillary

Kaneta, Takashi; Ueda, Takeshi; Hata, Kazuki; Imasaka, Totaro

doi:10.1016/j.chroma.2005.08.062

Cited by 63 publications

(59 citation statements)

References 18 publications

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“…3), we mixed 10 nM MB in the leading electrolyte (LE) buffer composed of 250 mM HCl, 500 mM Bistris, 2 mM MgCl 2 , and 0.1% polyvinylpyrrolidone (PVP). We used PVP for suppression of elctroosmotic flow (EOF) (36) and equal concentrations (in all experiments) of Mg 2þ to promote kinetic rates of hybridization (14). We used 1, 10, and 100 nM target DNA concentrations in the trailing electrolyte (TE) buffer composed of 100 mM Tricine, 100 mM Bistris.…”

Section: Methodsmentioning

confidence: 99%

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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: Methodsmentioning

confidence: 99%

“…We used six observation points located at distances of 4, 12, 20, 24, 28, and 32 mm from the TE well for 10 nM MBs and 20 nM target hybridization. We used 10 observation points at distances of 8,16,20,24,28,32,36,40,42, and 44 mm (total channel length was 50 mm for this case) for hybridization of 50 pM MBs with 500 pM target.…”

Section: Methodsmentioning

confidence: 99%

Rapid hybridization of nucleic acids using isotachophoresis

Bercovici

Han²,

Liao³

et al. 2012

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

150

View full text Add to dashboard Cite

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“…͓From these simulations, also the model parameters of Table I were derived to approximately match mobilities and conductivities.͔ To suppress electro-osmotic flow ͑EOF͒ in the channel, 1% ͑w/v͒ polyvinylpyrrolidone MG40000 ͑Merck 1.07370.0100͒ is added to both electrolytes. 26 The experiments for the bead collector were conducted with Fluoro MAX Green Fluorescent Polymer Particles of 5 m diameter ͑Thermo Scientific Particle Technology Catalog No. G0500͒.…”

Section: A Instrumentation Materials and Experimental Conditionsmentioning

confidence: 99%

Transport and separation of micron sized particles at isotachophoretic transition zones

2011

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Conventionally, isotachophoresis ͑ITP͒ is used for separation of ionic samples according to their electrophoretic mobilities. We demonstrate that the scope of ITP applications may be extended toward particle concentration and separation. Owing to the distributions of electrolyte concentration and electric field inside a transition zone between two electrolytes, a number of different forces act on a small particle. As far as possible, we provide estimates for the order of magnitude of these forces and analyze their scaling with the particle size and the electric-field strength. Furthermore, we experimentally demonstrate that polymer beads of 5 m diameter dispersed in a high mobility "leading" electrolyte are picked up and carried along by an ITP transition zone which is formed with a low mobility "trailing" electrolyte. By studying the particle positions and trajectories, we show that impurities in the electrolytes play a significant role in the experiments. Additionally, it is experimentally shown that different types of beads can be separated at an ITP transition zone. In particular, beads of 1 m diameter are not carried along with the transition zone, in contrast to the 5 m beads. The presented technique thus adds to the portfolio of electrokinetic transport, concentration, and separation methods in microfluidics.

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“…Because adsorption may cause broad and tailing peaks, 26 the capillary inner wall was treated to eliminate electroosmotic flow and minimize adsorption by dynamically coating it with 1.0% (w/v) PVP (Mr = 1,000,000). In a CGE system, the composition and concentration of the separation media have an effect on the separation of the DNA molecules.…”

Section: Resultsmentioning

confidence: 99%

Capillary Gel Electrophoretic Analysis of Cattle Breeds Based on Difference of DNA Mobility of Microsatellite Markers

Lee¹,

Yoon²,

Jeon³

et al. 2009

Bulletin of the Korean Chemical Society

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A breed of cattle, i.e., Korean cattle (Hanwoo), was identified based on the DNA mobilities of their microsatellites (MSs) by capillary gel electrophoresis (CGE) with a laser-induced fluorescence (LIF) detector. The MS markers were used for the accurate identification of species-specific genes. The DNA mobilities of the MS markers of Hanwoo and Holstein were measured using a CGE system with a fused-silica capillary (inner diameter of 75 µm, outer diameter of 365 µm, and total length of 50 cm). The capillary was dynamically coated with 1.0% (w/v) polyvinylpyrrolidone (Mr = 1,000,000) and then filled with a mixture of 1.3% (w/v) poly(ethylene oxide) (Mr = 600,000) and 1.9% (w/v) poly(ethylene oxide) (Mr = 8,000,000) as a sieving gel matrix. The species-specific genes of Hanwoo and Holstein were clearly distinguished within 33 min. This CGE assay technique is expected to be a useful analytical method for the fast and accurate identification of breeds of cattle.

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Suppression of electroosmotic flow and its application to determination of electrophoretic mobilities in a poly(vinylpyrrolidone)-coated capillary

Cited by 63 publications

References 18 publications

Rapid hybridization of nucleic acids using isotachophoresis

Rapid hybridization of nucleic acids using isotachophoresis

Transport and separation of micron sized particles at isotachophoretic transition zones

Capillary Gel Electrophoretic Analysis of Cattle Breeds Based on Difference of DNA Mobility of Microsatellite Markers

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