Leland Stanford Junior University

Lawrence Livermore National Security, LLC

Alexandre Persat

Juan G Santiago

Mostafa Ronaghi

Robert D Chambers

Reto B Schoch

Giancarlo Garcia

Charbel Eid

Klint Aaron Rose

Maxim Shusteff

Byoungsok Jung

Paul J Utz

Michael G Kattah

Isotachophoresis (ITP) is exploited to control various aspects of chemical reactions. In a first aspect, at least one of the reactants of a chemical reaction is confined to an ITP zone, but the resulting product of the chemical reaction is separated from this ITP zone by the ITP process. In a second aspect, one or more reactants of a chemical reaction are confined to an ITP zone, and one or more other reactants of the chemical reaction are not confined to this ITP zone. In a third aspect, ITP is employed to confine at least one reactant of a chemical reaction to an ITP zone, and at least one reactant of the chemical reaction is delivered to the ITP zone in two or more discrete doses. These aspects are especially relevant to performing polymerase chain reactions using chemical denaturants as opposed to thermal cycling.

Control of chemical reactions using isotachophoresis

Techniques for enhanced isotachophoresis assays using additives with spatial gradients include forming a concentration gradient of an additive along a channel from an input port to an output port. The channel is used for isotachophoresis with ions of a leading electrolyte having a first mobility greater than a mobility of an analyte, and ions of a trailing electrolyte having a second mobility less than the mobility of the analyte. The additive is different from both the leading electrolyte and the trailing electrolyte; and the additive has a third mobility that assures the analyte will encounter the additive. The method further comprises introducing a mixture of the trailing electrolyte and a sample including the analyte. The method further comprises applying an electric field to the channel; and, measuring the analyte.

Enhanced isotachophoresis assays using additives with spatial gradients

A method and system are presented for fast and efficient isolation, purification and quantitation of nucleic acids from complex biological samples using isotachophoresis in microchannels. In an embodiment, a sieving medium may be used to enhance selectivity. In another embodiment, PCR-friendly chemistries are used to purify nucleic acids from complex biological samples and yield nucleic acids ready for further analysis including for PCR. In another embodiment, small RNAs from biological samples are extracted, isolated, preconcentrated and quantitated using on-chip ITP with a high efficiency sieving medium. The invention enables fast concentration and separation (takes 10s to 100s of seconds) of nucleic acids with high selectivity and using lower volumes of reagents (order of 10s of &#x3bc;L to focus less than 1 &#x3bc;g/&#x3bc;L of nucleic acid).

Isotachophoretic focusing of nucleic acids

The present invention provides a mechanism for separating or isolating charged particles under the influence of an electric field without metal electrodes being in direct contact with the sample solution. The metal electrodes normally in contact with the sample are replaced with high conductivity fluid electrodes situated parallel and adjacent to the sample. When the fluid electrodes transmit the electric field across the sample, particles within the sample migrate according to their electrophoretic mobility.

Liquid and gel electrodes for transverse free flow electrophoresis

A method and system are presented for fast and efficient isolation, purification and quantitation of nucleic acids from complex biological samples using isotachophoresis in microchannels. In an embodiment, a sieving medium may be used to enhance selectivity. In another embodiment, PCR-friendly chemistries are used to purify nucleic acids from complex biological samples and yield nucleic acids ready for further analysis including for PCR. In another embodiment, small RNAs from biological samples are extracted, isolated, preconcentrated and quantitated using on-chip ITP with a high efficiency sieving medium. The invention enables fast concentration and separation (takes 10s to 100s of seconds) of nucleic acids with high selectivity and using lower volumes of reagents (order of 10s of &#x3bc;L to focus less than 1 pg/&#x3bc;L of nucleic acid).

Isotachophoresis (ITP) can be employed to simultaneously focus the target and ligand of an assay into the same ITP focus zone. The target and ligand can bind to each other in the ITP focus zone, and then the resulting bound complex can be detected (e.g., by fluorescence). The sensitivity of this approach can be greatly increased by the enhanced concentration of both target and ligand that ITP provides in the focus zone. Since ITP can be performed quickly, the resulting assay is both rapid and sensitive. Markers of bacterial urinary tract infections have been experimentally detected at clinically relevant concentrations with this approach. MicroRNA sequences have also been profiled with this approach, which is clinically relevant because MicroRNA is expected to provide useful markers for disease. In one experiment, miR-in human kidney and liver was detected and quantified.

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Princeton, NJ, United States of America

Alexandre Persat