Tandem isotachophoresis/zone electrophoresis method and system
First Claim
1. A method of separating components having a given negative or positive charge and contained in a sample, comprising(a) loading a separation microchannel having, in an upstream to downstream direction, an upstream end, an upstream channel region, a sample-volume channel region, a downstream separation channel region, and a downstream end, so as to fill (i) the upstream channel region with a trailing-edge electrolyte containing a trailing edge ion having an electrophoretic mobility lower than that of any of the sample components, and containing a selected concentration of a titratable species, (ii) the sample-volume channel region with the dilute sample, and (iii) the separation channel region with a leading-edge electrolyte, containing a leading edge ion having an electrophoretic mobility greater than that of any of the sample components;
- and (b) creating an electrical field potential across said microchannel, by applying a voltage potential across electrodes in contact with said upstream and downstream channel ends, thereby causing charged components in said sample to stack by isotachophoresis, and subsequently causing hydroxyl or hydrogen ions to migrate into the trailing-edge electrolyte, titrating said titratable species therein, under conditions that permit the sample to stack into a relatively small sample volume before hydroxyl- or hydrogen-ion migration into and through the sample-volume region is effective to overtake the charged sample components, wherein continued application of an electric potential across the channel ends causes charged sample components in the stacked sample volume to separate by zone electrophoresis.
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Abstract
A method of separating components having a given negative or positive charge and contained in a sample is disclosed. The method involves, in one embodiment, loading a microchannel with a sample, placed between a trailing-edge electrolyte having a selected concentration of a titratable species, and a leading-edge electrolyte. With the application of a voltage potential across the microchannel, charged components in the sample stack by isotachophoresis, and electrolytic hydroxyl or hydrogen ions formed by electrolysis at the upstream-end electrode migrate into the trailing-edge ion buffer, titrating the titratable species therein, where the concentration of the titratable species in the trailing-edge electrolyte is selected, in relation to the lengths of the upstream channel region and sample-loading volume, to permit the sample to stack into a relatively small sample volume before electrolytic-ion migration from the upstream electrode into and through the sample-volume region is effective to overtake the charged sample components. With continued application of an electric potential across the channel ends, charged sample components in the stacked sample volume separate by zone electrophoresis.
150 Citations
14 Claims
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1. A method of separating components having a given negative or positive charge and contained in a sample, comprising
(a) loading a separation microchannel having, in an upstream to downstream direction, an upstream end, an upstream channel region, a sample-volume channel region, a downstream separation channel region, and a downstream end, so as to fill (i) the upstream channel region with a trailing-edge electrolyte containing a trailing edge ion having an electrophoretic mobility lower than that of any of the sample components, and containing a selected concentration of a titratable species, (ii) the sample-volume channel region with the dilute sample, and (iii) the separation channel region with a leading-edge electrolyte, containing a leading edge ion having an electrophoretic mobility greater than that of any of the sample components; - and
(b) creating an electrical field potential across said microchannel, by applying a voltage potential across electrodes in contact with said upstream and downstream channel ends, thereby causing charged components in said sample to stack by isotachophoresis, and subsequently causing hydroxyl or hydrogen ions to migrate into the trailing-edge electrolyte, titrating said titratable species therein, under conditions that permit the sample to stack into a relatively small sample volume before hydroxyl- or hydrogen-ion migration into and through the sample-volume region is effective to overtake the charged sample components, wherein continued application of an electric potential across the channel ends causes charged sample components in the stacked sample volume to separate by zone electrophoresis. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
where the concentration of said titratable species in the trailing-edge electrolyte is selected, in relation to the lengths of the upstream channel region and sample-loading volume, to permit the sample to stack into a relatively small sample volume before electrolytic-ion migration from the upstream electrode into and through the sample-volume region is effective to overtake the charged sample components, and wherein continued application of an electric potential across the channel ends causes charged sample components in the stacked sample volume to separate by zone electrophoresis. -
4. The method of claim 3, wherein the trailing-edge electrolyte includes a trailing-edge ion and a titratable counter-ion buffer at said selected concentration.
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5. The method of claim 4, wherein the electrolytic ions formed at the upstream-end electrode are hydroxyl ions, and the titratable counter-ion buffer is a TRIS buffer.
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6. The method of claim 1, for use in detecting charged sample components present at nanomolar concentrations or less, wherein the ratio of sample volume before and after isotachophoretic stacking is at least about 10:
- 1.
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7. The method of claim 6, wherein the ratio of sample volume before and after isotachophoretic stacking is at least about 50:
- 1.
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8. The method of claim 7, for use in detecting charged sample components present at picomolar or less concentrations, wherein the ratio of sample volume before and after isotachophoretic stacking is at least about 100:
- 1.
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9. The method of claim 1, wherein the ratio of the lengths of the sample-volume region to the separation channel is between about 1:
- 50 to 1;
1.
- 50 to 1;
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10. The method of claim 1, wherein said ratio is between about 1:
- 10 to 1;
2.
- 10 to 1;
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11. The method of claim 3, wherein the leading-edge electrolyte contains a negatively charged leading-edge ion having an effective conductivity greater than that of the sample ions and a concentration between 1-50 mM, the trailing-edge electrolyte contains a negatively charged trailing-edge ion having an effective conductivity lower than that of the sample ions and a concentration of between 1-50 mM, and both electrolytes have a positively charged buffer at a selected concentration between about 2 and 50 mM.
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12. The method of claim 1, wherein the separation microchannel is part of a channel network in a microfluidics device, which also includes first and second side channels which intersect the separation channel at axially spaced intersections, defining said sample-volume region between said intersections, and said loading includes moving said sample from a sample reservoir connected to the first side channel through the sample-volume region and into the second side channel.
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13. The method of claim 1, for use in separating a plurality of electrophoretic tags contained in a sample, each tag having a detectable moiety and a mobility modifier that confers on the tag, a unique electrophoretic mobility, which further includes the steps, after separating the tags electrophoretically, of detecting the separated tags, and determining from their electrophoretic mobilities, the identify of probe from which the tags were cleaved.
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14. The method of claim 13, wherein said tags are contained on branched polymer structures and linked thereto through photo-labile linkages, and said method further includes, after permitting branched structures in the sample to stack into a relatively small sample volume, irradiating the branched structures to release the tags therefrom, wherein continued application of an electric potential across the channel ends causes said tags in the stacked sample volume to separate by zone electrophoresis.
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Specification