Microfluidic devices, solid supports for reagents and related methods

Patent metadata

Field	Value
Publication number	US-11345947-B2
Application number	US-201715445217-A
Country	US
Kind code	B2
Filing date	Feb 28, 2017
Priority date	May 25, 2012
Publication date	May 31, 2022
Grant date	May 31, 2022

Abstract

A microfluidic device includes a plurality of reaction wells; and a plurality of solid supports, and each of the solid supports has a reagent attached thereto. The reagent is attached to the solid support via a labile reagent/support bond such that the reagent is configured to be cleaved from the support via a cleaving operation.

First claim

That which is claimed is:

1. A method of conducting a reaction using the solid supports in a microfluidic device, the microfluidic device comprising: a substrate having a plurality of solid support containment regions that are sized and configured to capture a plurality of solid supports having a diameter d, wherein each of the solid supports has a reagent attached via a labile reagent/support bond and the reagent is configured to be cleaved from the support via a cleaving operation, and wherein the reagent comprises a DNA primer or probe for a PCR reaction, and the solid support containment regions have a cylindrical shape having a diameter of 1.05d to 1.55d and a depth of 1.2d to 1.8d, the solid supports comprising magnetic solid supports, the method comprising:
loading the solid supports in the plurality of solid support containment regions by applying a magnet or magnetic field, wherein the magnetic field has a field component parallel to the substrate and is applied translationally across the substrate to move the solid supports across the substrate and into the plurality of solid support containment regions;
washing the plurality of solid support containment regions with a blocking buffer configured to reduce or prevent bead adsorption, multiple bead loading, and chain formation; and
loading a sample and a PCR master mix in the plurality of solid support containment regions; and
after applying the magnetic field to move the solid supports across the substrate and into the containment regions and loading the sample and the PCR master mix, flowing a sealing fluid comprising oil, liquid polymer, or fluorocarbon liquid over the plurality of solid support containment regions to seal the plurality of containment regions, wherein the sealing fluid isolates reagents in the plurality of solid support containment regions and fluidically isolates each of the plurality of solid support containment regions during a reaction.

2. The method of claim 1 , wherein the solid supports comprise super paramagnetic supports.

3. The method of claim 1 , wherein the substrate comprises channels and an array of the containment regions, and loading the solid supports comprises moving at least one of the solid supports across at least one of the channels and into one of the containment regions.

4. The method of claim 1 , the method further comprising:
performing a cleaving operation to release the reagent into a solution in the plurality of containment regions.

5. The method of claim 1 , wherein the plurality of solid supports comprises at least a first plurality of solid supports having a first reagent attached thereto and a second plurality of solid supports having a second reagent attached thereto, wherein the first and second plurality of solid supports further comprise respective first and second markers configured to identify a property of the solid support and/or reagent.

6. The method of claim 5 , further comprising decoding the device by identifying whether each of the plurality of solid support containment regions is occupied by the first plurality of solid supports or the second plurality of solid supports.

7. The method of claim 6 , wherein decoding the device comprises identifying the first and second markers of the plurality of solid supports.

8. The method of claim 1 , wherein the solid supports are a polymer, magnetic material, or a combination thereof.

9. The method of claim 1 , wherein the solid supports comprise beads about 3.3 μm in diameter.

10. The method of claim 4 , wherein the cleaving operation is an addition of a chemical, the addition of an enzyme, application of an electric potential, and/or an application of light, or ionizing radiation to the reagent/support bond.

11. The method of claim 4 , wherein the cleaving operation comprises a thermal operation.

12. The method of claim 4 , wherein the reagent comprises a nucleic acid sequence for a nucleic acid transcription and/or amplification reaction.

13. A method of conducting a reaction using the solid supports in a microfluidic device, the microfluidic device comprising: a substrate having a plurality of solid support containment regions that are sized and configured to capture a plurality of solid supports having a diameter d, wherein each of the solid supports has a reagent attached via a labile reagent/support bond and the reagent is configured to be cleaved from the support via a cleaving operation, and wherein the reagent comprises a DNA primer or probe for a PCR reaction, and the solid support containment regions have a cylindrical shape having a diameter of 1.05d to 1.55d and a depth of 1.2d to 1.8d, the solid supports comprising magnetic solid supports, the method comprising:
loading the solid supports in the plurality of solid support containment regions by applying a magnet or magnetic field, wherein the magnet or magnetic field is applied to move the solid supports across the substrate and into the plurality of solid support containment regions, wherein the plurality of solid support containment regions and the plurality of solid supports are sized and configured such that a single one of the plurality of solid supports is retained in a corresponding single one of the plurality of solid support containment regions; and
loading a sample and a PCR master mix in the plurality of solid support containment regions; and
after applying the magnetic field to move the solid supports across the substrate and into the containment regions and loading the sample and the PCR master mix, flowing a sealing fluid comprising oil, liquid polymer, or fluorocarbon liquid over the plurality of solid support containment regions to seal the plurality of containment regions, wherein the sealing fluid isolates reagents in the plurality of solid support containment regions and fluidically isolates each of the plurality of solid support containment regions during a reaction.

14. The method of claim 1 , wherein the buffer comprises bovine serum albumin (BSA).

Assignees

• Univ North Carolina Chapel Hill

Inventors

• Ramsey John Michael
• Henley William
• Oblath Emily

Classifications

• B01J2219/00619

  using hydrophilic or hydrophobic regions · CPC title

• C12Q1/6853

  using modified primers or templates · CPC title

• B01L2300/0864

  comprising only one inlet and multiple receiving wells, e.g. for separation, splitting · CPC title

• B01J2219/00317

  Microwell devices, i.e. having large numbers of wells · CPC title

• B01J2219/00454

  by chemical cleavage from the solid support · CPC title