Addressable flow cell using patterned electrodes

• US 10,537,889 B2
• Filed: 12/30/2014
• Issued: 01/21/2020
• Est. Priority Date: 12/31/2013
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Hou, Chenlu, Jung, Byoungsok, Wu, Yir-Shyuan, Khurana, Tarun
• Primary Examiner(s): Bunker, Amy M
• Application Number:
  US15/107,882
• Publication Number:
  US 20160318016A1
• Time in Patent Office: 1,848 Days
• CPC Class Codes: B01L 2200/141 Preventing contamination, tampering
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

Disclosed are methods and systems concerning flow cells for sequencing a nucleic acid sample that may be characterized by the following components: (a) a substrate having an inner surface facing a library sequencing region, and an outer surface; (b) a plurality of a plurality of forward and reverse amplification primers immobilized over the inner surface and providing a nucleic acid library capture surface of the library sequencing region; (c) a plurality of electrodes disposed along the inner surface directly under at least some of the forward and reverse amplification primers, and configured to provide, when charged, an electric field through the library capture surface and into the library sequencing region; (d) electrical leads connected to the plurality of electrodes to permit the electrodes to be independently addressable; and (e) fluidic couplings configured to deliver a plurality of nucleic acid libraries to the flow cell during different time periods.

Claims

1. A flow cell for nucleic acid sequencing, the flow cell comprising:

(a) a plurality of inlet ports and a plurality of outlet ports,

(b) a top plate that defines an upper surface of the flow cell and a boundary of the library sequencing region, wherein the top plate is overlaid upon a channel layer, wherein the top plate is coated with a counter electrode;

(c) the channel layer overlaid upon a bottom plate and defining multiple substantially parallel channels that form a library sequencing region, wherein the multiple substantially parallel channels comprise one to twenty enclosed channels and each of the multiple substantially parallel channels is positioned between one of the plurality of inlet ports and one of the plurality of outlet ports, and wherein each of the multiple substantially parallel channels of the channel layer comprises;

(i) a library capture surface comprising an inner surface facing a library sequencing region, and

(ii) a plurality of forward amplification primers having a first sequence and a plurality of reverse amplification primers having a second sequence, wherein both the plurality of forward amplification primers and the plurality of reverse amplification primers are directly immobilized to the inner surface of each of the multiple substantially parallel channels to provide a nucleic acid library capture surface of the library sequencing region; and

(d) the bottom plate, wherein the bottom plate comprises;

(i) an inner surface;

(ii) a plurality of electrodes disposed along the inner surface of the bottom plate and arranged such that each channel of the multiple substantially parallel channels of the channel layer passes over the plurality of electrodes;

wherein the plurality of electrodes comprises two to twelve electrodes;

each electrode of the plurality of electrodes having a width that spans more than half or all of the width of the channel, or that spans the width of the flow cell, wherein the width of each electrode, the width of the channel, and the width of the flow cell are perpendicular to the direction of flow through the flow cell, wherein the plurality of electrodes comprise at least;

(a) a first electrode directly under a plurality of forward amplification primers and a plurality of reverse amplification primers, and (b) a second electrode directly under a plurality of forward amplification primers and a plurality of reverse amplification primers, wherein the first electrode and the second electrode are configured to provide, when charged, an electric field through the library capture surface and into the library sequencing region; and

(ii) electrical leads connected to the plurality of electrodes to permit each of plurality of electrodes to be independently addressable.

2. The flow cell of claim 1, wherein the top plate comprises a film of transparent electrically conductive film attached to an electrical lead.

3. The flow cell of claim 1, further comprising an attachment layer over the plurality of electrodes to which the plurality of forward amplification primers and the plurality of reverse amplification primers are attached.

4. The flow cell of claim 1, wherein the nucleic acid library capture surface occupies an area of between about 60 mm² and about 2400 mm², and wherein polynucleotides are hybridized to the plurality of forward amplification primers and the plurality of reverse amplification primers at a density of 10,000/mm² to 2,000,000/mm².

5. The flow cell of claim 1, wherein the plurality of electrodes are disposed in an array.

6. The flow cell of claim 1, wherein the plurality of electrodes are made of a conductor selected from the group consisting of gold, indium-doped tin oxide (ITO), silver, tin, titanium, copper, platinum, palladium, polysilicon, and carbon.

7. The flow cell of claim 3, wherein the plurality of first amplification primers are configured to hybridize to a first gene sequence and are attached to a first region of the flow cell, and wherein the plurality of second amplification primers are configured to hybridize to a second gene sequence and are attached to a second, spatially separate, region of the flow cell.

8. The flow cell of claim 3, wherein the plurality of forward amplification primers and the plurality of reverse amplification primers are disposed on one or more beads immobilized on the attachment layer.

9. The flow cell of claim 1, wherein each channel of the multiple substantially parallel channels passes over at least five electrodes of the plurality of electrodes.

10. The flow cell of claim 1, further comprising fluidic couplings at each inlet port and each outlet port that are configured to deliver a plurality of nucleic acid libraries to the flow cell.

11. The flow cell of claim 1, wherein the width of each electrode of the plurality of electrodes spans the width of the flow cell.