Nucleic acid sequencing by electrochemical detection

Patent metadata

Field	Value
Publication number	US-9670538-B2
Application number	US-201214237328-A
Country	US
Kind code	B2
Filing date	Aug 3, 2012
Priority date	Aug 5, 2011
Publication date	Jun 6, 2017
Grant date	Jun 6, 2017

Abstract

Provided herein is technology relating to sequencing nucleic acids and particularly, but not exclusively, to devices, methods, and systems for sequencing-by-synthesis using changes in pH to monitor base addition. In some embodiments the electrochemical hydrogen ion sensor is a microfabricated mixed metal oxide electrode and in some embodiments the electrochemical hydrogen ion sensor is a membrane electrode. Moreover, in some embodiments the device further comprises a reference electrode. Performing the sequencing reaction involves moving solutions and other fluids (e.g., samples, nucleotide solutions, wash solutions) into and out of the reaction vessel. Thus, in some embodiments, the device further comprises a tube or other transport mechanism or pathway attached to the reaction vessel.

First claim

We claim:

1. A device for determining the sequence of a nucleic acid, the device comprising a reaction vessel comprising a hydrogen ion-sensitive electrode in contact with a sample comprising the nucleic acid, wherein the hydrogen ion-sensitive electrode is either:
a) a microfabricated metal oxide electrode comprising:
i) an insulating material sensitive to hydrogen ions; and
ii) a conductive metal oxide,
wherein the microfabricated metal oxide electrode does not exhibit the bulk conductive properties of the conductive metal oxide; or

b) a membrane electrode comprising:
i) a photolithographic base layer; and
ii) a hydrogen ion-sensitive membrane;

wherein the nucleic acid coats the surface of the hydrogen ion-sensitive electrode.

2. The device of claim 1 further comprising a reference electrode.

3. The device of claim 1 wherein the reaction vessel is a cylinder having a diameter of approximately 200 μm or less and a height of approximately 30 μm or less.

4. The device of claim 1 wherein the hydrogen ion-sensitive electrode detects changes in pH greater than or equal to 0.1.

5. The device of claim 1 comprising a plurality of hydrogen ion-sensitive electrodes.

6. The device of claim 1 comprising a plurality of nucleic acids covering the hydrogen ion-sensitive electrode at a density equal to or greater than 2.2×10 10 molecules/cm 2 .

7. The device of claim 1 wherein the insulating material has a density of hydrogen ion binding sites that is greater than 10 13 /cm 2 .

8. The device of claim 1 wherein the insulating material is selected from the group consisting of tantalum oxide, zirconium oxide, and aluminum oxide.

9. The device of claim 1 wherein the conductive metal oxide is selected from the group consisting of iridium oxide, ruthenium oxide, platinum oxide, palladium oxide, rhodium oxide, and osmium oxide.

10. The device of claim 1 comprising a clonal plurality of nucleic acids.

11. The device of claim 1 wherein the hydrogen ion sensitive membrane comprises:
a) a solvent mixture comprising cyclohexanone and propiophenone;
b) sodium tetraphenylborate
c) tridodecyl amine;
d) dibutyl sebacate or o-nitrophenyloctylether; and
e) high-molecular weight polyvinylchloride.

12. A system for sequencing a nucleic acid comprising:
i) a device according to claim 1 comprising a cartridge and
ii) a hand-held reading apparatus.

13. The system of claim 12 wherein the cartridge comprises:
1) a hydrogen ion-sensitive electrode; and
2) a first interface component configured to mate with the reading apparatus and communicate with the reading apparatus; and
wherein the reading apparatus comprises:
1) a second interface component configured to mate with the cartridge and communicate with the cartridge; and
2) a microprocessor configured to receive data from the cartridge.

14. The system of claim 13 wherein the data is raw data or transformed data.

15. The system of claim 13 wherein the data indicates the presence of a medical condition in a subject.

16. The system of claim 13 wherein the data indicates the absence of a medical condition in a subject.

17. The system of claim 12 further comprising a user interface.

18. A method for determining the sequence of a nucleic acid, the method comprising:
a) providing a reaction solution comprising:
1) the nucleic acid;
2) a polymerase; and
3) an oligonucleotide complementary to the nucleic acid;

b) adding a deoxynucleotide to the reaction solution; and
c) monitoring the pH of the reaction solution using a device according to claim 1 ,
wherein a change in the pH of the reaction solution indicates that the deoxynucleotide was polymerized to the 3′ end of the oligonucleotide.

19. The method of claim 18 further comprising:
d) removing the deoxynucleotide from the reaction solution or inactivating the deoxynucleotide.

20. A device for determining the sequence of a nucleic acid, the device comprising a reaction vessel comprising a hydrogen ion-sensitive electrode in contact with a sample comprising the nucleic acid, wherein the hydrogen ion-sensitive electrode is either:
a) a microfabricated metal oxide electrode comprising:
i) an insulating material sensitive to hydrogen ions; and
ii) a conductive metal oxide,
wherein the microfabricated metal oxide electrode does not exhibit the bulk conductive properties of the conductive metal oxide; or

b) a membrane electrode comprising:
i) a photolithographic base layer; and
ii) a hydrogen ion-sensitive membrane;

wherein the nucleic acid comprises a plurality of nucleic acids covering the hydrogen ion-sensitive electrode at a density equal to or greater than 2.2×10 10 molecules/cm 2 .

Assignees

• Huff Jeffrey
• Davis Graham
• Hayden Mark
• Ecker David J
• Wang Dan
• Collier Gordon Bruce
• Ibis Biosciences Inc

Inventors

• Huff Jeffrey
• Davis Graham
• Hayden Mark
• Ecker David J
• Wang Dan
• Collier Gordon Bruce

Classifications

• G01N27/447

  using electrophoresis · CPC title

• G01N27/44756

  Apparatus specially adapted therefor · CPC title

• G01N27/3275

  Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction · CPC title

• G01N27/333

  Ion-selective electrodes or membranes (glass electrodes G01N27/36) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title