Nucleic acid sequencing by electrochemical detection

• US 9,670,538 B2
• Filed: 08/03/2012
• Issued: 06/06/2017
• Est. Priority Date: 08/05/2011
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Davis, Graham, Huff, Jeffrey, Ecker, David J., Wang, Dan, Collier, Gordon Bruce, Hayden, Mark
• Primary Examiner(s): NOGUEROLA, ALEXANDER STEPHAN
• Application Number:
  US14/237,328
• Publication Number:
  US 20140238859A1
• Time in Patent Office: 1,768 Days
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 27/3275 Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
• CPC Class Codes: G01N 27/333 Ion-selective electrodes or membranes glass electrodes G01N27/36
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 27/44756 Apparatus specially adapted therefor
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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.

Claims

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 μ

m or less and a height of approximately 30 μ

m 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×

10¹⁰ molecules/cm².

7. The device of claim 1 wherein the insulating material has a density of hydrogen ion binding sites that is greater than 10¹³/cm².

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×

10¹⁰ molecules/cm².