Nucleic acid sequencing by electrochemical detection

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.

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 one or more microparticles in an electrode well compartment.   
     
     
         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 one or more particles in an electrode well compartment 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 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.   
     
     
         13 . The method of  claim 12  further comprising:
 d) removing the deoxynucleotide from the reaction solution or inactivating the deoxynucleotide. 
 
     
     
         14 . 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.   
     
     
         15 . The system of  claim 14  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. 
 
     
     
         16 . The system of  claim 15  wherein the data is raw data or transformed data. 
     
     
         17 . The system of  claim 14  further comprising a user interface. 
     
     
         18 . The system of  claim 15  wherein the data indicates the presence of a medical condition in a subject. 
     
     
         19 . The system of  claim 15  wherein the data indicates the absence of a medical condition in a subject. 
     
     
         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 one or more microparticles in an electrode well compartment.