Methods and apparatus for measuring analytes

Abstract

Methods and apparatus relating to FET arrays including large FET arrays for monitoring chemical and/or biological reactions such as nucleic acid sequencing-by-synthesis reactions. Some methods provided herein relate to improving signal (and also signal to noise ratio) from released hydrogen ions during nucleic acid sequencing reactions.

1 . A method for sequencing a nucleic acid comprising
 contacting and incorporating known nucleotides into a plurality of identical nucleic acids in a reaction chamber in contact with or capacitively coupled to an ISFET, wherein the nucleic acids are covalently bound to a single bead in the reaction chamber, and   detecting hydrogen ions released upon nucleotide incorporation in the presence of no or limited buffering activity.   
     
     
         2 . The method of  claim 1 , wherein the ISFET is in an ISFET array that comprises 256 ISFET. 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1 ,  2  or  3 , wherein the reaction chamber comprises a solution having a buffering inhibitor. 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the nucleic acids are sequencing primers. 
     
     
         7 . The method of  claim 6 , wherein the nucleic acids are hybridized to template nucleic acids. 
     
     
         8 . The method of  claim 6 , wherein the nucleic acids are hybridized to concatemers of identical template nucleic acids. 
     
     
         9 . The method of  claim 1 , wherein the nucleic acids are self-priming template nucleic acids. 
     
     
         10 .- 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the nucleotides are unblocked. 
     
     
         13 . The method of  claim 1 , wherein the nucleotides are not extrinsically labeled. 
     
     
         14 .- 15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein the ISFET is present in an ISFET array having a center-to-center spacing of 1-10 microns. 
     
     
         17 . The method of  claim 16 , wherein the center-to-center spacing is about 9 microns, about 5.1 microns, or about 2.8 microns. 
     
     
         18 .- 23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein the bead has a diameter of about 1-7 microns. 
     
     
         25 . A method for determining incorporation of a nucleotide triphosphate into a newly synthesized nucleic acid comprising
 combining a known nucleotide triphosphate, a template/primer hybrid, a buffering inhibitor and a polymerase, in a solution in contact with or capacitively coupled to an ISFET, and   detecting a signal at the ISFET,   wherein detection of the signal indicates incorporation of the known nucleotide triphosphate into the newly synthesized nucleic acid.   
     
     
         26 . The method of  claim 25 , wherein the signal indicates release of hydrogen ions as a result of nucleotide incorporation. 
     
     
         27 . A method for synthesizing a nucleic acid comprising
 incorporating nucleotides into a nucleic acid in the presence of a buffering inhibitor.   
     
     
         28 . The method of  claim 27 , wherein the method further comprises detecting incorporation of nucleotides by detecting hydrogen ion release. 
     
     
         29 . The method of  claim 25 , wherein the buffering inhibitor is a sulfonic acid surfactant. 
     
     
         30 . The method of  claim 29 , wherein the sulfonic acid surfactant is poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether (PNSE) or a salt thereof. 
     
     
         31 . The method of  claim 25 , wherein the buffering inhibitor is poly(styrenesulfonic acid), poly(diallydimethylammonium), or tetramethyl ammonium, or a salt thereof. 
     
     
         32 . The method of  claim 25 , wherein the nucleic acid is a plurality of identical nucleic acids. 
     
     
         33 .- 154 . (canceled)