Methods and Apparatus for Measuring Analytes Using Large Scale FET Arrays

Abstract

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis.

1 - 106 . (canceled) 
     
     
         107 . A method for nucleic acid sequencing comprising:
 (a) disposing a plurality of nucleic acid templates at discrete locations on a substrate associated with a semiconductor based sensor array, the discrete locations associated with at least one sensor comprising a field-effect transistor configured to provide output signals representative of a sequencing reaction byproducts proximate thereto;   (b) introducing a known nucleotides, primer, and enzyme into the reaction chambers;   (c) providing conditions that allow the nucleic acid templates, nucleotide, primer, and enzyme to react;   (d) detecting incorporation of the nucleotides by sequencing reaction byproduct accumulation proximate to the at least one sensor;   (e) washing unincorporated known nucleotides from the discrete locations;   (f) outputting signals from respective sensors in response to detecting the at least one sequencing reaction byproducts; and   (g) repeating steps (b) through (d) until a portion of the plurality of nucleic acid templates are at least partially sequenced.   
     
     
         108 . The method of  claim 107  wherein the sequencing reaction byproducts comprise hydrogen ions. 
     
     
         109 . The method of  claim 107  wherein the sequencing reaction byproducts resulting from incorporation are of chemically similar composition for each of the known nucleotides. 
     
     
         110 . The method of  claim 109  wherein the sequence of the nucleic acid templates are identified on the basis of the identity of the known nucleotides and detection of the sequencing byproduct. 
     
     
         111 . The method of  claim 107  wherein the sensors of the sensor array have a chemically sensitive portion responsive to sequencing reaction byproducts and disposed in proximity to the substrate such that the at least one sample reaction byproducts diffuse or contact the sensors to thereby be detected. 
     
     
         112 . The method of  claim 111  wherein the chemically sensitive portion of the sensors of the sensor array is responsive to a plurality of different sample reaction byproducts. 
     
     
         113 . The method of  claim 107  wherein the discrete locations are disposed within respective reaction chambers. 
     
     
         114 . The method of  claim 113  wherein the reaction chambers provide weak buffer conditions. 
     
     
         115 . The method of  claim 107  wherein the sensors are configured to output signals being similar for each sensor of the array in response to similar amounts of sequencing reaction byproduct proximate thereto. 
     
     
         116 . The method of  claim 107  wherein the nucleic acid templates are hybridized to the primer and the enzyme comprises a polymerase capable of incorporating the known nucleotides at the 3′ end of the primer. 
     
     
         117 . The method of  claim 116  wherein a respective incorporated known nucleotide is complementary to a corresponding nucleotide in the nucleic acid template. 
     
     
         118 . The method of  claim 107  wherein the sensors of the array occupy an approximate area of 100 um or less. 
     
     
         119 . The method of  claim 107  wherein the sensors of the array have an approximate pitch of 10 um or less. 
     
     
         120 . The method of  claim 107  wherein the discrete locations on the substrate have an approximate volume in the range of 1500 um 3  to 150 um 3 . 
     
     
         121 . The method of  claim 107  wherein the discrete locations on the substrate have an approximate volume in the range of 150 um 3  to 15 um 3 . 
     
     
         122 . The method of  claim 107  wherein the discrete locations on the substrate have an approximate volume in the range of 15 um 3  to 1.5 um 3 . 
     
     
         123 . The method of  claim 107  wherein the discrete locations on the substrate have an approximate volume of 1 um 3  or less. 
     
     
         124 . The method of  claim 107  wherein the step of detecting includes measuring a change in the hydrogen ion concentration within respective discrete locations. 
     
     
         125 . The method of  claim 107  wherein a respective discrete location contains at least 10 5  copies of a respective nucleic acid template. 
     
     
         126 . The method of  claim 107  wherein a respective nucleic acid template is coupled to a solid support. 
     
     
         127 . The method of  claim 126  wherein the solid support comprises a portion of the substrate. 
     
     
         128 . The method of  claim 126  wherein the solid support comprises a particle. 
     
     
         129 . The method of  claim 127  wherein respective nucleic acid templates are amplified to create a clonal population coupled to the solid support. 
     
     
         130 . The method of  claim 126  wherein the array of sensors comprises at least 10 3  sensors. 
     
     
         131 . A method for sequencing a nucleic acid comprising:
 disposing a plurality of template nucleic acids in a plurality of reaction chambers, wherein the plurality of reaction chambers are disposed proximate to a chemical-sensitive field effect transistor (chemFET) array comprising at least one chemFET for respective reaction chambers, hybridizing the template nucleic acids to primers and subjecting to enzyme activity to synthesize a complementary nucleic acid strand by incorporating one or more known nucleotides sequentially at the 3′ end of the primer; and   detecting incorporation of the one or more known nucleotides by a change in current in the at least one chemFET of the chemFET array.   
     
     
         132 . The method of  claim 131 , wherein the chemFET array comprises at least 256 chemFETs. 
     
     
         133 . A method for sequencing a nucleic acid comprising:
 disposing a plurality of template nucleic acids into a plurality of reaction chambers, wherein the plurality of reaction chambers are coupled to a chemical-sensitive field effect transistor (chemFET) array comprising at least one chemFET for respective reaction chambers;   incorporating one or more known nucleotides sequentially into newly synthesized nucleic acid strands complementary to the template nucleic acids; and   detecting the incorporation of the one or more known nucleotides by detecting the generation of sequencing reaction byproduct by the chemFETs.   
     
     
         134 . The method of  claim 133  wherein the sequencing reaction byproducts comprise hydrogen ions. 
     
     
         135 . The method of  claim 133  wherein the sequencing reaction byproducts comprise inorganic pyrophosphate (PPi).