Sensing apparatus and method

Abstract

Provided is a sensing apparatus comprising a chip for integrated amplification and sequencing of a template polynucleotide in a sample. The apparatus comprises a chip with at least one ISFET in a well or chamber, amplification means for amplifying the template polynucleotide on a surface of said chip and comprising at least one heating means suitable for conducting amplification of the template polynucleotide at temperatures elevated with respect to room temperature, and sequencing means for sequencing the amplified template polynucleotide in said well or chamber. Methods of use are also provided.

1 - 20 . (canceled) 
     
     
         21 . A method of determining the sequence of a plurality of template polynucleotides, the method comprising:
 providing the templates to a plurality of wells, each well exposed to an ISFET;   providing a seal;   amplifying the templates;   immobilizing at least one template within each well; then   creating a clonal population within each well; then   removing the seal; and then   sequencing the template colonies in the wells.   
     
     
         22 . The method according to  claim 21 , further comprising monitoring an output signal from the ISFET to detect byproducts of hydrolysis reactions involving said templates in said wells. 
     
     
         23 . The method according to  claim 21 , wherein a nucleic acid in a sample is fragmented prior to amplification to create the plurality of template polynucleotides. 
     
     
         24 . The method according to  claim 21 , wherein the templates in the wells are provided in a ratio of one template to between 0.4 and 2 wells, preferably one template to about every one well. 
     
     
         25 . The method according to  claim 21 , wherein a single template binding site is provided in each well to immobilize a single template. 
     
     
         26 . The method according to  claim 25 , wherein the single template binding site is on a bead, preferably wherein either (a) the bead is bound to a surface of each well by a binding site only large enough to bind one bead or (b) said bead is sized so that only one such bead can fit into each well. 
     
     
         27 . The method according to  claim 21 , further comprising controlling charges on electrodes exposed to each well to attract templates to each well, preferably wherein the charge on individual electrodes in each well is removed or reversed upon detection of a template binding to a surface of that well. 
     
     
         28 . The method according to  claim 21 , wherein sequence specific oligonucleotides immobilized within the well bind one or more strands of a target template type to a single solid substrate. 
     
     
         29 . The method according to  claim 21 , wherein oligonucleotides immobilized within the well are sequestered, preferably by coating the oligonucleotides with wax, preferably wherein the sequestration of immobilized oligonucleotides is removed by heating the wax, preferably after washing unbound template from the wells. 
     
     
         30 . The method according to  claim 21 , further comprising providing to each well a plurality of beads having further binding sites for clonal amplification of the template bound in that well, the step preferably performed after washing unbound template from the wells. 
     
     
         31 . A method of determining the sequence of a plurality of template polynucleotides, the method comprising:
 providing the plurality of templates to a plurality of wells, each well exposed to an ISFET;   providing a seal to the wells to isolate adjacent wells;   clonally amplifying the templates on a solid substrate in each well;   removing the seal; and   sequencing the amplified templates in the wells.   
     
     
         32 . A sensing apparatus for amplification and sequencing of a plurality of template polynucleotides, the apparatus comprising:
 a semiconductor chip having a plurality of ISFETs;   a microfluidic structure defining a plurality of wells, wherein each well is exposed to at least one of the ISFETs and the well is arranged to create a clonal population within each well;   a removable seal arranged to cover a surface of the microfluidic structure thus substantially isolating each well from adjacent wells;   amplification means for amplifying the template polynucleotides and comprising at least one heating means suitable for conducting amplification of the template polynucleotide at temperatures elevated with respect to room temperature; and   sequencing means for sequencing the template colonies in each well.   
     
     
         33 . The sensing apparatus according to  claim 32 , wherein there is at least one oligonucleotide immobilized within each well, which oligonucleotide is unbound to a polynucleotide but adapted to bind to the template polynucleotide. 
     
     
         34 . The sensing apparatus according to  claim 32 , wherein the removable seal is a liquid, such as a mineral oil. 
     
     
         35 . The sensing apparatus according to  claim 32 , wherein the removable seal is a solid, such as a flexible membrane, preferably wherein the seal comprises an adhesive, such as a pressure sensitive adhesive. 
     
     
         36 . The sensing apparatus according to  claim 32 , wherein a surface within each well is modified with means to immobilize oligonucleotides. 
     
     
         37 . The sensing apparatus according to  claim 32 , wherein only a single oligonucleotide is available in each of said wells to hybridize with the template polynucleotide in the sample. 
     
     
         38 . The sensing apparatus according to  claim 36 , wherein beads provide the surface for immobilization of oligonucleotides, preferably wherein the beads are streptavidin-coated polystyrene beads. 
     
     
         39 . The sensing apparatus according to  claim 32 , wherein the heating means comprises an on-chip heating element and, optionally, also an on-chip temperature sensor and/or on-chip temperature control circuitry, preferably wherein the on-chip heater is a resistive heating element integrated into the chip, preferably the element formed as a path from one or more metal layers in the chip. 
     
     
         40 . A sensing apparatus for amplification and sequencing of a plurality of template polynucleotides, the apparatus comprising:
 a semiconductor chip having a plurality of ISFETs;   a microfluidic structure defining a plurality of wells, each well exposed to at least one of the ISFETs;   a removable seal arranged to cover a surface of the microfluidic structure thus substantially isolating each well from adjacent wells;   amplification means for immobilizing and amplifying the template polynucleotides within the wells and comprising at least one heating means suitable for conducting amplification of the template polynucleotide at temperatures elevated with respect to room temperature; and   sequencing means for sequencing the amplified template polynucleotides in the wells.