Patent Publication Number: US-9852919-B2

Title: Methods and systems for point of use removal of sacrificial material

Description:
RELATED APPLICATION 
     This application is a Divisional application of U.S. patent application Ser. No. 13/734,696 filed Jan. 4, 2013, the entire contents of which is herein incorporated by reference. 
    
    
     FIELD 
     This application generally relates to methods and systems for nucleic acid sequencing. More specifically, the specification relates to methods and systems for processing and/or analyzing nucleic acid sequencing data and/or signals. 
     BACKGROUND 
     Deoxyribonucleic acid, or DNA, is the genetic material in the nuclei of all cells. DNA is made of chemical building blocks called nucleotides. Nucleotides comprise three parts: a phosphate group, a sugar group and one of four types of nitrogen bases. The four types of nitrogen bases found in nucleotides are: adenine (A), thymine (T), cytosine (C) and guanine (G). To form a strand of DNA, nucleotides are linked into chains, with the phosphate and sugar groups alternating. 
     The structure of a DNA molecule was first described in 1953 by Francis Crick and James D. Watson as two strands wound around each other in a double helix to resemble a twisted ladder. The two strands of DNA contain complementary information: A forms hydrogen bonds only with T, C only with G. The precise order in which the four types of nitrogen bases (A, T, C, G) appear in the strand determines genetic characteristics of all life forms. The process of determining the precise order of nucleotides within a DNA molecule is termed DNA sequencing. DNA sequencing may be used to determine the sequence of individual genes, larger genetic regions (i.e. clusters of genes or operons), full chromosomes or entire genomes. Depending on the methods used, sequencing may provide the order of nucleotides in DNA or RNA isolated from cells of animals, plants, bacteria, archaea, or virtually any other source of genetic information. The resulting sequences may be used by researchers in molecular biology or genetics to further scientific progress or may be used by medical personnel to make treatment decisions. 
     Conventional methods of DNA sequencing comprise chemical (i.e. electrophoresis), optical, and electronic methods. As compared to other methods for detecting a DNA sequence, electronic sequencing methods differ from other sequencing technologies in that modified/labeled nucleotides and/or optics/optical measurements are not necessary. Instead, electronic sequencing methods rely on ion or other reaction byproducts to identify the relevant DNA sequence. 
     A type of electronic DNA sequencing, ion semiconductor sequencing is a method of DNA sequencing based on the detection of ions (for example, hydrogen ions) that are released during the polymerization of DNA. Ion semiconductor sequencing is a method of “sequencing by synthesis,” during which a complementary strand is built by incorporation (pairing of bases) based on the sequence of a template strand. 
     The incorporation of a deoxyribonucleotide triphosphate (dNTP) into a growing DNA strand involves the formation of a covalent bond and the release of pyrophosphate and a positively charged hydrogen ion. A dNTP will only be incorporated if it is complementary to the leading unpaired template nucleotide. Ion semiconductor sequencing leverages this process by detecting whether a hydrogen ion is released when a single species of dNTP is provided to the reaction. 
     Hydrogen ions may be detected by providing an array of microwells on a semiconductor chip. Beneath the layer of microwells is an ion sensitive layer, below which is an ion sensitive (ISFET) or a chemical sensitive (chemFET) sensor. Each microwell on the chip may contain a template DNA molecule to be sequenced. Each microwell containing a template strand DNA molecule also contains a DNA polymerase. A DNA polymerase is a cellular or viral enzyme that synthesizes DNA molecules from their nucleotide building blocks. A microfluidics device may be used to introduce a solution of unmodified A, T, C, or G dNTP into the microwells one after the other and one at a time. If an introduced dNTP is complementary to the next unpaired nucleotide on the template strand, a biochemical reaction occurs (which includes the release of a hydrogen ion) and the introduced dNTP is incorporated into the growing complementary strand by the DNA polymerase. If the introduced dNTP is not complementary there is no incorporation and no biochemical reaction. The release of a hydrogen ion during incorporation causes a change in the pH of the solution in the microwell. That change in the pH of the solution can be detected/measured by the ISFET or chemFET sensor and translated into an electrical pulse. 
     Before the next cycle of dNTP is introduced into the microwells, the microwells are flushed with a wash solution. Unattached dNTP molecules are washed out during the flush cycle. The detected series of electrical pulses are transmitted from the chip to a computer and are translated into a DNA sequence. Because nucleotide incorporation events are measured directly by electronics, intermediate signal conversion is not required. Signal processing and DNA assembly can then be carried out in software. 
     The chip may be fabricated by taking advantage of conventional semiconductor technology. However, the release of a hydrogen ion produces a small and transient signal that is difficult to measure. To further complicate detection and nucleic acid sequencing, ion detection is sensitive to various forms of contaminants on the chip surfaces. Accordingly, problems arise during manufacturing, packaging and exposure of the chip surfaces to the environment. Thus, there is a need for improved methods and an apparatus to prevent contamination of the chip surfaces for reliable ion detection and sequencing. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The accompanying drawings, which are incorporated into and form a part of the specification, illustrate one or more exemplary embodiments and serve to explain the principles of various exemplary embodiments. The drawings are exemplary and explanatory only and are not to be construed as limiting or restrictive in any way. 
         FIG. 1  illustrates components of a system for nucleic acid sequencing according to an exemplary embodiment. 
         FIG. 2  illustrates cross-sectional and expanded views of a flow cell for nucleic acid sequencing according to an exemplary embodiment. 
         FIGS. 3A-F  are schematic representations of a method of manufacturing a sensor with a protective layer according to an exemplary embodiment. 
         FIGS. 4A-C  are schematic representations of a sensor with a protective layer according to an exemplary embodiment. 
         FIG. 5  is flow diagram for performing a method of manufacturing a sensor with a protective layer according to an exemplary embodiment. 
         FIGS. 6A-B  are schematic representations of a sensor with ion-sensing layers according to different exemplary embodiments. 
     
    
    
     SUMMARY 
     The disclosure relates to methods of manufacturing a sensor for nucleic acid sequencing having a protective layer that prevents surface contamination of the sensor during various stages of fabrication. The disclosure further relates to a sensor for nucleic acid sequencing comprising an array of chemically-sensitive field effect transistors (chemFETs) and a conformal protective layer over the sensing surfaces, sidewalls of the cavities, and top surface of the array of chemFETs. The protective layer over the array of chemFETs may be removed by an end user before Nucleic acid sequencing is performed. 
     In one embodiment, the disclosure relates to a method of manufacturing a sensor for Nucleic acid sequencing. The method comprises forming an array of chemically-sensitive field effect transistors (chemFETs); depositing a dielectric layer over the chemFETs in the array; depositing a protective layer over the dielectric layer; etching the dielectric layer and the protective layer to form cavities corresponding to sensing surfaces of the chemFETs; and removing the protective layer. In another embodiment, the step of etching includes etching the dielectric layer and the protective layer together to form cavities corresponding to sensing surfaces of the chemFETs. In one embodiment, the protective layer is at least one of a polymer, photoresist material, noble metal, copper oxide, and zinc oxide. In another embodiment, the protective layer is removed using at least one of an acid, a base, and an oxidizing solution. For example, the protective layer may be removed using at least one of sodium hydroxide, organic solvent, aqua regia, ammonium carbonate, hydrochloric acid, acetic acid, and phosphoric acid. In another embodiment, the dielectric layer includes at least one of silicon oxide, silicon nitride and silicon oxynitride. In one embodiment, the method further comprises patterning a photosensitive etch mask and wherein the etching of the dielectric layer and the protective layer is a photolithographic process. 
     In another embodiment, the disclosure relates to a sensor for Nucleic acid sequencing. The sensor comprises an array of chemically-sensitive field effect transistors (chemFETs), each chemFET in the array having a floating gate structure including an upper surface; a dielectric layer over the upper surfaces of the floating gate structures of the chemFETs in the array, the dielectric layer including cavities extending to the upper surfaces of the floating gate structures and corresponding to sensing surfaces of the chemFETs; a conformal protective layer over the sensing surfaces, sidewalls of the cavities, and top surface of the array. In one embodiment, the protective layer defines a removable layer. In another embodiment, the protective layer is at least one of a polymer, photoresist, noble metal, copper oxide, and zinc oxide. In another embodiment, the protective layer is removed using at least one of sodium hydroxide, organic solvent, aqua regia, ammonium carbonate, hydrochloric acid, acetic acid, and phosphoric. In another embodiment, the dielectric layer includes at least one of silicon oxide, silicon nitride and silicon oxynitride. In another embodiment, the sensing material includes at least one of tantalum oxide, titanium oxide, and titanium nitride. In another embodiment, the sensing material is a metal oxide or metal nitride selected from one or more of the group of Al2O3, Ta2O5, HfO3, WO3, ZrO2, TiO2 or mixtures thereof. 
     DETAILED DESCRIPTION 
       FIG. 1  illustrates components of a system for nucleic acid sequencing according to an exemplary embodiment. The components include a flow cell and sensor array  100 , a reference electrode  108 , a plurality of reagents  114 , valve block  116 , wash solution  110 , valve  112 , fluidics controller  118 , lines  120 / 122 / 126 , passages  104 / 109 / 111 , waste container  106 , array controller  124 , and user interface  128 . The flow cell and sensor array  100  includes inlet  102 , outlet  103 , microwell array  107 , and flow chamber  105  defining a flow path of reagents over the microwell array  107 . The reference electrode  108  may be of any suitable type or shape, including a concentric cylinder with a fluid passage or a wire inserted into a lumen of passage  111 . The reagents  114  may be driven through the fluid pathways, valves, and flow cell by pumps, gas pressure, or other suitable methods, and may be discarded into the waste container  106  after exiting the flow cell and sensor array  100 . 
     Fluidics controller  118  may control driving forces for reagents  114  and the operation of valve  112  and valve block  116  with suitable software. Microwell array  107  may include an array of defined spaces or reaction confinement regions, such as microwells, for example, that is operationally associated with a sensor array  100  so that, for example, each microwell has a sensor suitable for detecting an analyte or reaction property of interest. Microwell array  107  may preferably be integrated with the sensor array as a single device or chip. The flow cell may have a variety of designs for controlling the path and flow rate of reagents over microwell array  107 , and may be a microfluidics device. Array controller  124  may provide bias voltages and timing and control signals to the sensor, and collect and/or process output signals. User interface  128  may display information from the flow cell and sensor array  100  as well as instrument settings and controls, and allow a user to enter or set instrument settings and controls. 
     In an exemplary embodiment, such a system may deliver reagents to the flow cell and sensor array in a predetermined sequence, for predetermined durations, at predetermined flow rates, and may measure physical and/or chemical parameters providing information about the status of one or more reactions taking place in defined spaces or reaction confinement regions, such as, for example, microwells (or in the case of empty microwells, information about the physical and/or chemical environment therein). In an exemplary embodiment, the system may also control a temperature of the flow cell and sensor array so that reactions take place and measurements are made at a known, and preferably, a predetermined temperature. 
     In an exemplary embodiment, such a system may be configured to let a single fluid or reagent contact reference electrode  108  throughout an entire multi-step reaction. Valve  112  may be shut to prevent any wash solution  110  from flowing into passage  109  as the reagents are flowing. Although the flow of wash solution may be stopped, there may still be uninterrupted fluid and electrical communication between the reference electrode  108 , passage  109 , and the sensor array  107 . The distance between reference electrode  108  and the junction between passages  109  and  111  may be selected so that little or no amount of the reagents flowing in passage  109  and possibly diffusing into passage  111  reach the reference electrode  108 . In an exemplary embodiment, wash solution  110  may be selected as being in continuous contact with the reference electrode  108 , which may be especially useful for multi-step reactions using frequent wash steps. 
       FIG. 2  is an illustration of expanded and cross-sectional views of an exemplary flow cell  200  and shows a portion of an exemplary flow chamber  206 . A reagent flow  208  flows across a surface of a microwell array  202 , in which reagent flow  208  flows over the open ends of the microwells. Microwell array  202  and sensor array  205  together can form an integrated unit forming a bottom wall (or floor) of flow cell  200 . Reference electrode  204  can be fluidly coupled to flow chamber  206 . Further, flow cell cover  230  encapsulates flow chamber  206  to contain reagent flow  208  within a confined region. 
     Example flow cell structures and associated components can be found in U.S. Pat. No. 7,948,015 (filed Dec. 14, 2007). 
       FIG. 2  also illustrates an expanded view of exemplary microwell  201 , dielectric layer  210 , and exemplary sensor  214 . The volume, shape, aspect ratio (such as base width-to-well depth ratio), and other dimensional characteristics of the microwells are design parameters that depend on a particular application, including the nature of the reaction taking place, as well as the reagents, byproducts, and labeling techniques (if any) that are employed. Sensor  214  can be an ion-sensitive field-effect transistor (ISFET) with a floating gate structure  218  having sensor plate  220  separated from the microwell interior by ion-sensing layer  216 . Ion-sensing layer  216  may cover the entire microwell or a portion thereof. Ion-sensing layer  216  may cover surfaces between microwells. (See, for example,  FIGS. 6A-B ). Ion-sensing layer  216  may be a metal oxide layer such as, for example and without limitation, silicon nitride, tantalum oxide, aluminum oxide, or a combination thereof. 
     Ion-sensing layer  216 , particularly in a region above floating gate structure  218  and sensor plate  220 , can alter the electrical characteristics of the ISFET so as to modulate a current flowing through a conduction channel of the ISFET. That is, sensor  214  can be responsive to (and generate an output signal related to) the amount of charge  224  present on ion-sensing layer  216  opposite of sensor plate  220 . Changes in charge  224  can cause changes in a current between source  221  and drain  222  of the ISFET. In turn, the ISFET can be used to provide a current-based output signal or indirectly with additional circuitry to provide a voltage-based output signal. Reactants, wash solutions, and other reagents can move in and out of the microwells by a diffusion mechanism  240 . 
     In an embodiment, reactions carried out in microwell  201  can be analytical reactions to identify or determine characteristics or properties of an analyte of interest. Such reactions can generate directly or indirectly byproducts that affect the amount of charge adjacent to sensor plate  220 . If such byproducts are produced in small amounts or rapidly decay or react with other constituents, then multiple copies of the same analyte can be analyzed in microwell  201  at the same time in order to increase the output signal ultimately generated. For instance, multiple copies of an analyte may be attached to solid phase support  212 , either before or after deposition into a microwell. The solid phase support  212  may be a microparticle, nanoparticle, bead, or the like. For nucleic acid analyte, multiple, connected copies may be made by rolling circle amplification (RCA), exponential RCA, and other similar techniques, to produce an amplicon without the need of a solid support. 
       FIGS. 3A-F  are schematic representations of a method of manufacturing a sensor with a protective layer according to one embodiment. Sensor  314  may be an ion sensitive (ISFET) or a chemical sensitive (chemFET) sensor with a floating gate  318  having sensor plate  320  separated from the microwell interior by an ion-sensing layer (not shown), and may be predominantly responsive to (and generate an output signal related to) an amount of charge present on the ion-sensing layer opposite of sensor plate  320 . Changes in the amount of charge cause changes in the current between source  321  and drain  322  of sensor  314 , which may be used directly to provide a current-based output signal or indirectly with additional circuitry to provide a voltage output signal. 
       FIG. 3A  shows a sensor  314  which may be an ion sensitive (ISFET) or a chemical sensitive (chemFET). Substrate  300  may comprise a silicon wafer, or other relevant materials for CMOS fabrication as appropriate. 
       FIG. 3B  shows dielectric layer  330  disposed on sensor  314 . Dielectric layer  330  may comprise silicon oxide, silicon nitride and silicon oxynitride. 
       FIG. 3C  shows protective layer  340  disposed on dielectric layer  330 . Protective layer  340  may comprise a polymer, photoresist material, noble metal, copper oxide, or zinc oxide. 
       FIG. 3D  shows sensor  314  after patterning of protective layer  340 . 
       FIG. 3E  shows cavity  350  extending to the upper surfaces of the floating gate structures and corresponding to sensing surfaces of the chemFETs after etching protective layer  340  and dielectric layer  330 . 
       FIG. 3F  shows sensor  314  with cavity  350  after removal of protective layer  340 . Protective layer  340  may be removed using sodium hydroxide, organic solvent, aqua regia, ammonium carbonate, hydrochloric acid, acetic acid, and phosphoric acid, for example. 
       FIGS. 4A-C  are schematic representations of a sensor with protective layer  440  according to one embodiment. Sensor  414  may be an ion sensitive (ISFET) or a chemical sensitive (chemFET) sensor  414  with floating gate  418  having sensor plate  420  separated from the microwell interior by an ion-sensing layer (not shown, see  FIGS. 6A and 6B  for example), and may be predominantly responsive to (and generate an output signal related to) an amount of charge present on the ion-sensing layer opposite of sensor plate  420 . Changes in the amount of charge cause changes in the current between source  421  and drain  422  of sensor  414 , which may be used directly to provide a current-based output signal or indirectly with additional circuitry to provide a voltage output signal. 
       FIG. 4A  shows sensor  414  with cavity  450  formed for use as a microwell, for example, as described above with reference to  FIG. 1 . Dielectric layer  430  extends to the upper surface of the floating gate structure. Dielectric layer  430  may include at least one of silicon oxide, silicon nitride and silicon oxynitride. Substrate  400  may comprise a silicon wafer, or other relevant materials for CMOS fabrication as appropriate. 
       FIG. 4B  shows a conformal protective layer  440  over the microwell structure of sensor  414 . Protective layer  440  may comprise a polymer, photoresist material, noble metal, copper oxide, or zinc oxide. 
       FIG. 4C  shows sensor  414  with cavity  450  formed for use as a microwell after removal of protective layer  440 . Protective layer  440  serves to protect sensor  414  from environmental contamination and is removed before performing nucleic acid sequencing according to an exemplary embodiment. Protective layer  440  may be removed using at least one of sodium hydroxide, organic solvent, aqua regia, ammonium carbonate, hydrochloric acid, acetic acid, or phosphoric acid. 
       FIG. 5  is flow diagram for performing a method of manufacturing a sensor with a protective layer  500  according to one embodiment. Step  510  includes forming an array of chemically-sensitive field effect transistors (chemFETs). Step  520  includes depositing a dielectric layer over the chemFETs in the array. Step  530  includes depositing a protective layer over the dielectric layer. Step  540  includes etching the dielectric layer and the protective layer to form cavities corresponding to sensing surfaces of the chemFETs. Step  550  includes removing the protective layer. The protective layer serves to protect the sensor from environmental contamination and is removed before performing nucleic acid sequencing according to an exemplary embodiment. 
     In one embodiment, the dielectric layer and the protective layer are etched together to form cavities corresponding to sensing surfaces of the chemFETs. In one embodiment, the protective layer comprises a polymer, photoresist material, noble metal, copper oxide, or zinc oxide. In one embodiment, the protective layer is removed using at least one of sodium hydroxide, organic solvent, aqua regia, ammonium carbonate, hydrochloric acid, acetic acid, or phosphoric acid. In one embodiment, the dielectric layer includes at least one of silicon oxide, silicon nitride and silicon oxynitride. In one embodiment, a further act includes patterning a photosensitive etch mask, wherein the etching of the dielectric layer and the protective layer is a photolithographic process. In one embodiment, the protective layer removal includes removal of photoresist residue contamination resulting from the etching step. 
       FIGS. 6A-B  are schematic representations of exemplary sensors with cavity  650  formed for use as a microwell, for example, as described above with reference to  FIG. 1 . Sensor  614  may be an ion sensitive (ISFET) or a chemical sensitive (chemFET) sensor  614  with floating gate  618  having sensor plate  620  separated from the microwell interior by an ion-sensing layer  616 , and may be predominantly responsive to (and generate an output signal related to) an amount of charge present on the ion-sensing layer opposite of sensor plate  620 . Changes in the amount of charge cause changes in the current between source  621  and drain  622  of sensor  614 , which may be used directly to provide a current-based output signal or indirectly with additional circuitry to provide a voltage output signal. Substrate  600  may comprise a silicon wafer, or other relevant materials for CMOS fabrication as appropriate.