Patent Description:
The present disclosure relates to sensors for chemical analysis, and to methods for manufacturing such sensors. The invention is directed to chemical sensors.

A variety of types of chemical sensors have been used in the detection of chemical processes. One type is a chemically-sensitive field effect transistor (chemFET). A chemFET includes a source and a drain separated by a channel region, and a chemically sensitive area coupled to the channel region. The operation of the chemFET is based on the modulation of channel conductance, caused by changes in charge at the sensitive area due to a chemical reaction occurring nearby. The modulation of the channel conductance changes the threshold voltage of the chemFET, which can be measured to detect and/or determine characteristics of the chemical reaction. The threshold voltage may for example be measured by applying appropriate bias voltages to the source and drain, and measuring a resulting current flowing through the chemFET. As another example, the threshold voltage may be measured by driving a known current through the chemFET, and measuring a resulting voltage at the source or drain.

An ion-sensitive field effect transistor (ISFET) is a type of chemFET that includes an ion-sensitive layer at the sensitive area. The presence of ions in an analyte solution alters the surface potential at the interface between the ion-sensitive layer and the analyte solution, due to the protonation or deprotonation of surface charge groups caused by the ions present in the analyte solution. The change in surface potential at the sensitive area of the ISFET affects the threshold voltage of the device, which can be measured to indicate the presence and/or concentration of ions within the solution.

Arrays of ISFETs may be used for monitoring chemical reactions, such as DNA sequencing reactions, based on the detection of ions present, generated, or used during the reactions. See, for example, <CIT> which is incorporated by reference herein. More generally, large arrays of chemFETs or other types of chemical sensors may be employed to detect and measure static and/or dynamic amounts or concentrations of a variety of analytes (e.g. hydrogen ions, other ions, compounds, etc.) in a variety of processes. The processes may for example be biological or chemical reactions, cell or tissue cultures or monitoring neural activity, nucleic acid sequencing, etc..

An issue that arises in the operation of large scale chemical sensor arrays is the susceptibility of the sensor output signals to noise. Specifically, the noise affects the accuracy of the downstream signal processing used to determine the characteristics of the chemical and/or biological process being detected by the sensors.

It is therefore desirable to provide devices including low noise chemical sensors, and methods for manufacturing such devices.

The invention is directed to an apparatus, comprising a flow cell containing a device comprising a microwell array of reaction regions each coupled to chemical sensors, a row select circuit, a column output circuit, a reference electrode, and an array controller, according to claim <NUM>.

Furthermore, but not forming part of the invention as claimed, a method for manufacturing a device is described. The method includes forming a material defining a reaction region. The method further includes forming a plurality of chemically-sensitive field effect transistors having a common floating gate in communication with the reaction region. The method further includes forming a circuit to obtain individual output signals from the chemically-sensitive field effect transistors indicating an analyte within the reaction region.

Particular aspects of one more implementations of the subject matter described in this specification are set forth in the drawings and the description below.

A chemical detection device is described that includes multiple chemical sensors for concurrently detecting a chemical reaction within the same, operationally associated reaction region. The multiple sensors can provide redundancy, as well as improved accuracy in detecting characteristics of the chemical reaction.

By utilizing multiple chemical sensors to separately detect the same chemical reaction, the individual output signals can be combined or otherwise processed to produce a resultant, low noise output signal. For example, the individual output signals can be averaged, such that the signal-to-noise ratio (SNR) of the resultant output signal is increased by as much as the square root of the number of individual output signals. In addition, the resultant output signal can compensate for differences among the values of the individual output signals, caused by variations in chemical sensor performance which could otherwise complicate the downstream signal processing. As a result of the techniques described herein, low-noise chemical sensor output signals can be provided, such that the characteristics of reactions can be accurately detected.

<FIG> illustrates a block diagram of components of a system for nucleic acid sequencing according to an exemplary embodiment. The components include a flow cell <NUM> on an integrated circuit device <NUM>, a reference electrode <NUM>, a plurality of reagents <NUM> for sequencing, a valve block <NUM>, a wash solution <NUM>, a valve <NUM>, a fluidics controller <NUM>, lines <NUM>/<NUM>/<NUM>, passages <NUM>/<NUM>/<NUM>, a waste container <NUM>, an array controller <NUM>, and a user interface <NUM>. The integrated circuit device <NUM> includes a microwell array <NUM> of reaction regions overlying groups of chemical sensors of a sensor array as described herein. The flow cell <NUM> includes an inlet <NUM>, an outlet <NUM>, and a flow chamber <NUM> defining a flow path of reagents over the microwell array <NUM>.

The reference electrode <NUM> 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 <NUM>. The reagents <NUM> may be driven through the fluid pathways, valves, and flow cell <NUM> by pumps, gas pressure, or other suitable methods, and may be discarded into the waste container <NUM> after exiting the outlet <NUM> of the flow cell <NUM>. The fluidics controller <NUM> may control driving forces for the reagents <NUM> and the operation of valve <NUM> and valve block <NUM> with suitable software.

The microwell array <NUM> includes reaction regions, also referred to herein as microwells, which are operationally associated with chemical sensors of the sensor array. As described in more detail below, each reaction region is operationally associated with multiple chemical sensors suitable for detecting an analyte or reaction of interest within that reaction region. These multiple chemical sensors can provide redundancy, as well as improved detection accuracy. The microwell array <NUM> is integrated in the integrated circuit device <NUM>, so that the microwell array <NUM> and the sensor array are part of a single device or chip.

In exemplary embodiments described below, groups of four chemical sensors are coupled to each of the reaction regions. Alternatively, the number of chemical sensors operationally associated with a single reaction region may be different than four. More generally, two or more chemical sensors may be operationally associated with a single reaction region.

The flow cell <NUM> may have a variety of configurations for controlling the path and flow rate of reagents <NUM> over the microwell array <NUM>. The array controller <NUM> provides bias voltages and timing and control signals to the integrated circuit device <NUM> for reading the chemical sensors of the sensor array as described herein. The array controller <NUM> also provides a reference bias voltage to the reference electrode <NUM> to bias the reagents <NUM> flowing over the microwell array <NUM>.

During an experiment, the array controller <NUM> collects and processes individual output signals from the chemical sensors of the sensor array through output ports on the integrated circuit device <NUM> via bus <NUM>. As described in more detail below, this processing can include calculating a resultant output signal for a group of sensors as a function of the individual output signals from the chemical sensors in the group. The array controller <NUM> may be a computer or other computing means. The array controller <NUM> may include memory for storage of data and software applications, a processor for accessing data and executing applications, and components that facilitate communication with the various components of the system in <FIG>.

In the illustrated embodiment, the array controller <NUM> is external to the integrated circuit device <NUM>. In some alternative embodiments, some or all of the functions performed by the array controller <NUM> are carried out by a controller or other data processor on the integrated circuit device <NUM>. In yet other embodiments, a combination of resources internal and external to the integrated circuit device <NUM> is used to obtain the individual output signals and calculate the resultant output signal for a group of sensors using the techniques described herein.

The value of a resultant output signal for a group of chemical sensors indicates physical and/or chemical characteristics of one or more reactions taking place in the corresponding reaction region. For example, in an exemplary embodiment, the values of the resultant output signals may be further processed using the techniques disclosed in <CIT>, and in No. <CIT>.

The user interface <NUM> may display information about the flow cell <NUM> and the output signals received from chemical sensors of the sensor array on the integrated circuit device <NUM>. The user interface <NUM> may also display instrument settings and controls, and allow a user to enter or set instrument settings and controls.

The fluidics controller <NUM> may control delivery of the individual reagents <NUM> to the flow cell <NUM> and integrated circuit device <NUM> in a predetermined sequence, for predetermined durations, at predetermined flow rates. The array controller <NUM> can then collect and analyze the output signals of the chemical sensors indicating chemical reactions occurring in response to the delivery of the reagents <NUM>.

During the experiment, the system may also monitor and control the temperature of the integrated circuit device <NUM>, so that reactions take place and measurements are made at a known predetermined temperature.

The system may be configured to let a single fluid or reagent contact the reference electrode <NUM> throughout an entire multi-step reaction during operation. The valve <NUM> may be shut to prevent any wash solution <NUM> from flowing into passage <NUM> as the reagents <NUM> are flowing. Although the flow of wash solution may be stopped, there may still be uninterrupted fluid and electrical communication between the reference electrode <NUM>, passage <NUM>, and the microwell array <NUM>. The distance between the reference electrode <NUM> and the junction between passages <NUM> and <NUM> may be selected so that little or no amount of the reagents flowing in passage <NUM> and possibly diffusing into passage <NUM> reach the reference electrode <NUM>. In an exemplary embodiment, the wash solution <NUM> may be selected as being in continuous contact with the reference electrode <NUM>, which may be especially useful for multi-step reactions using frequent wash steps.

<FIG> illustrates cross-sectional and expanded views of a portion of the integrated circuit device <NUM> and flow cell <NUM>. The integrated circuit device <NUM> includes the microwell array <NUM> of reaction regions operationally associated with sensor array <NUM>. During operation, the flow chamber <NUM> of the flow cell <NUM> confines a reagent flow <NUM> of delivered reagents across open ends of the reaction regions in the microwell array <NUM>. The volume, shape, aspect ratio (such as base width-to-well depth ratio), and other dimensional characteristics of the reaction regions may be selected based on the nature of the reaction taking place, as well as the reagents, byproducts, or labeling techniques (if any) that are employed.

The chemical sensors of the sensor array <NUM> are responsive to (and generate output signals related to) chemical reactions within associated reaction regions in the microwell array <NUM> to detect an analyte of interest. The chemical sensors of the sensor array <NUM> are chemically sensitive field-effect transistors (chemFETs), such as ion-sensitive field effect transistors (ISFETs). Examples of chemical sensors and array configurations that may be used in embodiments are described in <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, and <CIT>, and <CIT>. Devices in which multiple ISFETs share a common floating gate are described in International Patent Application Publication No. <CIT>.

<FIG> illustrates a schematic diagram of a portion of the integrated circuit device <NUM> including sensor array <NUM> having multiple chemical sensors coupled to the same reaction region. In the illustrated embodiment, sixteen chemical sensors and four reaction regions are illustrated, representing a small section of the sensor array <NUM> and microwell array <NUM> that can include millions of chemical sensors and reaction regions.

The integrated circuit device <NUM> includes an access circuit for accessing the chemical sensors of the sensor array <NUM>. In the illustrated example, the access circuit includes a row select circuit <NUM> coupled to the sensor array <NUM> via row lines <NUM> - <NUM>. The access circuit also includes column output circuit <NUM> coupled to the sensor array <NUM> via column lines <NUM> - <NUM>.

The row select circuit <NUM> and the column output circuit <NUM> are responsive to timing and control signals provided by the array controller <NUM> in <FIG> to select the various chemical sensors and operate the sensor array <NUM> as described below. The array controller <NUM> also provides a reference bias voltage to the reference electrode (See, <FIG>, reference numeral <NUM>) to bias the reagents flowing across open ends of the reaction regions <NUM>, <NUM>, <NUM>, <NUM> of the microwell array <NUM> during operation.

In the illustrated embodiment, groups of four chemical sensors are operationally associated with each of the reaction regions <NUM>, <NUM>, <NUM>, <NUM>. Alternatively, the number of chemical sensors operationally associated with a single reaction region may be different than four. More generally, two or more chemical sensors may be operationally associated with a single reaction region. In some embodiments, the number of chemical sensors operationally associated with a single reaction region may be greater than four, such as sixteen or more.

The group containing chemical sensors <NUM> - <NUM> is representative of the groups of sensors of the sensor array <NUM>. Each chemical sensor <NUM> - <NUM> includes a chemically-sensitive field effect transistor <NUM> - <NUM> and a row select switch <NUM> - <NUM>.

The chemically-sensitive field effect transistors <NUM> - <NUM> have a common floating gate <NUM> in communication with the reaction region <NUM>. That is, the common floating gate <NUM> is coupled to channels of each of the chemically-sensitive field effect transistors <NUM> - <NUM>. Thre chemically-sensitive field effect transistors <NUM> - <NUM> may each include multiple patterned layers of conductive elements within layers of dielectric material.

The common floating gate <NUM> may for example include an uppermost conductive element (referred to herein as a sensor plate) that defines a surface (e.g. a bottom surface) of the reaction region <NUM>. That is, there is no intervening deposited material layer between the uppermost electrical conductor and the surface of the reaction region <NUM>. In some alternative embodiments, the uppermost conductive element of the common floating gate <NUM> is separated from the reaction region <NUM> by a deposited sensing material (discussed in more detail below).

In operation, reactants, wash solutions, and other reagents may move in and out of the reaction region <NUM> by a diffusion mechanism. The chemical sensors <NUM> - <NUM> are each responsive to (and generate individual output signals related to) chemical reactions within the reaction region <NUM> to detect an analyte or reaction property of interest. Changes in the charge within the reaction region <NUM> cause changes in the voltage on the common floating gate <NUM>, which in turn changes the individual threshold voltages of each of the chemically-sensitive field effect transistors <NUM> - <NUM> of the sensors <NUM>-<NUM>.

In a read operation of a selected chemical sensor <NUM>, the row select circuit <NUM> facilitates providing a bias voltage to row line <NUM> sufficient to turn on row select transistor <NUM>. Turning on the row select transistor <NUM> couples the drain terminal of the chemically-sensitive transistor <NUM> to the column line <NUM>. The column output circuit <NUM> facilitates providing a bias voltage to the column line <NUM>, and providing a bias current on the column line <NUM> that flows through the chemically-sensitive transistor <NUM>. This in turn establishes a voltage at the source terminal of the chemically-sensitive transistor <NUM>, which is coupled to the column line <NUM>. In doing so, the voltage on the column line <NUM> is based on the threshold voltage of the chemically-sensitive transistor <NUM>, and thus based on the amount of charge within the reaction region <NUM>. Alternatively, other techniques may be used to read the selected chemical sensor <NUM>.

The column output circuit <NUM> produces an individual output signal for the chemically-sensitive transistor <NUM> based on the voltage on the column line <NUM>. The column output circuit <NUM> may include switches, sample and hold capacitors, current sources, buffers, and other circuitry used to operate and read the chemical sensors, depending upon the array configuration and read out technique. In some embodiments, the column output circuit <NUM> may include circuits such as those described in <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, and <CIT>, and <CIT>.

The individual output signals of the other chemical sensors <NUM> - <NUM> coupled to the reaction region <NUM> can be read out in a similar fashion. In doing so, the column output circuit <NUM> produces individual output signals for each of the chemical sensors <NUM> - <NUM>.

The individual output signals for each of the chemical sensors <NUM>-<NUM> can then be combined or otherwise processed by the array controller <NUM> (or other data processor) to calculate a resultant, low noise output signal for the group of chemical sensors <NUM>-<NUM>. For example, the resultant output signal may be an average of the individual output signals. In such a case, the SNR of the resultant output signal can be increased by as much as the square root of the number of individual output signals. In addition, the resultant output signal can compensate for differences among the values of the individual output signals, caused by variations in performance of the chemical sensors <NUM>-<NUM> which could otherwise complicate the downstream signal processing.

<FIG> is a flow chart of an example process for calculating a resultant output signal for a group of chemical sensors coupled to a single reaction region. Other embodiments may perform different or additional steps than the ones illustrated in <FIG>. For convenience, <FIG> will be described with reference to a system that performs a process. The system can be for example, the system of <FIG>.

At step <NUM>, a chemical reaction is initiated within a reaction region coupled to a group of two or more chemical sensors. The group of chemical sensors includes respective chemically-sensitive field effect transistors having a common floating gate in communication with the reaction region, as described above with respect to <FIG>. The chemical reaction may be a sequencing reaction, as described above.

At step <NUM>, individual output signals are obtained from the chemical sensors in the group. The individual output signals may for example be obtained by selecting and reading out the individual chemical sensors using the techniques described above. In some embodiments, flowing of reagent(s) causes chemical reactions within the reaction region that release hydrogen ions, and the amplitude of the individual output signals from the chemical sensors is related to the amount of hydrogen ions detected.

At step <NUM>, a resultant output signal for the group is calculated based on one or more of the individual output signals. The resultant output signal may for example be an average of the individual output signals. Alternatively, other techniques may be used to calculate the resultant output signal.

At step <NUM>, a characteristic of the chemical reaction is determined based on the resultant output signal. For example, the characteristic of the chemical reaction may be determined based on the value of the resultant output signal using the techniques disclosed in <CIT>, and in No. <CIT>.

<FIG> illustrates a cross-sectional view of portions of two groups of chemical sensors and their corresponding reaction regions according to a first embodiment. In <FIG>, the chemically-sensitive field effect transistors <NUM>, <NUM> of the chemical sensors <NUM>, <NUM> in the group of sensors <NUM>-<NUM> coupled to the reaction region <NUM> are visible. The chemically-sensitive field effect transistors <NUM>, <NUM> of the other chemical sensors <NUM>, <NUM> in the group lie behind this cross-section. Similarly, the cross-section of <FIG> shows the chemically-sensitive field effect transistors of two chemical sensors in the group of chemical sensors that is coupled to the adjacent reaction region <NUM>. In this illustration, the select switches of the chemical sensors, access lines and other connections are omitted for simplicity.

The chemical sensor <NUM> is representative of the group of chemical sensors <NUM>-<NUM>. In the illustrated example, the chemically-sensitive field effect transistor <NUM> of the chemical sensor <NUM> is an ion-sensitive field effect transistor (ISFET) in this example.

The chemically-sensitive field effect transistor <NUM> includes common floating gate <NUM> having a conductive element <NUM> coupled to the reaction region <NUM>. The conductive element <NUM> is the uppermost floating gate conductor (also referred to herein as a sensor plate) in the common floating gate <NUM>. In the embodiment illustrated in <FIG>, the common floating gate <NUM> includes multiple patterned layers of conductive material within layers of dielectric material <NUM>.

In <FIG>, the conductive element <NUM> electrically connects individual multilayer floating gate structures that extend over the channel regions of each of the chemically-sensitive field effect transistors <NUM> - <NUM> of the group of chemical sensors <NUM>-<NUM>. In doing so, the common floating gate <NUM> is shared among the chemical sensors <NUM>-<NUM>.

The chemically-sensitive field effect transistor <NUM> includes a source region <NUM> and a drain region <NUM> within a semiconductor substrate <NUM>. The source region <NUM> and the drain region <NUM> comprise doped semiconductor material having a conductivity type different from the conductivity type of the substrate <NUM>. For example, the source region <NUM> and the drain region <NUM> may comprise doped P-type semiconductor material, and the substrate may comprise doped N-type semiconductor material.

Channel region <NUM> separates the source region <NUM> and the drain region <NUM>. The common floating gate <NUM> includes a conductive element <NUM> separated from the channel region <NUM> by a gate dielectric <NUM>. The gate dielectric <NUM> may be for example silicon dioxide. Alternatively, other dielectrics may be used for the gate dielectric <NUM>.

As shown in <FIG>, the reaction region <NUM> is within an opening extending through dielectric material <NUM> to the upper surface of the conductive element <NUM>. The dielectric material <NUM> may comprise one or more layers of material, such as silicon dioxide or silicon nitride. The opening in the dielectric material <NUM> may for example have a circular cross-section. Alternatively, the opening may be non-circular. For example, the cross-section may be square, rectangular, hexagonal, or irregularly shaped. The dimensions of the openings within the dielectric material <NUM>, and their pitch, can vary from embodiment to embodiment.

In the illustrated embodiment, the upper surface <NUM> of the conductive element <NUM> is the bottom surface of the reaction region <NUM>. That is, there is no intervening deposited material layer between the upper surface <NUM> of the conductive element <NUM> and the reaction region <NUM>. As a result of this structure, the upper surface <NUM> of the conductive element <NUM> acts as the sensing surface for the group of chemical sensors <NUM>-<NUM>. The conductive element <NUM> may comprise one or more of a variety of different materials to facilitate sensitivity to particular ions (e.g. hydrogen ions).

During manufacturing and/or operation of the device, a thin oxide of the electrically conductive material of the conductive element <NUM> may be grown on the upper surface <NUM> which acts as a sensing material (e.g. an ion-sensitive sensing material) for the group of chemical sensors <NUM>-<NUM>. For example, in one embodiment the conductive element <NUM> may be titanium nitride, and titanium oxide or titanium oxynitride may be grown on the upper surface <NUM> during manufacturing and/or during exposure to solutions during use. Whether an oxide is formed depends on the conductive material, the manufacturing processes performed, and the conditions under which the device is operated.

In the illustrated example, the conductive element <NUM> is shown as a single layer of material. More generally, the conductive element <NUM> may comprise one or more layers of a variety of electrically conductive materials, such as metals or ceramics, depending upon the embodiment. The conductive material can be for example a metallic material or alloy thereof, or can be a ceramic material, or a combination thereof. An exemplary metallic material includes one of aluminum, copper, nickel, titanium, silver, gold, platinum, hafnium, lanthanum, tantalum, tungsten, iridium, zirconium, palladium, or a combination thereof. An exemplary ceramic material includes one of titanium nitride, titanium aluminum nitride, titanium oxynitride, tantalum nitride, or a combination thereof.

In some alternative embodiments, an additional conformal sensing material (not shown) is deposited on the sidewall of the opening in the dielectric material <NUM> and on the upper surface <NUM> of the sensor plate <NUM>. In such a case, an inner surface of the deposited sensing material defines the reaction region <NUM>. The sensing material may comprise one or more of a variety of different materials to facilitate sensitivity to particular ions. For example, silicon nitride or silicon oxynitride, as well as metal oxides such as silicon oxide, aluminum or tantalum oxides, generally provide sensitivity to hydrogen ions, whereas sensing materials comprising polyvinyl chloride containing valinomycin provide sensitivity to potassium ions. Materials sensitive to other ions such as sodium, silver, iron, bromine, iodine, calcium, and nitrate may also be used, depending upon the embodiment.

In operation, reactants, wash solutions, and other reagents may move in and out of the reaction region <NUM> by a diffusion mechanism <NUM>. Each of the chemical sensors <NUM>-<NUM> are responsive to (and generates an output signal related to) the amount of charge <NUM> proximate to the conductive element <NUM>. The presence of charge <NUM> in an analyte solution alters the surface potential at the interface between the analyte solution and the conductive element <NUM>, due to the protonation or deprotonation of surface charge groups. Changes in the charge <NUM> cause changes in the voltage on the floating gate structure <NUM>, which in turn changes in the threshold voltages of the chemically-sensitive transistors <NUM>-<NUM> of each of the chemical sensors <NUM>-<NUM>. The respective changes in threshold voltages can be measured by measuring the current through the respective channel regions (e.g. channel region <NUM> of sensor <NUM>). As a result, each of the chemical sensors <NUM>-<NUM> can be operated to provide individual current-based or voltage-based output signals on an array line connected to its corresponding source region or drain region.

In an embodiment, reactions carried out in the reaction region <NUM> can be analytical reactions to identify or determine characteristics or properties of an analyte of interest. Such reactions can directly or indirectly generate byproducts that affect the amount of charge <NUM> adjacent to the conductive element <NUM>. If such byproducts are produced in small amounts or rapidly decay or react with other constituents, multiple copies of the same analyte may be analyzed in the reaction region <NUM> at the same time in order to increase the individual output signals generated by the group of chemical sensors <NUM>-<NUM>. In an embodiment, multiple copies of an analyte may be attached to a solid phase support <NUM>, either before or after deposition into the reaction region <NUM>. The solid phase support <NUM> may be microparticles, nanoparticles, beads, solid or porous gels, or the like. For simplicity and ease of explanation, solid phase support <NUM> is also referred herein as a particle. For a nucleic acid analyte, multiple, connected copies may be made by rolling circle amplification (RCA), exponential RCA, Recombinase Polymerase Amplification (RPA), Polymerase Chain Reaction amplification (PCR), emulsion PCR amplification, or like techniques, to produce an amplicon without the need of a solid support.

The methods, systems, and computer readable media described herein may advantageously be used to process and/or analyze data and signals obtained from electronic or charged-based nucleic acid sequencing. In electronic or charged-based sequencing (such as, pH-based sequencing), a nucleotide incorporation event may be determined by detecting ions (e.g., hydrogen ions) that are generated as natural by-products of polymerase-catalyzed nucleotide extension reactions. This may be used to sequence a sample or template nucleic acid, which may be a fragment of a nucleic acid sequence of interest, for example, and which may be directly or indirectly attached as a clonal population to a solid support, such as a particle, microparticle, bead, etc. The sample or template nucleic acid may be operably associated to a primer and polymerase and may be subjected to repeated cycles or "flows" of deoxynucleoside triphosphate ("dNTP") addition (which may be referred to herein as "nucleotide flows" from which nucleotide incorporations may result) and washing. The primer may be annealed to the sample or template so that the primer's <NUM>' end can be extended by a polymerase whenever dNTPs complementary to the next base in the template are added. Then, based on the known sequence of nucleotide flows and on measured output signals from the chemical sensors indicative of ion concentration during each nucleotide flow, the identity of the type, sequence and number of nucleotide(s) associated with a sample nucleic acid present in a reaction region coupled to a group of chemical sensors can be determined.

<FIG> illustrate stages in a manufacturing process for forming a device including multiple chemical sensors coupled to the same reaction region according to a first embodiment. The manufacturing process however does not form part of the invention as claimed.

<FIG> illustrates a structure <NUM> formed in a first stage. The structure <NUM> includes partially completed floating gate structures for the field effect transistors of the chemical sensors. For example, the structure <NUM> includes partially completed floating gate structure <NUM> for the chemically-sensitive field effect transistor <NUM> of the chemical sensor <NUM>.

The structure <NUM> can be formed by depositing a layer of gate dielectric material on the semiconductor substrate <NUM>, and depositing a layer of polysilicon (or other electrically conductive material) on the layer of gate dielectric material. The layer of polysilicon and the layer gate dielectric material can then be etched using an etch mask to form the gate dielectric elements (e.g. gate dielectric <NUM>) and the lowermost conductive material element (e.g. conductive element <NUM>) of the floating gate structures. Following formation of an ion-implantation mask, ion implantation can then be performed to form the source and drain regions (e.g. source region 521and a drain region <NUM>) of the chemical sensors.

A first layer of the dielectric material <NUM> can then be deposited over the lowermost conductive material elements. Conductive plugs can then be formed within vias etched in the first layer of dielectric material <NUM> to contact the lowermost conductive material elements of the floating gate structures. A layer of conductive material can then be deposited on the first layer of the dielectric material <NUM> and patterned to form second conductive material elements electrically connected to the conductive plugs. This process can then be repeated multiple times to form the partially completed floating gate structures shown in <FIG>. Alternatively, other and/or additional techniques may be performed to form the structure.

Forming the structure <NUM> in <FIG> can also include forming additional elements such as array lines (e.g. row lines, column lines, etc.) for accessing the chemical sensors, additional doped regions in the substrate <NUM>, and other circuitry (e.g. select switches, access circuitry, bias circuitry etc.) used to operate the chemical sensors, depending upon the device and array configuration in which the chemical sensors are implemented.

Next, conductive material <NUM> is formed on the structure illustrated in <FIG> to contact the partially completed floating gate structures. An etch mask including mask elements <NUM>, <NUM> is then formed on the conductive material <NUM>, resulting in the structure illustrated in <FIG>.

The conductive material <NUM> includes one or more layers of electrically conductive material. For example, the conductive material <NUM> may include a layer of titanium nitride formed on a layer of aluminum, or a layer of titanium nitride formed on a layer of copper. Alternatively, the number of layers may be different than two, and other and/or additional conductive materials may be used. Examples of conductive materials that can be used in some embodiments include tantalum, aluminum, lanthanum, titanium, zirconium, hafnium, tungsten, palladium, iridium, etc., and combinations thereof.

The locations of the mask elements <NUM>, <NUM> define the locations of the sensor plates for the chemically-sensitive field effect transistors of the corresponding groups of chemical sensors. In the illustrated embodiment, the mask elements <NUM>, <NUM> comprise photoresist material which has been patterned using a lithographic process. Alternatively, other techniques and materials may be used.

Next, the conductive material <NUM> is etched using the mask elements <NUM>, <NUM> as a mask, resulting in the structure illustrated in <FIG>. The etching process forms conductive elements <NUM>, <NUM> in electrical contact with the partially completed floating gate structures of a corresponding group of sensors. The conductive element <NUM> electrically connects the partially completed floating gate structures for a group of sensors <NUM>-<NUM>, to complete the common floating gate <NUM> for this group of sensors. Similarly, the conductive material element <NUM> completes the common floating gate for the adjacent group of sensors.

Next, the mask elements <NUM>, <NUM> are removed and dielectric material <NUM> is formed, resulting in the structure illustrated in <FIG>. The dielectric material <NUM> may comprise one or more layers of deposited dielectric material, such as silicon dioxide or silicon nitride.

Next, the dielectric material <NUM> is etched to form openings defining reaction regions <NUM>, <NUM> extending to upper surfaces of the conductive material elements <NUM>, <NUM>, resulting in the structure illustrated in <FIG>.

<FIG> illustrates a cross-sectional view of portions of two groups of chemical sensors and their corresponding reaction regions according to a second embodiment. In contrast to the embodiment shown in <FIG>, the common floating gate for each group of chemical sensors includes a sensor plate that is smaller than the bottom surface of the corresponding reaction region.

In <FIG>, the floating gate structure <NUM> for the group of chemical sensors <NUM>-<NUM> includes conductive element <NUM> coupled to the reaction region <NUM>. The conductive element <NUM> is coupled to the conductive element <NUM> by conductive plug <NUM>. The conductive element <NUM> is the uppermost floating gate conductor in the floating gate structure <NUM>, and thus acts as the sensor plate for the group of chemical sensors <NUM>-<NUM>.

In <FIG>, an upper surface <NUM> of the conductive element <NUM> is a portion of the bottom surface of the reaction region <NUM>. That is, there is no intervening deposited material layer between the upper surface <NUM> of the conductive element <NUM> and the reaction region <NUM>. As a result of this structure, the upper surface <NUM> of the conductive element <NUM> acts as the sensing surface for the group of chemical sensors <NUM>-<NUM>. In the illustrated embodiment, the conductive element <NUM> is within the dielectric material <NUM>, such that the upper surface <NUM> of the conductive element <NUM> is co-planar with the upper surface of the dielectric material <NUM>. Alternatively, the conductive element <NUM> may be formed on the upper surface of dielectric material <NUM>, and thus protrude slightly into the reaction region <NUM>.

As shown in <FIG>, the upper surface <NUM> of the conductive element <NUM> has a width <NUM> that is the less than the width of the bottom surface of the reaction region <NUM>. As described in more detail below, having a small conductive element <NUM> as the sensor plate can enable the signal-to-noise ratio (SNR) of the individual output signals of the chemical sensors <NUM>-<NUM> to be maximized.

The amplitude of the desired signal detected by the chemical sensors <NUM>-<NUM> in response to the charge <NUM> in an analyte solution is a superposition of the charge concentration along the interface between the conductive element <NUM> and the analyte solution. Because the charge <NUM> is more highly concentrated at the bottom and middle of the reaction region <NUM>, the width <NUM> of the conductive element <NUM> is a tradeoff between the amplitude of the desired signal detected in response to the charge <NUM>, and the fluidic noise due to random fluctuation between the conductive element <NUM> and the analyte solution. Increasing the width <NUM> of the conductive element <NUM> increases the fluidic interface area for the chemical sensors <NUM>-<NUM>, which reduces fluidic noise. However, since the localized surface density of charge <NUM> decreases with distance from the middle of the reaction region <NUM>, the conductive element <NUM> detects a greater proportion of the signal from areas having lower charge concentration, which can reduce the overall amplitude of the detected signal. In contrast, decreasing the width <NUM> of the conductive element <NUM> reduces the sensing surface area and thus increases the fluidic noise, but also increases the overall amplitude of the detected signal.

For a very small sensing surface area, Applicants have found that the fluidic noise changes as a function of the sensing surface area differently than the amplitude of the desired signal. Because the SNR of an individual output signal is the ratio of these two quantities, there is an optimal width <NUM> at which the SNR of the individual output signals from the chemical sensors <NUM>-<NUM> is maximum.

The optimal width <NUM> can vary from embodiment to embodiment depending on the material characteristics of the conductive element <NUM> and the dielectric materials <NUM>, <NUM>, the volume, shape, aspect ratio (such as base width-to-well depth ratio), and other dimensional characteristics of the reaction regions, the nature of the reaction taking place, as well as the reagents, byproducts, or labeling techniques (if any) that are employed. The optimal width may for example be determined empirically.

<FIG> illustrate stages in a manufacturing process (not forming part of the invention as claimed) for forming a device including multiple chemical sensors coupled to the same reaction region according to a second embodiment.

<FIG> illustrates a first stage of forming conductive plugs <NUM>, <NUM> extending through dielectric material <NUM> to contact the conductive elements <NUM>, <NUM> of the structure illustrated in <FIG>. The structure in <FIG> can be formed by removing the mask elements <NUM>, <NUM> in <FIG> and forming dielectric material <NUM> on the resulting structure. Vias can then be etched through the dielectric material <NUM>, and metal deposited within the vias. A planarization process (e.g. chemical mechanical polishing) can then be performed to remove the deposited metal from the upper surface of the dielectric material <NUM> and form the plugs <NUM>, <NUM>. Alternatively, other techniques may be used.

Next, conductive material <NUM> is formed on the structure illustrated in <FIG>. An etch mask including mask elements <NUM>, <NUM> is then formed on the conductive material <NUM>, resulting in the structure illustrated in <FIG>.

The conductive material <NUM> may comprise one or more layers of conductive material, such as those described above with respect the conductive material <NUM> of <FIG>. The locations of the mask elements <NUM>, <NUM> define the locations of the sensor plates of the field effect transistors of the corresponding groups of chemical sensors. In the illustrated embodiment, the mask elements <NUM>, <NUM> comprise photoresist material which has been patterned using a lithographic process. Alternatively, other techniques and materials may be used.

Next, the conductive material <NUM> is etched using the mask elements <NUM>, <NUM> as a mask to form the conductive elements <NUM>, <NUM>. Dielectric material <NUM> is then formed between the conductive elements <NUM>, <NUM>, resulting in the structure illustrated in <FIG>.

Next, dielectric material <NUM> is formed on the structure illustrated in <FIG>. The dielectric material <NUM> is then be etched to form openings defining reaction regions <NUM>, <NUM> extending to upper surfaces of the conductive elements <NUM>, <NUM>, resulting in the structure illustrated in <FIG>. The dielectric material <NUM> may comprise material different than that of dielectric material <NUM>. For example, the dielectric material <NUM> may comprise material (e.g. silicon oxide) which can be selectively etched relative to the material (e.g. silicon nitride) of the dielectric material <NUM> when subjected to a chosen etch process. In such a case, the dielectric material <NUM> can act as an etch stop during the etching process used to form the reaction regions <NUM>, <NUM>. In doing so, the dielectric material <NUM> can prevent etching past the conductive elements <NUM>, <NUM>, and thus can define and maintain the desired shape of the reaction regions <NUM>, <NUM>.

<FIG> illustrate stages in a manufacturing process (not forming part of the invention as claimed) for forming a device including multiple chemical sensors coupled to the same reaction region according to a third embodiment.

<FIG> illustrates a first stage of forming dielectric material <NUM> on the structure illustrated in <FIG>. An etch mask including mask elements <NUM>, <NUM>, <NUM> is then formed on the dielectric material <NUM>, resulting in the structure illustrated in <FIG>. As described in more detail below, openings between the mask elements <NUM>, <NUM>, <NUM> define the locations of the sensor plates of the field effect transistors of the corresponding groups of chemical sensors.

Next, the dielectric material <NUM> is etched using the mask elements <NUM>, <NUM>, <NUM> as an etch mask to form openings <NUM>, <NUM> within the dielectric material <NUM>, resulting in the structure illustrated in <FIG>. As shown in <FIG>, the openings extend to the upper surfaces of the conductive plugs <NUM>, <NUM>.

Next, the mask elements <NUM>, <NUM>, <NUM> are removed and conductive material <NUM> is deposited on the structure illustrated in <FIG>, resulting in the structure illustrated in <FIG>. The conductive material <NUM> may comprise one or more layers of conductive material, such as those described above with respect the conductive material <NUM> of <FIG>.

Next, a planarization process (e.g. CMP) is performed to remove the conductive material <NUM> from the upper surface of the dielectric material <NUM>, resulting in the structure illustrated in <FIG>. The planarization process leaves remaining conductive material within the openings <NUM>, <NUM> to form the conductive elements <NUM>, <NUM>.

Next, dielectric material <NUM> is formed on the structure illustrated in <FIG>. The dielectric material <NUM> can then be etched to form openings defining reaction regions <NUM>, <NUM> extending to upper surfaces of the conductive elements <NUM>, <NUM>, resulting in the structure illustrated in <FIG>.

<FIG> illustrate stages in a manufacturing process (not forming part of the invention as claimed) for forming a device including multiple chemical sensors coupled to the same reaction region according to a fourth embodiment.

<FIG> illustrates a first stage of forming conductive plugs <NUM>, <NUM> extending through dielectric material <NUM> to contact the conductive elements <NUM>, <NUM> of the structure illustrated in <FIG>. As described in more detail below, the dielectric material <NUM>, comprising one or more layers of dielectric material, acts an etch stop during the subsequent formation of the reaction regions <NUM>, <NUM>. The structure in <FIG> can be formed by removing the mask elements <NUM>, <NUM> illustrated in <FIG> and forming the dielectric material <NUM> on the resulting structure. The plugs <NUM>, <NUM> can then be formed using the techniques described above with reference to <FIG>. Alternatively, other techniques may be used.

Next, conductive elements <NUM>, <NUM> are formed on the upper surface of the dielectric material <NUM>, resulting in the structure illustrated in <FIG>. The conductive elements <NUM>, <NUM> may be formed by depositing conductive material, forming an etch mask including mask elements defining the locations of the conductive elements <NUM>, <NUM>, and etching the conductive material using the mask elements as an etch mask.

Next, dielectric material <NUM> is formed on the structure illustrated in <FIG>. The dielectric material <NUM> is then be etched to form openings defining reaction regions <NUM>, <NUM> extending to upper surfaces of the conductive elements <NUM>, <NUM>, resulting in the structure illustrated in <FIG>. As shown in <FIG>, in this embodiment the reaction regions <NUM>, <NUM> extend below the upper surfaces of the conductive elements <NUM>, <NUM> to expose their side surfaces.

The dielectric material <NUM> may comprise material different than that of dielectric material <NUM>. For example, the dielectric material <NUM> may comprise material (e.g. silicon oxide) which can be selectively etched relative to the material (e.g. silicon nitride) of the dielectric material <NUM> when subjected to a chosen etch process. In such a case, the dielectric material <NUM> can act as an etch stop during the etching process used to form the reaction regions <NUM>, <NUM>. In doing so, the dielectric material <NUM> can prevent etching below the conductive elements <NUM>, <NUM>, and thus can define and maintain the shape of the reaction regions <NUM>, <NUM>.

Various aspects of the embodiments may be implemented using hardware elements, software elements, or a combination of both.

Some aspects of the embodiments may be implemented, for example, using a computer-readable medium or article which may store an instruction or a set of instructions that, if executed by a machine, may cause the machine to perform a method and/or operations in accordance with said aspects of the embodiments. Such a machine may include, for example, any suitable processing platform, computing platform, computing device, processing device, computing system, processing system, computer, processor, or the like, and may be implemented using any suitable combination of hardware and/or software. The computer-readable medium or article may include, for example, any suitable type of memory unit, memory device, memory article, memory medium, storage device, storage article, storage medium and/or storage unit, for example, memory, removable or nonremovable media, erasable or non-erasable media, writeable or re-writeable media, digital or analog media, hard disk, floppy disk, read-only memory compact disc (CD-ROM), recordable compact disc (CD-R), rewriteable compact disc (CD-RW), optical disk, magnetic media, magneto-optical media, removable memory cards or disks, various types of Digital Versatile Disc (DVD), a tape, a cassette, or the like. The instructions may include any suitable type of code, such as source code, compiled code, interpreted code, executable code, static code, dynamic code, encrypted code, and the like, implemented using any suitable high-level, low-level, object-oriented, visual, compiled and/or interpreted programming language.

Claim 1:
An apparatus, comprising:
a flow cell (<NUM>) containing a device (<NUM>) comprising a microwell array (<NUM>) of reaction regions (<NUM>; <NUM>, <NUM>; <NUM>); wherein each reaction region is coupled to a group of two or more chemical sensors (<NUM> - <NUM>), wherein each chemical sensor comprises a chemically sensitive field effect transistor, chemFET (<NUM> - <NUM>), and a row select transistor (<NUM> - <NUM>), wherein each group of two or more chemFETs has a common floating gate (<NUM>) in communicatior with a respective reaction region;
a row select circuit (<NUM>) for providing a bias voltage to each row select transistor of each chemFET sensor of the group of two or more chemFET sensors for each reaction region in the array of reaction regions, wherein the bias voltage is sufficient to turn on the row select transistor to couple the respective chemFET sensor to a column line (<NUM> - <NUM>);
a column output circuit (<NUM>) for providing a bias current to a respective column line for each chemFET sensor of the group of two or more chemFET sensors for each reaction region in the array of reaction regions in response to biasing each respective row select transistor for producing an individual output signal for each chemFET sensor;
a reference electrode (<NUM>) in fluid communication with the device:
and an array controller (<NUM>) to provide:
a reference bias voltage to the reference electrode; and timing and control signals to the row select circuit and the column output circuit to collect and process output signals for each group of two or more chemical sensors coupled to a reaction region.