Patent Description:
Increasingly, biological and medical research is turning to nucleic acid sequencing for enhancing biological studies and medicine. For example, biologists and zoologists are turning to sequencing to study the migration of animals, the evolution of species, and the origins of traits. The medical community is using sequencing for studying the origins of disease, sensitivity to medicines, and the origins of infection. As such, sequencing has wide applicability in many aspects of biology, therapeutics, diagnostics, forensics and research.

Nevertheless, the use of sequencing can be limited by assay availability, sequencing run time, preparation time, and cost. Additionally, quality sequencing has historically been an expensive process, thus limiting its practice. <CIT> discloses a system including a communication interface to communicatively couple to a sensor cartridge, a fluidic subsystem to exchange a reagent solution with the sensor cartridge, and a computational circuitry communicatively coupled to the communication interface and the fluidic subsystem. <CIT> discloses methods and apparatuses relating to large scale FET arrays for analyte detection and measurement. The present invention provides a sensor device according to independent claim <NUM> and a method of preparing a sensor device according to independent claim <NUM>.

In an embodiment, a sensor device, such as a biosensor, includes a die secured to a substrate. The die has a plurality of sensors cooperatively associated with an array of wells. The die is attached to a substrate providing structural support and an electronic interface to the sensors of the die. A flow cell is attached to the substrate. In an example not forming part of the present invention, the flow cell defines a unified space over the die and includes a single inlet and a single outlet. According to the invention, the flow cell defines a plurality of separate volumes or lanes, each having an associated inlet and outlet. The separate volumes or lanes are separated by dividers. When the flow cell is attached to the substrate, the dividers are disposed over a set of wells of the array of wells on the die. In particular, the dividers can isolate a set of wells from the separate volumes and thus prevent the use of associated sensors. According to the invention, the dividers are attached to the die using an adhesive. The adhesive can further limit access to the set of wells on the die.

In a further embodiment, a method for manufacturing a sensor device includes providing a substrate having a die having an array of wells. The method further includes selecting a flow cell. In an example not forming part of the present invention, the flow cell can be selected from a flow cell defining a single volume or a flow cell defining lanes over the die. According to the invention, the selected flow cell defines lanes separated by dividers, and the dividers are disposed over and in proximity to a set of wells of the die. The dividers can limit access to the set of wells, preventing use of associated sensors. According to the invention, attaching the flow cell to the substrate includes using an adhesive to secure the flow cell dividers to the die. In an alternative example not forming part of the present invention, the flow cell can include elastomeric components that are pressed against the set of wells of the die.

Such sensor devices find particular use in sequencing systems. For example, in <FIG>, a system <NUM> containing fluidics circuit <NUM> is connected by inlets to at least two reagent reservoirs (<NUM>, <NUM>, <NUM>, <NUM>, or <NUM>), to waste reservoir <NUM>, and to biosensor <NUM> by fluid pathway <NUM> that connects fluidics node <NUM> to inlet <NUM> of biosensor <NUM> for fluidic communication. Reagents from reservoirs (<NUM>, <NUM>, <NUM>, <NUM>, or <NUM>) can be driven to fluidic circuit <NUM> by a variety of methods including pressure, pumps, such as syringe pumps, gravity feed, and the like, and are selected by control of valves <NUM>. Reagents from the fluidics circuit <NUM> can be driven through the valves <NUM> receiving signals from control system <NUM> to waste container <NUM>. Reagents from the fluidics circuit <NUM> can also be driven through the biosensor <NUM> to the waste container <NUM>. The control system <NUM> includes controllers for valves, which generate signals for opening and closing via electrical connection <NUM>.

The control system <NUM> also includes controllers for other components of the system, such as wash solution valve <NUM> connected thereto by electrical connection <NUM>, and reference electrode <NUM>. Control system <NUM> can also include control and data acquisition functions for biosensor <NUM>. In one mode of operation, fluidic circuit <NUM> delivers a sequence of selected reagents <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> to biosensor <NUM> under programmed control of control system <NUM>, such that in between selected reagent flows, fluidics circuit <NUM> is primed and washed, and biosensor <NUM> is washed. Fluids entering biosensor <NUM> exit through outlet <NUM> and are deposited in waste container <NUM> via control of pinch valve regulator <NUM>. The valve <NUM> is in fluidic communication with the sensor fluid output <NUM> of the biosensor <NUM>.

The sensor device including a dielectric layer defining a well formed from the first access and second access and exposing a sensor pad finds particular use in detecting chemical reactions and byproducts, such as detecting the release of hydrogen ions in response to nucleotide incorporation, useful in genetic sequencing, among other applications. In a particular embodiment, a sequencing system includes a flow cell in which a sensory array is disposed, includes communication circuitry in electronic communication with the sensory array, and includes containers and fluid controls in fluidic communication with the flow cell. In an example, <FIG> illustrates an expanded and cross-sectional view of a flow cell <NUM> and illustrates a portion of a flow chamber <NUM>. A reagent flow <NUM> flows across a surface of a well array <NUM>, in which the reagent flow <NUM> flows over the open ends of wells of the well array <NUM>. The well array <NUM> and a sensor array <NUM> together may form an integrated unit forming a lower wall (or floor) of flow cell <NUM>. A reference electrode <NUM> may be fluidly coupled to flow chamber <NUM>. Further, a flow cell cover <NUM> encapsulates flow chamber <NUM> to contain reagent flow <NUM> within a confined region.

<FIG> illustrates an expanded view of a well <NUM> and a sensor <NUM>, as illustrated at <NUM> of <FIG>. The volume, shape, aspect ratio (such as base width-to-well depth ratio), and other dimensional characteristics of the wells 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 sensor <NUM> can be a chemical field-effect transistor (chemFET), more specifically an ion-sensitive FET (ISFET), with a floating gate <NUM> having a sensor plate <NUM> optionally separated from the well interior by a material layer <NUM>. The sensor <NUM> can be responsive to (and generate an output signal related to) the amount of a charge <NUM> present on the material layer <NUM> opposite the sensor plate <NUM>. The material layer <NUM> can be a ceramic layer, such as an oxide of zirconium, hafnium, tantalum, aluminum, or titanium, among others, or a nitride of titanium. Alternatively, the material layer <NUM> can be formed of a metal, such as titanium, tungsten, gold, silver, platinum, aluminum, copper, or a combination thereof. In an example, the material layer <NUM> can have a thickness in a range of <NUM> to <NUM>, such as a range of <NUM> to <NUM>, a range of <NUM> to <NUM>, or even a range of <NUM> to <NUM>.

While the material layer <NUM> is illustrated as extending beyond the bounds of the illustrated FET component, the material layer <NUM> can extend along the bottom of the well <NUM> and optionally along the walls of the well <NUM>. The sensor <NUM> can be responsive to (and generate an output signal related to) the amount of a charge <NUM> present on the material layer <NUM> opposite the sensor plate <NUM>. Changes in the charge <NUM> can cause changes in a current between a source <NUM> and a drain <NUM> of the chemFET. In turn, the chemFET can be used directly 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 may move in and out of the wells by a diffusion mechanism <NUM>.

The well <NUM> can be defined by a wall structure, which can be formed of one or more layers of material. In an example, the wall structure can have a thickness extending from the lower surface to the upper surface of the well in a range of <NUM> micrometers to <NUM> micrometers, such as a range of <NUM> micrometers to <NUM> micrometers, a range of <NUM> micrometers to <NUM> micrometers, a range of <NUM> micrometers to <NUM> micrometers, or a range of <NUM> micrometers to <NUM> micrometers. In particular, the thickness can be in a range of <NUM> micrometers to <NUM> micrometer, such as a range of <NUM> micrometers to <NUM> micrometers, or a range of <NUM> micrometers to <NUM> micrometers. The wells <NUM> of array <NUM> can have a characteristic diameter, defined as the square root of <NUM> times the cross-sectional area (A) divided by Pi (e.g., sqrt(<NUM>*A/?)), of not greater than <NUM> micrometers, such as not greater than <NUM> micrometers, not greater than <NUM> micrometers, not greater than <NUM> micrometers, not greater than <NUM> micrometers, not greater than <NUM> micrometers or even not greater than <NUM> micrometers. In an example, the wells <NUM> can have a characteristic diameter of at least <NUM> micrometers. In a further example, the well <NUM> can define a volume in a range of <NUM> fL to <NUM> pL, such as a volume in a range of <NUM> fL to <NUM> pL, a range of <NUM> fL to <NUM> fL, a range of <NUM> fL to <NUM> fL, or even a range of <NUM> fL to <NUM> fL.

In an embodiment, reactions carried out in the well <NUM> 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 the sensor plate <NUM>. If such byproducts are produced in small amounts or rapidly decay or react with other constituents, then multiple copies of the same analyte may be analyzed in the well <NUM> at the same time in order to increase the output signal generated. In an embodiment, multiple copies of an analyte may be attached to a solid phase support <NUM>, either before or after deposition into the well <NUM>. The solid phase support <NUM> may be microparticles, nanoparticles, beads, solid or porous comprising gels, or the like. For simplicity and ease of explanation, solid phase support <NUM> is also referred herein as a particle or bead. For a nucleic acid analyte, multiple, connected copies may be made by rolling circle amplification (RCA), exponential RCA, or like techniques, to produce an amplicon without the need of a solid support.

In an example, the biosensor is an example of a sensor device. <FIG> and <FIG> illustrate an example sensor device <NUM>, such as a microchip including a flow cell. For example, the sensor device <NUM> includes a substrate <NUM> securing a die <NUM> that has a plurality of microwells in fluid communication with a sensor array. A flow cell <NUM> is secured over the substrate, providing a volume over the die <NUM>.

In an example, the flow cell <NUM> includes a set of fluid inlets <NUM> and a set of fluid outlets <NUM>. In particular, the flow cell can be divided into lanes <NUM>. Each lane <NUM> is individually accessed by a respective fluid inlet <NUM> and fluid outlet <NUM>.

As illustrated, the sensor device <NUM> includes four lanes <NUM>. Alternatively, the sensor device <NUM> can include less than four lanes or more than four lanes. For example, the sensor device <NUM> can include between <NUM> and <NUM> lanes, such as between <NUM> and <NUM> lanes, or <NUM> to <NUM> lanes. The lanes <NUM> can be fluidically isolated from each other. As such, the lanes <NUM> can be used at separate times, concurrently, or simultaneously, depending upon aspects of a run plan.

The sensor device <NUM> can further include guide structures <NUM>, for example, formed as part of the flow cell <NUM>, to engage complementary structures on a fluidic coupler. Such guide structures <NUM> assist with aligning the fluid inlets <NUM> and fluid outlet <NUM> with associated ports on a fluidic coupler.

<FIG> includes a further example of a flow cell attached to a substrate. For example, a substrate <NUM> is attached to a die <NUM>. In an example, the substrate <NUM> can be secured to the die <NUM> utilizing semiconductor encapsulation techniques. The die <NUM> includes a plurality of wells <NUM> cooperatively associated with sensors, for example, as illustrated in <FIG>.

Further, substrate <NUM> can provide an electronic interface to the die <NUM>, for example, as illustrated in <FIG>. In an example, the substrate <NUM> includes interface pads <NUM> distributed around a central area <NUM> free from interface pads. The interface pads <NUM> are on a side of the substrate <NUM> opposite the flow cell <NUM>. In particular, the interface pads <NUM> are in electrical communication with interface contacts of the die <NUM>. For example, the interface pads <NUM> can communicate instructions to circuits within the die <NUM> and can receive data from the circuits within the die <NUM>.

The flow cell <NUM> can be coupled to the substrate <NUM>. In an example, the flow cell <NUM> can define a single volume and have a single inlet and a single outlet providing access to the wells <NUM> of the die <NUM>. According to the invention, the flow cell <NUM> can include dividers <NUM> that separate a space defined over the die <NUM> into a plurality of separate volumes <NUM> isolated from one another. In particular, the volumes <NUM> can form lanes across the die <NUM>.

In particular, the dividers <NUM> extend to the die <NUM>. For example, the dividers <NUM> are disposed over a set of wells, such as wells <NUM>, and at least partially isolate such wells <NUM> from the separate volumes <NUM>. As such, the use of sensors associated with such wells <NUM> is limited.

The flow cell <NUM> can be formed of a variety of polymeric materials. For example, the polymeric material can include polycarbonate, polyethylene, polypropylene, polyamides, ABS, polytetrafluoroethylene, polyvinylidene fluoride, or polyvinylchloride, among other polymeric materials. Optionally, the dividers <NUM> can include elastomeric material disposed at the tip of the dividers <NUM>. In an example, the elastomeric material can include ABS, butylene rubber, or other elastomers.

The flow cell <NUM> can be secured to the substrate utilizing mechanical methods. According to the invention, the flow cell <NUM> is secured to the substrate <NUM> using an adhesive <NUM>. More particularly, the adhesive is further used to secure the dividers <NUM> to the die <NUM>. Portions of the adhesive <NUM> obscure or enter wells of the array of wells <NUM> disposed on the die <NUM>, further limiting use of the obscured wells and associated sensors. Example adhesives include silicone adhesives, epoxy, or urethane adhesive, among others.

<FIG> includes a block flow diagram illustrating an example method <NUM> for forming a sensor device. For example, the method <NUM> includes forming a die with an array of wells disposed in cooperation with an array of sensors, as illustrated at block <NUM>. For example, the die can be formed as part of a wafer using conventional semiconductor processing techniques to include sensors on array of sensors, such as those described in relation to <FIG>. One or more insulative layers be disposed over the die, and an array of wells can be defined through the one or more insulative layers to provide access to electrodes of the array of sensors.

As illustrated at block <NUM>, the die is attached to a substrate. For example, a wireframe or ball mount be defined or secured to the die and the die encapsulated on the substrate, thus providing an electronic interface through substrate to the sensors of the die.

Optionally, a flow cell can be selected, as illustrated at block <NUM>. For example and said alternative not forming part of the present invention, a flow cell defining a single volume and having a single inlet and a single outlet can be selected for attachment over the substrate. According to the invention, a flow cell is selected that is configured to define a plurality of volumes, each volume of the plurality of volumes having an associated inlet and an associated output. The flow cell utilizes dividers that separate the volumes. For example, the volumes can form lanes when attached to the substrate over the die.

As illustrated at block <NUM>, the flow cell is attached to the substrate. For example and said option not forming part of the present invention, the flow cell can include mechanical features that interact with complementary features of the substrate to secure the flow cell to the substrate.

According to the invention, the flow cell is adhered to the substrate using an adhesive.

Optionally and said case not forming part of the present invention, the dividers of the flow cell can include elastomeric material compressed into the surface of the die. According to the invention, adhesive is used to secure the dividers of the flow cell to die. In either case, portions of the wells of the die are blocked and the associated sensors have limited use.

In particular, the above method allows for standardization of a semiconductor process and subsequent selection of a configuration of flow cell. Accordingly, only a single type of semiconductor chip may be designed which can have different flow cell configurations associated therewith in contrast to adapting the design of the semiconductor die to a configuration of the flow cell. The different flow cell configurations can include a flow cell with multiple lanes or dividers and optionally (not forming part of the present invention), a flow cell configuration without dividers.

In the foregoing specification, the concepts have been described with reference to specific embodiments.

For example, a process, method, article, or apparatus that comprises a list of features is not necessarily limited only to those features but may include other features not expressly listed or inherent to such process, method, article, or apparatus.

Claim 1:
A sensor device (<NUM>) comprising:
a substrate (<NUM>) having a die (<NUM>) attached to the substrate (<NUM>), the die (<NUM>) including an array of sensors and an array of wells (<NUM>) cooperatively disposed over the array of sensors, the array of wells (<NUM>) exposed by the substrate (<NUM>); and
a flow cell (<NUM>) secured to the substrate (<NUM>) and defining a flow space disposed over the die and accessible to the array of wells, wells (<NUM>), the flow cell (<NUM>) defining a plurality of separate volumes (<NUM>),
each volume of the plurality of separate volumes (<NUM>) having an inlet (<NUM>) and an outlet (<NUM>), the plurality of separate volumes (<NUM>) separated by dividers (<NUM>), the dividers (<NUM>) covering a set of wells (<NUM>) of the array of wells (<NUM>);
the sensor device (<NUM>) being characterized in that an adhesive (<NUM>) secures the flow cell (<NUM>) to the substrate (<NUM>), wherein the adhesive (<NUM>) secures the dividers (<NUM>) to the die (<NUM>), the adhesive (<NUM>) covering the set of wells (<NUM>).