Device comprising a multi-channel flow cell

Abstract

The sensor device includes a substrate having a die attached thereto. The die includes a sensor array and a pool array disposed cooperatively on the sensor array. The pool array is exposed by the substrate. The sensor device further includes a flow cell fastened to the substrate and defining a flow space disposed on the die and accessible to the pool array. The flow cell defines a plurality of individual volumes. Each of the plurality of individual volumes has an inlet and an outlet. The plurality of individual volumes are separated by a separator. The separator covers a set of pools of the pool array.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 890,060, filed Aug. 21, 2019, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure generally relates to systems and methods for manipulating and analyzing nucleic acids. Background Art

[0004] Biological and medical research is increasingly turning to nucleic acid sequencing for enhancing biological research and medicine. For example, biologists and zoologists are turning to sequencing to study animal migration, species evolution, and the origin of traits. Medical communities are using sequencing to study the causes of diseases, sensitivity to drugs, and the origin of infections. Thus, sequencing has broad applicability in many aspects of biology, therapeutics, diagnostics, forensics, and research.

[0005] However, the use of sequencing is limited by assay availability, sequencing run time, preparation time, and cost. Additionally, quality sequencing has historically been an expensive process, thus limiting its practice. Summary of the Invention

[0006] The present invention discloses a sensor device, comprising: a substrate having a die attached thereto, the die including a sensor array and a pool array cooperatively disposed on the sensor array, the pool array being exposed by the substrate; and a flow cell fastened to the substrate and defining a flow space disposed on the die and accessible to the pool array, the flow cell defining a plurality of individual volumes, each of the plurality of individual volumes having an inlet and an outlet, the plurality of individual volumes being separated by a separator covering a set of pools of the pool array.

[0007] In a preferred embodiment of the sensor device, the sensors of the sensor array comprise chemically sensitive field effect transistors.

[0008] In a preferred embodiment of the sensor device, the chemically sensitive field effect transistor is an ion sensitive field effect transistor.

[0009] In a preferred embodiment of the sensor device, the sensor device further comprises an adhesive fastening the flow cell to the substrate.

[0010] In a preferred embodiment of the sensor device, the adhesive fastens the separator to the die, the adhesive covering the set of pools.

[0011] In a preferred embodiment of the sensor device, the set of pools is fluidically isolated from the plurality of individual volumes.

[0012] In a preferred embodiment of the sensor device, the flow cell comprises a guiding structure.

[0013] In a preferred embodiment of the sensor device, the plurality of individual volumes define channels across the die.

[0014] In a preferred embodiment of the sensor device, the number of channels is at least 2 and no greater than 10.

[0015] In a preferred embodiment of the sensor device, the number of channels is at least 3 and no greater than 6.

[0016] The present invention also discloses a method of preparing a sensor device, the method comprising: providing a die attached to a substrate, the die comprising a sensor array and an array of pools disposed cooperatively on the sensor array, the array of pools being exposed by the substrate; selecting a flow cell to be applied to the substrate to form a flow space disposed on the die and accessible to the array of pools, the flow cell defining a plurality of individual volumes, each of the plurality of individual volumes having an inlet and an outlet, the plurality of individual volumes being separated by dividers; and applying the flow cell to the substrate, the dividers covering a set of pools of the array of pools.

[0017] In a preferred embodiment of the method of preparing a sensor device, the sensors of the sensor array comprise chemically sensitive field effect transistors.

[0018] In a preferred embodiment of the method of preparing a sensor device, the chemically sensitive field effect transistors are ion sensitive field effect transistors.

[0019] In a preferred embodiment of the method of preparing a sensor device, applying the flow cell comprises adhering the flow cell to the substrate with an adhesive.

[0020] In a preferred embodiment of the method of preparing a sensor device, the adhesive secures the dividers to the die, the adhesive covering the set of pools.

[0021] In a preferred embodiment of the method of preparing a sensor device, the set of pools is fluidically isolated from the plurality of individual volumes.

[0022] In a preferred embodiment of the method of preparing a sensor device, the flow cell comprises a guiding structure.

[0023] In a preferred embodiment of the method of preparing a sensor device, the plurality of individual volumes define channels across the die.

[0024] In a preferred embodiment of the method of preparing a sensor device, the number of channels is at least 2 and not greater than 10.

[0025] In a preferred embodiment of the method of preparing a sensor device, the number of channels is at least 3 and not greater than 6. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present disclosure may be better understood by reference to the accompanying drawings, and many of the features and advantages thereof will be apparent to those of ordinary skill in the art.

[0027] Figure 1 Diagrams of an exemplary sequencing system are included.

[0028] Figure 2 Diagrams of an exemplary system including a sensor array are included.

[0029] Figure 3 Diagrams of an exemplary sensor and associated wells are included.

[0030] Figure 4 and Figure 5 Diagrams of an exemplary sensor device are included.

[0031] Figure 6 Diagrams of a cross-section of an exemplary sensor device are included.

[0032] Figure 7 Diagrams of an electronic interface of an exemplary sensor device are included.

[0033] Figure 8 Block flowcharts showing exemplary methods for forming a sensor device are included.

[0034] Like reference symbols are used in different drawings to indicate similar or identical items. DETAILED DESCRIPTION

[0035] In one embodiment, a sensor device, such as a biosensor, includes a die fastened to a substrate. The die has a plurality of sensors associated in cooperation with an array of wells. The die is attached to the substrate, which provides structural support and an electronic interface to the sensors of the die. A flow cell may be attached to the substrate. In one example, the flow cell defines a unified space above the die and includes a single inlet and a single outlet. In another example, the flow cell defines a plurality of separate volumes or channels, each having an associated inlet and outlet. The separate volumes or channels are separated by dividers. When the flow cell is attached to the substrate, the dividers are positioned over a set of wells of the well array on the die. Specifically, the dividers may isolate a set of wells from the separate volumes and thus avoid the use of the associated sensors. Optionally, an adhesive may be used to attach the dividers to the die. The adhesive may further restrict access to the set of wells on the die.

[0036] In yet another embodiment, a method for fabricating a sensor device includes providing a substrate having dies, the dies having an array of wells. The method may also include selecting a flow cell. In one example, the flow cell may be selected from a flow cell that defines a single volume or a flow cell that defines a channel on the die. When the selected flow cell defines channels separated by a septum, the septum may be disposed over and proximate a set of wells on the upper die. The septum may restrict access to the set of wells, thereby avoiding use of the associated sensors. In one example, attaching the flow cell to the substrate includes securing the flow cell septum to the die using an adhesive. In an alternative example, the flow cell may include an elastomeric component that presses against the set of wells of the die.

[0037] Such sensor devices are particularly useful in sequencing systems. For example, in Figure 1 system 100 that includes a fluidic control loop 102 is connected by a fluid path 132 from an inlet to at least two reagent reservoirs (104, 106, 108, 110, or 112), to a waste reservoir 120, and to a biosensor 134. The fluid path connects a fluidic control node 130 to an inlet 138 of the biosensor 134 for fluid communication. Reagents from the reservoirs (104, 106, 108, 110, or 112) can be driven into the fluidic control circuit 102 by a variety of methods including pressure, pumps (such as syringe pumps), gravity feed, and the like, and are selected by control of valves 114. Reagents from the fluidic control loop 102 can be driven to the waste container 120 by valves 114 that receive signals from a control system 118. Reagents from the fluidic control loop 102 can also be driven to the waste container 136 by the biosensor 134. The control system 118 includes a controller for the valves that generates signals for opening and closing via an electrical connection 116.

[0038] The control system 118 also includes a controller for other components of the system, such as a wash solution valve 124 and a reference electrode 128 connected thereto via an electrical connection 122. The control system 118 may also include control and data acquisition functions for the biosensor 134. In one operating mode, the fluidic control loop 102 delivers a sequence of selected reagents 1, 2, 3, 4, or 5 to the biosensor 134 under programmed control of the control system 118 such that between selected reagent flows, the fluidic control loop 102 is primed and washed, and the biosensor 134 is washed. Under control of a pinch valve regulator 144, fluid entering the biosensor 134 exits through an outlet 140 and is deposited in the waste container 136. The valve 144 is in fluid communication with a sensor fluid output 140 of the biosensor 134.

[0039] A sensor device including a dielectric layer that defines a pool formed by a first inlet and a second inlet and exposes a sensor pad is particularly suitable for detecting chemical reactions and by-products, such as detecting the release of hydrogen ions in response to nucleotide incorporation, and can be used for genetic sequencing and other applications. In a particular embodiment, a sequencing system includes a flow cell in which a sensing array is disposed, includes a communication circuit in electronic communication with the sensing array, and includes a container and a fluid controller in fluid communication with the flow cell. In one example, Figure 2 An exploded view and a cross-sectional view of the flow cell 200 are shown and a portion of the flow chamber 206 is shown. A reagent flow 208 flows over the surface of the pool array 202, where the reagent flow 208 flows over the open ends of the pools of the pool array 202. The pool array 202 and the sensor array 205 together can form an integrated unit that forms the lower wall (or bottom plate) of the flow cell 200. The reference electrode 204 can be fluidly coupled to the flow chamber 206. Additionally, the flow cell lid 230 encapsulates the flow chamber 206 to contain the reagent flow 208 within a restricted area.

[0040] Figure 3 As shown in Figure 2 An exploded view of the pool 301 and the sensor 314 as shown at 210 is shown. The volume, shape, aspect ratio (such as the ratio of the substrate width to the pool depth), and other dimensional characteristics of the pool can be selected based on the nature of the reaction occurring and the reagents, by-products, or labeling techniques (if any) employed. The sensor 314 can be a chemical field effect transistor (chemFET), more specifically an ion-sensitive FET (ISFET), having a floating gate 318 that has a sensor plate 320 optionally separated from the interior of the pool by a material layer 316. The sensor 314 can respond to the amount of charge 324 present on the material layer 316 opposite the sensor plate 320 (and generate an output signal related to the amount). The material layer 316 can be a ceramic layer, particularly an oxide of zirconium, hafnium, tantalum, aluminum, or titanium, or a nitride of titanium. Alternatively, the material layer 316 can be formed of a metal such as titanium, tungsten, gold, silver, platinum, aluminum, copper, or a combination thereof. In one example, the thickness of the material layer 316 can be in the range of 5 nm to 100 nm, such as in the range of 10 nm to 70 nm, 15 nm to 65 nm, or even 20 nm to 50 nm.

[0041] Although the material layer 316 is shown as extending beyond the boundaries of the illustrated FET assembly, the material layer 316 may extend along the bottom of the cell 301 and optionally along the walls of the cell 301. The sensor 314 may respond to the amount of charge 324 present on the material layer 316 opposite the sensor plate 320 (and generate an output signal related to that amount). A change in the charge 324 may cause a change in the current between the source 321 and drain 322 of the chemFET. Next, the chemFET may 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 enter and leave the cell through the diffusion mechanism 340.

[0042] The cell 301 may be defined by a wall structure, which may be formed of one or more material layers. In one example, the wall structure may have a thickness ranging from 0.01 μm to 10 μm, such as from 0.05 μm to 10 μm, from 0.1 μm to 10 μm, from 0.3 μm to 10 μm, or from 0.5 μm to 6 μm, extending from the lower surface to the upper surface of the cell. Specifically, the thickness may range from 0.01 micrometer to 1 micrometer, such as from 0.05 micrometer to 0.5 micrometer, or from 0.05 micrometer to 0.3 micrometer. The cells 301 of the array 202 may have a feature diameter of no more than 5 μm, such as no more than 3.5 μm, no more than 2.0 μm, no more than 1.6 μm, no more than 1.0 μm, no more than 0.8 μm, or even no more than 0.6 μm, where the feature diameter is defined as the square root of 4 times the cross-sectional area (A) divided by π (e.g., sqrt(4*A / π)). In one example, the cell 301 may have a feature diameter of at least 0.01 μm. In another example, the cell 301 may define a volume in the range of 0.05 fL to 10 pL, such as from 0.05 fL to 1 pL, from 0.05 fL to 100 fL, from 0.05 fL to 10 fL, or even from 0.1 fL to 5 fL.

[0043] In one embodiment, the reaction conducted in cell 301 can be an analytical reaction used to identify or determine the characteristics or properties of an analyte of interest. Such reactions can directly or indirectly produce by-products that affect the amount of charge near sensor plate 320. If such by-products are produced in small amounts or decay rapidly or react with other components, then multiple copies of the same analyte can be analyzed simultaneously in cell 301 to increase the output signal generated. In one embodiment, multiple copies of the analyte can be attached to solid-phase carrier 312 before or after deposition into cell 301. Solid-phase carrier 312 can be micron-sized particles, nano-sized particles, beads, solid or porous gels, etc. For the sake of simplicity and ease of description, solid-phase carrier 312 is also referred to herein as a particle or bead. For nucleic acid analytes, multiple linked copies can be prepared by rolling circle amplification (RCA), exponential RCA, or similar techniques to generate amplicons without the need for a solid support.

[0044] In one example, the biosensor is an example of a sensor device. Figure 4 and Figure 5 An example sensor device 400 is shown, such as a microchip including a flow cell. For example, sensor device 400 includes a substrate 402 that secures die 404, which has a plurality of microcells in fluid communication with a sensor array. Flow cell 406 is secured above the substrate, providing a volume above die 404.

[0045] In one example, flow cell 406 includes a set of fluid inlets 408 and a set of fluid outlets 410. Specifically, the flow cell can be divided into channels 412. Each channel 412 is individually accessed by a corresponding fluid inlet 408 and fluid outlet 410.

[0046] As illustrated, sensor device 400 includes four channels 412. Alternatively, sensor device 400 can include fewer than four channels or more than four channels. For example, sensor device 400 can include between 1 and 10 channels, such as between 2 and 8 channels, or 4 to 6 channels. Channels 412 can be fluidically isolated from each other. Thus, depending on aspects of the operation plan, channels 412 can be used at separate times, concurrently, or simultaneously.

[0047] Sensor device 400 can further include a guiding structure 414, such as being formed as part of flow cell 406, to engage complementary structures on a fluid connector. Such guiding structure 414 helps align fluid inlets 408 and fluid outlets 410 with associated ports on the fluid connector.

[0048] Figure 6Another example includes a flow cell attached to a substrate. For example, substrate 602 is attached to die 604. In one example, substrate 602 can be fastened to die 604 using semiconductor encapsulation techniques. Die 604 includes a plurality of cells 608 that are cooperatively associated with sensors such as Figure 3 shown in

[0049] In addition, substrate 602 can provide an electronic interface to die 604, such as Figure 7 illustrated. In one example, substrate 602 includes interface pads 712 distributed around a central region 710 that does not include interface pads. Interface pads 712 are on the side of substrate 602 opposite flow cell 606. Specifically, interface pads 712 are in electrical communication with interface contacts of die 604. For example, interface pads 712 can transmit instructions to circuitry within die 604 and can receive data from circuitry within die 604.

[0050] Flow cell 606 can be coupled to substrate 602. In one example, flow cell 606 can define a single volume and have a single inlet and a single outlet, providing access to cells 608 of die 604. In another example, flow cell 606 can include a separator 610 that divides the space defined above die 604 into a plurality of separate volumes 612 that are isolated from each other. Specifically, volumes 612 can form channels across die 604.

[0051] Specifically, separator 610 extends to die 604. For example, separator 610 can be placed on a group of cells such as cell 614 and at least partially isolate these cells 614 from separate volumes 612. Thus, the use of sensors associated with these cells 614 is restricted.

[0052] Flow cell 606 can be formed from a variety of polymeric materials. For example, the polymeric material can include polycarbonate, polyethylene, polypropylene, polyamide, ABS, polytetrafluoroethylene, polyvinylidene fluoride, or polyvinyl chloride, among other polymeric materials. Optionally, separator 610 can include an elastomeric material placed at the top of separator 610. In one example, the elastomeric material can include ABS, butene rubber, or other elastomers.

[0053] Flow cell 606 can be fastened to the substrate using mechanical methods. Alternatively, flow cell 606 can be fastened to substrate 602 using an adhesive 616. In certain examples, the adhesive can also be used to fasten separator 610 to die 604. Some portions of adhesive 616 will block or enter cells of cell array 608 placed on die 604, further restricting the use of the blocked cells and associated sensors. Example adhesives include silicone adhesives, epoxy resins, or urethane adhesives, among others.

[0054] Figure 8 A block flow diagram including an example method 800 for forming a sensor device is shown. By way of example, method 800 includes forming a die having a pool array disposed in cooperation with a sensor array, as illustrated at block 802. By way of example, the die can be formed as part of a wafer using conventional semiconductor processing techniques to include sensors on the sensor array, such as with respect to Figure 3 those described. One or more insulating layers are disposed on the die, and the pool array can be defined through the one or more insulating layers to provide access to electrodes of the sensor array.

[0055] As illustrated at block 804, the die can be attached to a substrate. By way of example, a wireframe or ball seat is defined or fastened to the die and the die is encapsulated on the substrate, thus providing an electrical interface through the substrate to the sensors of the die.

[0056] Optionally, a flow cell can be selected, as illustrated at block 806. By way of example, a flow cell defining a single volume and having a single inlet and a single outlet can be selected for attachment above the substrate. In another example, a flow cell configured to define multiple volumes can be selected, each of the multiple volumes having an associated inlet and an associated output. The flow cell can utilize partitions that separate the volumes. By way of example, the volumes can form channels when attached above the die to the substrate.

[0057] As illustrated at block 808, the flow cell can be attached to the substrate. By way of example, the flow cell can include mechanical features that interact with complementary features of the substrate to fasten the flow cell to the substrate. Alternatively, the flow cell can be bonded to the substrate using an adhesive. Optionally, the partitions of the flow cell can include an elastic material that is compressed into the surface of the die. Alternatively, an adhesive can be used to fasten the partitions of the flow cell to the die. In either case, portions of the die's pools are blocked and the use of the associated sensors is limited.

[0058] Specifically, the above method allows for the standardization of semiconductor processes and subsequent selection of flow cell configurations. Thus, only a single type of semiconductor chip can be designed, which can have different flow cell configurations associated therewith, rather than adapting the design of the semiconductor die to the flow cell configuration. Different flow cell configurations can include flow cells having multiple channels or partitions, and optionally can include a flow cell configuration without partitions.

[0059] In a first aspect, a sensor device includes: a substrate having a die attached thereto, the die including a sensor array and a pool array disposed cooperatively on the sensor array, the pool array being exposed by the substrate; and a flow cell fastened to the substrate and defining a flow space disposed on the die and accessible to the pool array, the flow cell defining a plurality of individual volumes, each of the plurality of individual volumes having an inlet and an outlet, the plurality of individual volumes being separated by a separator that covers a set of pools of the pool array.

[0060] In an example of the first aspect, the sensors of the sensor array include chemically sensitive field effect transistors. For example, the chemically sensitive field effect transistors are ion sensitive field effect transistors.

[0061] In another example of the first aspect and the above example, the sensor device further includes an adhesive that fastens the flow cell to the substrate. For example, the adhesive fastens the separator to the die, and the adhesive covers the set of pools.

[0062] In another example of the first aspect and the above example, the set of pools is fluidically isolated from the plurality of individual volumes.

[0063] In an additional example of the first aspect and the above example, the flow cell includes a guiding structure.

[0064] In another example of the first aspect and the above example, the plurality of individual volumes define channels across the die. For example, the number of channels is at least 2 and no greater than 10. In one example, the number of channels is at least 3 and no greater than 6.

[0065] In a second aspect, a method of preparing a sensor device includes: providing a die attached to a substrate, the die including a sensor array and a pool array disposed cooperatively on the sensor array, the pool array being exposed by the substrate; selecting a flow cell to apply to the substrate to form a flow space disposed on the die and accessible to the pool array, the flow cell defining a plurality of individual volumes, each of the plurality of individual volumes having an inlet and an outlet, the plurality of individual volumes being separated by a separator; and applying the flow cell to the substrate, the separator covering a set of pools of the pool array.

[0066] In an example of the second aspect, the sensors of the sensor array include chemically sensitive field effect transistors. For example, the chemically sensitive field effect transistors are ion sensitive field effect transistors.

[0067] In another example of the second aspect and the above example, applying the flow cell includes adhering the flow cell to the substrate with an adhesive. For example, the adhesive fastens the separator to the die, and the adhesive covers the set of pools.

[0068] In another example of the second aspect and the above examples, the set of pools is isolated from the plurality of individual volume fluids.

[0069] In additional examples of the second aspect and the above examples, the flow cell includes a guide structure.

[0070] In another example of the second aspect and the above examples, the plurality of individual volumes define channels across the die. For example, the number of channels is at least 2 and no greater than 10. In one example, the number of channels is at least 3 and no greater than 6.

[0071] Note that not all of the activities described above in the general description or examples are required, some particular activities may not be required, and one or more further activities may be performed in addition to those described. Also, the order of the listed activities is not necessarily the order in which they are performed.

[0072] In the foregoing specification, concepts have been described with reference to specific embodiments. However, one of ordinary skill in the art will appreciate that various modifications and changes can be made without departing from the scope of the invention as set forth in the appended claims. Accordingly, the specification and drawings are to be regarded as illustrative rather than restrictive, and all such modifications are intended to be included within the scope of the invention.

[0073] As used herein, the terms “comprises / comprising,” “includes / including,” “has / having” or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of features is not necessarily limited to only those features but may include other features not expressly listed or inherent to such process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not an exclusive or. For example, any one of the following satisfies the condition A or B: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0074] Also, the use of “a / an” is employed to describe elements and components herein. This is done for convenience only and to give a general sense of the scope of the invention. This description should be read to include one or at least one, and the singular also includes the plural unless it is obvious otherwise.

[0075] Benefits, other advantages, and solutions to problems have been described above with respect to specific embodiments. However, any benefits, advantages, problem solutions, and any features that may cause any benefit, advantage, or solution to arise or become more apparent should not be construed as critical, required, or essential features of any or all of the claims.

[0076] After reading this specification, those skilled in the art should understand that, for clarity, certain features described herein in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, the various features described in the context of a single embodiment may also be provided separately or in any sub-combination. Additionally, values recited in ranges include each value within that range.

Claims

1. A method of preparing a sensor device, the method comprising: Providing a die attached to a substrate, the die comprising a sensor array and a pool array disposed cooperatively on the sensor array, the pool array being exposed by the substrate; Selecting a flow cell to apply to the substrate to form a flow space disposed on the die and accessible to the pool array, the flow cell defining a plurality of individual volumes, each of the plurality of individual volumes having an inlet and an outlet, the plurality of individual volumes being separated by partitions; Applying the flow cell to the substrate, the partitions covering a set of pools of the pool array; And Wherein applying the flow cell comprises adhering the flow cell to the substrate and the die with an adhesive, and wherein the adhesive secures the partitions to the die, the adhesive covering the set of pools.

2. The method according to claim 1, wherein the sensors of the sensor array comprise chemically sensitive field effect transistors.

3. The method according to claim 2, wherein the chemically sensitive field effect transistor is an ion sensitive field effect transistor.

4. The method according to any one of claims 1 to 3, wherein the set of pools is fluidically isolated from the plurality of individual volumes.

5. The method according to any one of claims 1 to 3, wherein the flow cell comprises a guiding structure.

6. The method according to any one of claims 1 to 3, wherein the plurality of individual volumes define a channel across the die.

7. The method according to claim 6, wherein the number of channels is at least 2 and not more than 10.

8. The method according to claim 7, wherein the number of channels is at least 3 and not more than 6.

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