WO2025072642A1

WO2025072642A1 - Fluorescence sensor with multiple reaction sites per pixel and an electrochromic layer below each reaction site

Info

Publication number: WO2025072642A1
Application number: PCT/US2024/048814
Authority: WO
Inventors: Xu Liu
Original assignee: Illumina Inc
Current assignee: Illumina Inc
Priority date: 2023-09-29
Filing date: 2024-09-27
Publication date: 2025-04-03
Anticipated expiration: 2026-03-29

Abstract

There is set forth herein, in one example, an apparatus (100). The apparatus can comprise, for example: a biosensor (200) comprising pixel(s) (201), a first reaction site (206, 206a-206f) to emit light responsive to a cluster (C1, C2) at the site being exposed to excitation light (101); the emitted light is a first optical signal propagated through the biosensor on a first signal path to a pixel. The apparatus includes a second reaction site (206, 206a-206f) to emit light responsive to a cluster (C1, C2) at this site being exposed to the excitation light; the emitted light is a second optical signal propagated through the biosensor on a second signal path to the pixel. The apparatus includes a first layer (252) and a second layer (252) both situated in the signal paths and associated to the pixel, the first, to selectively open the signal path when a first voltage is applied, and the second, to selectively open the signal path when a second voltage is applied.

Description

MATERIAL BASED SENSING

BACKGROUND

[0001] The present application relates generally to sensing and specifically to light sensing.

[0002] Various protocols in biological or chemical research involve performing controlled reactions. The designated reactions can then be observed or detected and subsequent analysis can help identify or reveal properties of chemicals involved in the reaction. Image sensors are utilized for biological and chemical analysis. Various protocols in biological or chemical research involve performing controlled reactions on local support surfaces or within predefined reaction chambers. The designated reactions may then be observed or detected, and subsequent analysis may help identify or reveal properties of chemicals involved in the reaction. For example, in some multiplex assays, an unknown analyte having an identifiable label (e.g., fluorescent label) may be exposed to thousands of known probes under controlled conditions. Each known probe may be deposited into a corresponding well of a flow cell channel. Observing any chemical reactions that occur between the known probes and the unknown analyte within the wells may help identify or reveal properties of the analyte. Other examples of such protocols include known DNA sequencing processes, such as sequencing-by- synthesis (SBS) or cyclic-array sequencing.

[0003] In some conventional fluorescent-detection protocols, an optical system is used to direct an excitation light onto fluorescently labeled analytes and to also detect the fluorescent signals that may emit from the analytes. Such optical systems may include an arrangement of lenses, filters, and light sources. In other detection systems, the controlled reactions occur immediately over a solid-state imager (e.g., charged coupled device (CCD) or a complementary metal-oxide-semi conductor (CMOS) detector) that does not require a large optical assembly to detect the fluorescent emissions.

SUMMARY

[0004] One can increase throughout in a flow cell with a sensor and reaction sites (e.g., nano wells) by fitting at least two clusters in one pixel (above the pixels of the sensor and below the reaction sites of the flow cell). Utilizing two (or more) clusters to direct optical signals to a common pixel can cause interference between the individual signals and thus, separating or otherwise differentiating the signals enables the utilization of this configuration. To this end, the examples herein disclose a flow cell as well as a method of making and using a flow cell that includes voltage-switchable materials (e.g., electrochromic materials). In the examples herein, the opacity structures below the reaction sites changes based on the applied voltage, so that one can select which reaction site feeds the signal to the pixel and/or if the signals are received at the same time, one can differentiate the signals from each other based on signal strength (number of photons).

[0005] Thus, shortcomings of the prior art can be overcome and benefits as described later in this disclosure can be achieved through the provision of an apparatus. Various examples of the apparatus are described below, and the apparatus, including and excluding the additional examples enumerated below, in any combination (provided these combinations are not inconsistent), overcome these shortcomings. In some examples herein, the apparatus comprises: a biosensor comprising a plurality of pixels; a first reaction site configured to emit light responsive to a first cluster at the first reaction site being exposed to excitation light, wherein the emitted light from the first reaction site is propagated through the biosensor on a first signal path to a pixel of the plurality of pixels as a first optical signal; a second reaction site configured to emit light responsive to a second cluster at the second reaction site being exposed to the excitation light, wherein the emitted light from the second reaction site is propagated through the biosensor on a second signal path to the pixel of the plurality of pixels as a second optical signal; a first layer associated to the pixel, the first layer situated in the first signal path, wherein the first layer is configured to selectively open the first signal path to the first optical signal when a first voltage is applied to the first layer; and a second layer associated to the pixel, the second layer situated in the second signal path, wherein the second layer is configured to selectively open the second signal path to the second optical signal when a second voltage is applied to the second layer.

[0006] Thus, shortcomings of the prior art can be overcome and benefits as described later in this disclosure can be achieved through the provision of a method for forming a flow cell. Various examples of the method are described below, and the method, including and excluding the additional examples enumerated below, in any combination (provided these combinations are not inconsistent), overcome these shortcomings. In some examples herein, the method comprises: forming one or more layers over a sensor, the sensor comprising a plurality of pixels, the one or more layers comprising at least one material that changes in opacity when exposed to a given range of voltages; forming a filter layer over the one or more layers, wherein the one or more layers are situated between the sensor and the filter layer; forming trenches through the one or more layers and the filter layer, wherein the forming creates stacks separated from each other by the trenches, each stack comprising a portion of the filter layer and a portion of the one or more layers, wherein based on forming the trenches, two stacks of the stacks are situated on each pixel of the plurality of pixels; filling the trenches with a material that reduces crosstalk between the pixels; planarizing the filled trenches to define a contiguous surface with a top surface of the stacks; and forming reaction sites atop each stack such that each pixel is associated with a first reaction site and a second reaction site.

[0007] As aforementioned, shortcomings of the prior art can be overcome and benefits as described later in this disclosure can be achieved through the provision of a method for utilizing a biosensor. Various examples of the method are described below, and the method, including and excluding the additional examples enumerated below, in any combination (provided these combinations are not inconsistent), overcome these shortcomings. In some examples herein, the method comprises: detecting, using a pixel sensor of a plurality of pixels sensors, a cluster signal, the cluster signal being dependent on a first cluster signal emitted from a first reaction site associated to the pixel sensor, wherein the cluster signal passes from the first reaction site through a first layer based on a first voltage being applied to the first layer; detecting, using a pixel sensor of a plurality of pixels sensors, a second cluster signal, the second cluster signal being dependent on a second cluster signal emitted from a second reaction site associated to the pixel sensor, wherein the cluster signal passes from the second reaction site through a second layer based on a second voltage being applied to the second layer; determining an identity of a first analyte of interest in the first reaction site in dependence on the cluster signal detected using the pixel sensor; and determining an identity of a second analyte of interest in the second reaction site in dependence on the second cluster signal detected using the pixel sensor.

[0008] As aforementioned, shortcomings of the prior art can be overcome and benefits as described later in this disclosure can be achieved through the provision of a method for utilizing a biosensor. Various examples of the method are described below, and the method, including and excluding the additional examples enumerated below, in any combination (provided these combinations are not inconsistent), overcome these shortcomings. In some examples herein, the method comprises: obtaining an apparatus, the apparatus comprising: a biosensor comprising a plurality of pixels; a first reaction site configured to emit light responsive to a first cluster at the first reaction site being exposed to excitation light, wherein the emitted light from the first reaction site is propagated through the biosensor on a first signal path to a pixel of the plurality of pixels as a first optical signal; a second reaction site configured to emit light responsive to a second cluster at the second reaction site being exposed to the excitation light, wherein the emitted light from the second reaction site is propagated through the biosensor on a second signal path to the pixel of the plurality of pixels as a second optical signal; a first layer associated to the pixel, the first layer situated in the first signal path, wherein the first layer is configured to selectively open the signal path of the first optical signal when a first voltage is applied to the first layer; and a second layer associated to the pixel, the second layer situated between the second reaction site and the pixel in a signal path of the second optical signal, wherein the second layer is configured to selectively open the signal path of the second optical signal when a second voltage is applied to the second layer; exposing the first reaction and on the second reaction site to light from a light source; selectively applying the first voltage to the first layer and the second voltage to the second layer; based on the first voltage being applied to the first layer, receiving the first optical signal at the pixel; and based on the second voltage being applied to the second layer, receiving the second the second optical signal at the pixel.

[0009] Thus, shortcomings of the prior art can be overcome and benefits as described later in this disclosure can be achieved through the provision of an biosensor. Various examples of the biosensor are described below, and the biosensor, including and excluding the additional examples enumerated below, in any combination (provided these combinations are not inconsistent), overcome these shortcomings. In some examples herein, the biosensor comprises: a plurality of pixels; a first reaction site, wherein light emitted from the first reaction site propagates on a first signal path to a pixel of the plurality of pixels as a first optical signal; a second reaction site, wherein light emitted from the second reaction site propagates on a second signal path to the pixel of the plurality of pixels as a second optical signal; a first layer situated in the first signal path, wherein the first layer is comprised of a first electrochromic material, where the opacity of the first electrochromic material changes in response to a first voltage; and a second layer situated in the second signal path, wherein the second layer is comprised of a second electrochromic material, where the opacity of the second electrochromic material changes in response to a second voltage.

[0010] Additional features are realized through the techniques described herein. Other examples and aspects are described in detail herein and are considered a part of the claimed aspects. These and other objects, features and advantages of this disclosure will become apparent from the following detailed description of the various aspects of the disclosure taken in conjunction with the accompanying drawings.

[0011] It should be appreciated that all combinations of the foregoing aspects and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter and to achieve the benefits advantages disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] One or more aspects are particularly pointed out and distinctly claimed as examples in the claims at the conclusion of the specification. The foregoing and objects, features, and advantages of one or more aspects are apparent from the following detailed description taken in conjunction with the accompanying drawings in which:

[0013] FIG. 1 is a side cross-sectional view of an apparatus for use in analysis;

[0014] FIG. 2 is a top cross-sectional view of the apparatus shown in FIG. 1 ;

[0015] FIG. 3 is a top cross-sectional view of the voltage switching layers on the apparatus shown in FIG. 1 and of the detector of FIG. 5;

[0016] FIG. 4 is a top cross-sectional view of the voltage switching layers on the apparatus shown in FIG. 1 and of the detector of FIG. 5;

[0017] FIGS. 5A-5B are cross-sectional views of a portion of a detector that includes various aspects of the detector in the apparatus shown in FIG. 1 ;

[0018] FIGS. 6A-6F illustrate various states in a workflow for forming the detector of FIG. 5 A;

[0019] FIG. 7 illustrates the formation of the nano wells when forming the detector of FIG. 5 A; [0020] FIG. 8 illustrates the functionality of an electrochromic layer; [0021] FIG. 9 illustrates the transmittance behavior of the electrochromic layer detailed in Table

2;

[0022] FIG. 10 illustrates the transmittance behavior of the electrochromic layer detailed in Table 3;

[0023] FIG. 11 provides an illustration of aspects of various workflows that include utilizing various examples of the apparatuses described herein; and

[0024] FIG. 12 provides an illustration of aspects of various workflows that include utilizing various examples of the apparatuses described herein.

DETAILED DESCRIPTION

[0025] The accompanying figures, in which like reference numerals refer to identical or functionally similar elements throughout the separate views and which are incorporated in and form a part of the specification, further illustrate the present implementation(s) and, together with the detailed description of the implementation(s), serve to explain the principles of the present implementation(s). As understood by one of skill in the art, the accompanying figures are provided for ease of understanding and illustrate aspects of certain examples of the present implementation(s). The implementation(s) is/are not limited to the examples depicted in the figures.

[0026] The terms “connect,” “connected,” “contact,” “coupled,” and/or the like are broadly defined herein to encompass a variety of divergent arrangements and assembly techniques. These arrangements and techniques include, but are not limited to (1) the direct joining of one component and another component with no intervening components therebetween (i.e., the components are in direct physical contact); and (2) the joining of one component and another component with one or more components therebetween, provided that the one component being “connected to” or “contacting” or “coupled to” the other component is somehow in operative communication (e.g., electrically, fluidly, physically, optically, etc.) with the other component (notwithstanding the presence of one or more additional components therebetween). It is to be understood that some components that are in direct physical contact with one another may or may not be in electrical contact and/or fluid contact with one another. Moreover, two components that are electrically connected, electrically coupled, optically connected, optically coupled, fluidly connected or fluidly coupled may or may not be in direct physical contact, and one or more other components may be positioned therebetween.

[0027] The terms “including” and “comprising”, as used herein, mean the same thing.

[0028] The terms “substantially,” “approximately,” “about,” “relatively,” or other such similar terms that may be used throughout this disclosure, including the claims, are used to describe and account for small fluctuations, such as due to variations in processing, from a reference or parameter. Such small fluctuations include a zero fluctuation from the reference or parameter as well. For example, they can refer to less than or equal to ± 10%, such as less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1%, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%. If used herein, the terms “substantially,” “approximately,” “about,” “relatively,” or other such similar terms may also refer to no fluctuations, that is, ± 0%.

[0029] As used herein, a “flow cell” can include a device optionally having a lid extending over a reaction structure to form a fluidic channel therebetween that is in communication with a plurality of reaction sites (e.g., nano wells) of the reaction structure, and can optionally include a detection device that detects designated reactions that occur at or proximate to the reaction sites. A flow cell may include a solid-state light detection or “imaging” device, such as a Charge-Coupled Device (CCD) or Complementary Metal-Oxide Semiconductor (CMOS) (light) detection device. For example, the image sensor structure of a sensor system can include an image layer disposed over a base substrate. The image layer may be a dielectric layer, such as SiN and may contain an array of light detectors disposed therein. A light detector as used herein may be, for example, a semiconductor, such as a photodiode, a complementary metal oxide semiconductor (CMOS) material, or both. The light detectors detect light photons of emissive light that is emitted from the fluorescent tags attached to the strands supported in or on the reaction sites, for example, in nanowells. The base substrate may be glass, silicon or other like material. As another specific example, a flow cell can fluidically and electrically couple to a cartridge (optionally having an integrated pump), which can fluidically and/or electrically couple to a bioassay system. A cartridge and/or bioassay system may deliver a reaction solution to reaction sites of a flow cell according to a predetermined protocol (e.g., sequencing -by-synthesis), and perform a plurality of imaging events. For example, a cartridge and/or bioassay system may direct one or more reaction solutions through the fluidic channel of the flow cell, and thereby along the reaction sites. At least one of the reaction solutions may include four types of nucleotides having the same or different fluorescent labels. In some examples, the nucleotides bind to the reaction sites of the flow cell, such as to corresponding oligonucleotides at the reaction sites. The cartridge and/or bioassay system in these examples then illuminates the reaction sites using an excitation light source (e.g., solid-state light sources, such as light-emitting diodes (LEDs), and lasers). In some examples, the excitation light has a predetermined wavelength or wavelengths, including a range of wavelengths. The fluorescent labels excited by the incident excitation light may provide emission signals (e.g., light of a wavelength or wavelengths that differ from the excitation light and, potentially, each other) that may be detected by the light sensors of the flow cell.

[0030] Flow cells described herein perform various biological or chemical processes. More specifically, the flow cells described herein may be used in various processes and systems where it is desired to detect an event, property, quality, or characteristic that is indicative of a designated reaction. For example, flow cells described herein may include or be integrated with light detection devices, sensors, including but not limited to, biosensors, and their components, as well as bioassay systems that operate with sensors, including biosensors.

[0031] The flow cells facilitate a plurality of designated reactions that may be detected individually or collectively. The flow cells may perform numerous cycles in which the plurality of designated reactions occur in parallel. For example, the flow cells may be used to sequence a dense array of DNA features through iterative cycles of enzymatic manipulation and light or image detection/acquisition. As such, the flow cells may be in fluidic communication with one or more microfluidic channels that deliver reagents or other reaction components in a reaction solution to a reaction site of the flow cells. The reaction sites may be provided or spaced apart in a predetermined manner, such as in a uniform or repeating pattern. Alternatively, the reaction sites may be randomly distributed. Each of the reaction sites may be associated with one or more light guides and one or more light sensors that detect light from the associated reaction site. In one example, light guides include one or more filters for filtering certain wavelengths of light. The light guides may be, for example, an absorption filter (e.g., an organic absorption filter) such that the filter material absorbs a certain wavelength (or range of wavelengths) and allows at least one predetermined wavelength (or range of wavelengths) to pass therethrough. In some flow cells, the reaction sites may be located in reaction recesses or chambers, which may at least partially compartmentalize the designated reactions therein.

[0032] As used herein, a “designated reaction” includes a change in at least one of a chemical, electrical, physical, or optical property (or quality) of a chemical or biological substance of interest, such as an analyte-of-interest. In particular flow cells, a designated reaction is a positive binding event, such as incorporation of a fluorescently labeled biomolecule with an analyte-of-interest, for example. More generally, a designated reaction may be a chemical transformation, chemical change, or chemical interaction. A designated reaction may also be a change in electrical properties. In particular flow cells, a designated reaction includes the incorporation of a fluorescently labeled molecule with an analyte. The analyte may be an oligonucleotide and the fluorescently labeled molecule may be a nucleotide. A designated reaction may be detected when an excitation light is directed toward the oligonucleotide having the labeled nucleotide, and the fluorophore emits a detectable fluorescent signal. In another example of flow cells, the detected fluorescence is a result of chemiluminescence or bioluminescence. A designated reaction may also increase fluorescence (or Forster) resonance energy transfer (FRET), for example, by bringing a donor fluorophore in proximity to an acceptor fluorophore, decrease FRET by separating donor and acceptor fluorophores, increase fluorescence by separating a quencher from a fluorophore, or decrease fluorescence by co-locating a quencher and fluorophore.

[0033] As used herein, “electrically coupled” and “optically coupled” refers to a transfer of electrical energy and light waves, respectively, between any combination of a power source, an electrode, a conductive portion of a substrate, a droplet, a conductive trace, wire, waveguide, nanostructures, other circuit segment and the like. The terms electrically coupled and optically coupled may be utilized in connection with direct or indirect connections and may pass through various intermediaries, such as a fluid intermediary, an air gap and the like.

[0034] As used herein, a “reaction solution,” “reaction component” or “reactant” includes any substance that may be used to obtain at least one designated reaction. For example, potential reaction components include reagents, enzymes, samples, other biomolecules, and buffer solutions, for example. The reaction components may be delivered to a reaction site in the flow cells disclosed herein in a solution and/or immobilized at a reaction site. The reaction components may interact directly or indirectly with another substance, such as an analyte-of-interest immobilized at a reaction site of the flow cell.

[0035] As used herein, the term “reaction site” is a localized region where at least one designated reaction may occur. Reaction sites in the context of the biosensors described herein can also be referred to as nanowells. Although nanowells are often used in the examples herein for illustrative purposes, the examples of flow cells depicted herein can include various functionalized sensor surfaces, including but not limited to surfaces of reaction recesses, such as nanowells, reaction protrusions, such as posts, and flat surfaces. A reaction site may include support surfaces of a reaction structure or substrate where a substance may be immobilized thereon. For example, a reaction site may include a surface of a reaction structure (which may be positioned in a channel of a flow cell) that has a reaction component thereon, such as a colony of nucleic acids thereon. In some flow cells, the nucleic acids in the colony have the same sequence, being for example, clonal copies of a single stranded or double stranded template. However, in some flow cells a reaction site may contain only a single nucleic acid molecule, for example, in a single stranded or double stranded form.

[0036] The terms “active surface” and “active area” and “active detection area” are used herein to characterize a surface or area of a reaction structure which operates to support one or more designation reactions. Throughout this disclosure, the terms die and wafer are also used in reference to certain examples herein, as a die can include a sensor and the die is fabricated from a wafer. The words wafer and substrate are also used interchangeably herein.

[0037] Examples described herein may be used in various biological or chemical processes and systems for academic or commercial analysis. More specifically, examples described herein may be used in various processes and systems where it is desired to detect an event, property, quality, or characteristic that is indicative of a designated reaction. For instance, examples described herein include cartridges, biosensors, and their components as well as bioassay systems that operate with cartridges and biosensors. In particular examples, the cartridges and biosensors include a flow cell and one or more image sensors that are coupled together in a substantially unitary structure.

[0038] The bioassay systems may be configured to perform a plurality of designated reactions that may be detected individually or collectively. The biosensors and bioassay systems may be configured to perform numerous cycles in which the plurality of designated reactions occurs in parallel. For example, the bioassay systems may be used to sequence a dense array of DNA features through iterative cycles of enzymatic manipulation and image acquisition. Alternatively, rather than iterative cycles, the bioassay system can also be used to sequence a dense array of DNA features utilizing continuous observation without stepwise enzymatic action. The cartridges and biosensors may include one or more microfluidic channels that deliver reagents or other reaction components to a well or reaction site. Some examples discussed herein utilize wells and/or nano-wells as reactions sites. However, as used herein, the term “reaction site” is not limited to wells or nano-wells and contemplates various structures on a surface of the examples described herein.

[0039] In some examples, the wells or reaction sites are randomly distributed across a substantially planar surface. For example, the wells or reaction sites may have an uneven distribution in which some wells or reaction sites are located closer to each other than other wells or reaction sites. In other examples, the wells or reaction sites are patterned across a substantially planar surface in a predetermined manner. Each of the wells or reaction sites may be associated with one or more image sensors that detect light from the associated reaction site. Yet in other examples, the wells or reaction sites are located in reaction chambers that compartmentalize the designated reactions therein.

[0040] In some examples, image sensors may detect light emitted from wells (e.g., nanowells) or reaction sites and the signals indicating photons emitted from the wells or reaction sites and detected by the individual image sensors may be referred to as those sensors' illumination values. These illumination values may be combined into an image indicating photons as detected from the wells or reaction sites. Such an image may be referred to as a raw image. Similarly, when an image is composed of values which have been processed, such as to computationally correct for crosstalk, rather than being composed of the values directly detected by individual image sensors, that image may be referred to as a sharpened image.

[0041] In some examples, image sensors (e.g., photodiodes) are associated with corresponding wells or reaction sites. An image sensor that is associated with a reaction site is configured to detect light emissions from the associated reaction site when a designated reaction has occurred at the associated reaction site. In some cases, a plurality of image sensors (e.g., several pixels of a camera device) may be associated with a single reaction site. In other cases, a single image sensor (e.g., a single pixel) may be associated with a single reaction site or with a group of wells or reaction sites. The image sensor, the reaction site, and other features of the biosensor may be configured so that at least some of the light is directly detected by the image sensor without being reflected. In the examples herein, two reaction sites are often associated with a common single pixel.

[0042] Depending on the context, the term “image sensor” is utilized interchangeably herein to refer to both an array of individual pixels/photodiodes and/or an individual light sensor or pixel (which the array comprises). In the context of the examples described herein, an image sensor, which is an array, generates one or more signals. The sensors discussed in the examples herein may include image sensors such as front side illuminated sensors (FSIs) and back-side illuminated sensors (BSIs).

[0043] As used herein, the term “adjacent” when used with respect to two wells or reaction sites means no other reaction site is located between the two wells or reaction sites. The term "adjacent" may have a similar meaning when used with respect to adjacent detection paths and adjacent image sensors (e.g., adjacent image sensors have no other image sensor therebetween). In some cases, a reaction site may not be adjacent to another reaction site; but may still be within an immediate vicinity of the other reaction site. A first reaction site may be in the immediate vicinity of a second reaction site when fluorescent emission signals from the first reaction site are detected by the image sensor associated with the second reaction site. More specifically, a first reaction site may be in the immediate vicinity of a second reaction site when the image sensor associated with the second reaction site detects, for example, crosstalk from the first reaction site. Adjacent wells or reaction sites may be contiguous, such that they abut each other, or the adjacent sites may be non-contiguous, having an intervening or interstitial space between.

[0044] As used here, the term “electrochromic” as used in the phrase “electrochromic material” is a material that changes color or opacity when placed in an electric field. Hence, electrochromic material (also referred to as a chromophore) vary in opacity (colored vs. bleached) when exposed to specific voltages (behavior varies based on the material and the thickness). When voltage is applied to these materials, the optical color or opacity of a surface is affected. A common example of a component comprised of an electrochromic material is the liquid crystal display (LCD) of many calculators. Although the various examples herein utilize electrochromic materials, other voltage switching materials can be substituted in all examples. Electrochromic materials in the examples herein can include, but are not limited to WO3 (tungsten trioxide), molybdenum, titanium, and niobium oxides. In general, electrochromism occurs in some transition metal oxides which conduct both electrons and ions (e.g., WO3). These oxides have octahedral structures of oxygen which surround a central metal atom and are joined together at the comers. This arrangement produces a three-dimensional nanoporous structure with tunnels between individual octahedral segments. These tunnels allow dissociated ions to pass through the substance when they are motivated by an electric field.

[0045] The term chemical vapor deposition (CVD) refers to a vacuum deposition method used to produce high quality, and high-performance, solid materials, including, in some of the examples herein, films. In some examples, a substrate (e.g., a silicon wafer) wafer (substrate) is exposed to one or more volatile precursors, which react and/or decompose on the substrate surface to produce a desired deposit. As discussed herein, plasma-enhanced chemical vapor deposition (PECVD) is a chemical vapor deposition process used to deposit thin films from a gas state (vapor) to a solid state on a substrate. In the context of the examples herein, CVD and/or specifically PECVD is utilized to deposit an oxide layer with a low index of refraction (referred to also as a low index oxide layer, e.g., SiO (silicon monoxide)) on certain of the apparatuses discussed. This description also includes references to high index oxide materials, which refer to materials with a high index of refraction, including but not limited to SiN (silicon nitride).

[0046] The term chemical mechanical polishing or planarization (CMP) is a process (both polishing and planarization being options under the umbrella term) applied to selectively remove materials for topography planarization and device structure formation. CMP uses chemical oxidation and mechanical abrasion to remove material and achieve planarity. In some examples, CMP includes using a chemical reaction and mechanical abrasion with slurries containing unique chemical formulations and large numbers of abrasive particles. During polishing, chemical reaction products and mechanical wear debris are generated. Slurry particles and polishing byproducts are pressed onto wafer surface. During wafer transferring from polisher to cleaner, contaminants are adhered onto wafer surface. This process can include a cleanup of the surface that is polished and/or planarized to remove particles including organic residues. Certain of the workflows disclosed herein incorporate a CMP aspect to planarize surfaces. CMP can be utilized in the examples herein, for example, after depositions into high aspect ratio topography, which may impact the topography of the deposited top film (i.e., layer). However, even when incorporated into the examples herein, in some circumstances, this aspect can be omitted.

[0047] As used herein the term “emission filter ” refers to a filter that suitably prevents/blocks transmission of excitation wavelengths while suitably allowing transmission of emission wavelengths. For example, an emission filter can be a high quality optical-glass filter commonly used in fluorescence microscopy and spectroscopic applications for selection of the excitation wavelength of light from the light source. An excitation wavelength is a wavelength in the excitation spectrum, a range of light wavelengths that add energy to a fluorochrome, causing it to emit wavelengths of light (e.g., the emission spectrum).

[0048] As used herein, the term “crosstalk” refers to any phenomenon by which a signal transmitted on one circuit or channel of a transmission system creates an undesired effect in another circuit or channel. Crosstalk is usually caused by undesired capacitive, inductive, or conductive coupling from one circuit or channel to another. Crosstalk can be a significant issue in structured cabling, audio electronics, integrated circuit design, wireless communication, and other communications systems. In the context of certain of the examples herein, crosstalk includes a proportion of optical signals from a given reaction site reaching light sensors or pixels that do not form a sensing pair with the reaction site. In examples where each image sensor represents a single pixel, crosstalk may be understood to mean the proportion of optical signals reaching all pixels other than the center pixel. However, in the examples, herein, because each pixel is associated with two reaction sites, crosstalk can also refer to interference between signals targeted to the same pixel. Attenuation, or signal loss, can result from crosstalk. Additionally, crosstalk increases noise in pixels within an immediate vicinity of a reaction center. [0049] As used herein, the term “common voltage” when applied to the apparatuses described herein, refers to a similar amount of volts. As voltage is applied to electrochromic materials herein, a window that utilizes an electrochromic layer to regulate light would be, for example, approximately 5 V. Electrochromic materials, in general, reversibly respond to one or more surrounding stimuli (e.g., chemical, electrical, light, temperature, and mechanical force). In the examples herein, the stimulus employed in electrical. When electrochromic materials are noted as responding to a common voltage, this means that the opacity response of the two or more materials encompassed in the electrochromic materials described have a similar response to being stimulated by the same or a similar voltage (e.g., pressure from an electrical circuit's power source).

[0050] Reference is made below to the drawings, which are not drawn to scale for ease of understanding, wherein the same reference numbers are used throughout different figures, in some cases, to designate the same or similar components. The following detailed description of certain examples will be better understood when read in conjunction with the appended drawings. The various examples are not limited to the arrangements and instrumentality shown in the drawings.

[0051] As will be discussed in greater detail herein, the examples herein include an apparatus, which includes a flow cell, and methods of manufacturing and utilizing this apparatus where the apparatus can more efficiently produce results when compared to other apparatuses utilized for the same purpose. The examples herein include electrochromic materials. By integrating electrochromic materials into the apparatus, and doubling the number of reaction sites at a given location (e.g., doubling the number of nanowells if nanowells are included in the reaction sites), the apparatus can produce approximately double the output of a similar apparatus that does not include this feature. Specifically, while existing versions of this apparatus can test a single cluster of biological or chemical samples at a reaction site that is electrically coupled to a given pixel of a sensor comprising a plurality of pixels, in the examples herein, each pixel is electrically coupled to two reaction sites and hence, the apparatus can test twice as many clusters per pixel.

[0052] FIG. 1 is an apparatus 100 that incorporates various aspects described in examples herein. The apparatus 100 can include light energy exciter 10 and flow cell 282 Various aspects of the flow cell 282 of the apparatus 100 enable the throughput to be doubled when compared to a traditional flow cell. In many flow cells, a given reaction site is assigned to a given pixel sensor (which includes a pixel) in a semiconductor layer. Thus, the pixel sensor (the singular pixel) helps identify the contents of a cluster in the reaction site 206 when the apparatus is in use. In the apparatus 100 of FIG. 1, a single pixel sensor (which includes a pixel) can help identify two clusters as two reaction sites are assigned to this single pixel sensor. FIG. 2 is a partial cross-sectional top view of taken along the elevation of pixel sensors 202, which are components of apparatus 100 of FIG. 1. The remaining figures highlight various aspects of examples of the type of apparatus of which the apparatus 100 of FIG. 1 is an example.

[0053] In FIG. 1 there is shown an apparatus 100 for use in analysis, such as biological or chemical analysis. The apparatus 100 can include light energy exciter 10 and flow cell 282. Flow cell 282 can include detector 200 and an area above detector 200. Detector 200 can include a plurality of pixels 201 and detector surface 209 for supporting clusters Cl and C2; the clusters are biological or chemical samples subject to test. Sidewalls 284 and flow cover 288, as well as detector 200 having detector surface 209, can define and delimit flow channel 283. Elevation 130 can define a cluster supporting surface of reaction structure 260 according to one example. Where reaction structure 260 includes nano wells 208, elevation 130 can define an elevation of a plane that extends coextensively with respective cluster supporting bottom surfaces of such nanowells. A nanowell 208 is a non-limiting example of a type of reaction site that can be utilized in a flow cell 282. As aforementioned, each respective pixel 201 (and ultimately each pixel sensor 202) in the flow cell 282 obtains optical signals via two reaction sites 206. Respective pixels 201 can include a light guide 214 and a pixel sensor 202. Some flow cells 282 included in the types of apparatus 100 herein do not include light guides 214 (e.g., FIGS. 5A-5B). Respective pixels 201 and pixel sensors 202 can include respective vertically extending center axes 219.

[0054] Clusters Cl, C2 (e.g., biological or chemical samples subject to test) can include one or more strands, such as one or more DNA strand. Strands herein, according to one example can include monoclonal DNA strands.

[0055] In a further aspect, detector surface 209 can be configured to define reaction sites 206 which, in one example, can be provided by nanowells 208. According to one example, each reaction site 206 can be associated to a certain pixel 201 and certain pixel sensor 202 of the certain pixel 201. In FIG. 1, each of cluster (e.g., Cl and C2) can be supported on a respective reaction site 206, which can be defined by a nanowell 208; each pixel 201 (including its pixel sensor 202) supports two reaction sites 206 and hence, two clusters Cl, C2. Detector surface 209 can be defined by surfaces defining nano wells, as well as surfaces intermediate of nano wells as is indicated by FIG. 1. In this apparatus 100, two reaction sites 206 can be associated with a certain pixel sensor 201 and a certain pixel sensor 202, can accommodate two clusters.

[0056] Detector 200 can include, according to one example, dielectric stack 213, semiconductor layer 212, and light guides 214 disposed in a light path between detector surface 209 and pixel sensors 202, and isolation structures 218 defining and delimiting pixel areas above respective ones of pixel sensors 202. While isolation structure 218 is shown extended through the semiconductor layer 212, other examples may include isolation structures 218 that stop at or short of the semiconductor layer 212. Dielectric stack 213 can, in one example, include metallization layers defining various circuitry, e.g., circuitry for readout of signals from sensing pixels, digitization, storage, and signal processing. Metallization layers defining such circuitry can additionally or alternatively be incorporated into isolation structures 218.

[0057] Pixel sensors 202, in one example, can be provided by sensing photodiodes. Sensing photodiodes, in one example, can be defined by doped areas of semiconductor layer 212. Examples herein recognize that “area” as referred to herein can refer to a volumetric space (in other words, not limited to a 2-dimensional space).

[0058] According to one example, detector 200 can be provided by a solid-state integrated circuit detector, such as complementary metal oxide semiconductor (CMOS) integrated circuit detector or a charge coupled device (CCD) integrated circuit detector. Specifically, a CMOS or a CCD can comprise the semiconductor layer 212. Pixel sensors 202, in one example, can be provided in a two-dimensional pixel array having rows and columns of pixels arranged in a grid pattern that is shown in the cross- sectional top view of FIG. 2 taken along the elevation of pixel sensors 202. In one example, such pixel array can include at least IM pixels, or can include fewer pixels.

[0059] In one aspect, pixels 201 herein can include respective pixel sensors 202 and light guides 214. Light guides 214 can be disposed in an area above respective pixel sensors 202 and can be bounded by isolation structures 218 and reaction structure 260.

[0060] According to one example, apparatus 100 can be used for performance of biological or chemical testing with use of analytes provided by fluorophores. For example, a fluid having one or more fluorophores can be caused to flow into and out of flow cell 282 through an inlet port using inlet port 289 and outlet port 290. Analytes provided by fluorophores can attract to various clusters Cl, C2 and thus, by their detection, analytes provided by fluorophores can act as markers for the clusters Cl, C2, e.g., biological or chemical analytes to which they attract.

[0061] To detect the presence of an analyte provided by a fluorophore within flow cell 282, light energy exciter 10 can be energized so that excitation light 101 in an excitation wavelength range is emitted by light energy exciter 10. On receipt of excitation light 101, fluorophores attached to clusters Cl, C2 can radiate emission light 501, which is the signal of interest for detection by pixel sensors 202. Emission light 501 owing to fluorescence of a fluorophore attached to a cluster Cl, C2 can have a wavelength range red shifted relative to a wavelength range of excitation light 101.

[0062] Light energy exciter 10 can include at least one light source and at least one optical component to illuminate clusters Cl, C2. Examples of light sources can include, but are not limited to, lasers, arc lamps, LEDs, or laser diodes. The optical components can include, for example, reflectors, dichroics, beam splitters, collimators, lenses, filters (including emission filters), wedges, prisms, mirrors, detectors, and the like. In examples that use an illumination system, the light energy exciter 10 can be configured to direct excitation light 101 to reaction sites 206. As one example, fluorophores can be excited by light in the green wavelength range, e.g., can be excited using excitation light 101 having a center (peak) wavelength of about 523 nm.

[0063] Referring to FIG. 1, as aforementioned, the throughput of the flow cell 282 in the apparatus 100 is approximately twice the throughput of a flow cell where each pixel 201 (and its respective pixel sensor 202) is assigned to only a single reaction site; in flow cell 282 each pixel 201 can receive an optical signal from two reaction sites 206. To avoid optical signal interference (and to enable aspects of the flow cell 282 to separately identify the cluster Cl C2 at each reaction site 206 assigned to a common pixel 201 and pixel sensor 202, the flow cell 282 includes one or more layers 252 comprising voltage-switchable materials, referred to herein (as a non-limiting example) as electrochromic materials (and electrochromic material is an example of a voltage-switchable material).

[0064] In the recitation of certain workflows of forming portions of the flow cell 282, the one or more layers 252 are referred to as voltage switchable layers. The one or more layers 252 are communicatively coupled to the reaction sites 206 (which can include a nanowell 208) so that each material comprising the one or more layers 252 can (selectively, based on voltage) transmit a signal to the respective pixel 202 for the given reaction site 206, where each pixel is assigned to two reaction sites 206. In some examples, two or more electrochromic materials comprising the one or more layers 252 are selected to vary in opacity at a common voltage, meaning that the same voltage produces the same or similar effects (e.g., opacity in the layer(s)) when applied to the two or more electrochromic materials comprising the one or more layers 252. In some examples, each electrochromic material (of the at least two electrochromic materials) is an individual layer of the one or more layers 252 and the one or more layers 252 are patterned such that although no single layer or material covers an entire surface of the flow cell 282, and instead, a portion of each layer of each material is in electrical contact with each reaction site 206. In some examples (which will be illustrated in the additional figures), a first layer of electrochromic material (of the one or more layers 252) is configured to selectively transmit a signal to a pixel 201 when a first voltage is applied to a first reaction site 206 and a second layer (of the one or more layers 252) is configured to selectively transmit the signal to this same pixel 201 when a second voltage is applied to the a second reaction site 206. The first voltage and the second voltage can be different because the voltage switching (which separates the signals and enables the doubling of the clusters at each reaction site 206) can function based on the materials being of differing opacities at a common voltage - when a layer is not opaque, it can block a signal or a portion of a signal (a portion of photons) from the reaction site 206 to the pixel 201 and pixel sensor 202. In other examples, a common voltage is selectively applied to a layer (of the one or more layers 252) and its respective reaction site 206, while voltage is not applied to another layer (of the one or more layers 252) and its respective reaction site 206, wherein both these reaction sites are electrically coupled to a common pixel 201. The opacity of the reaction sites changes based on the applied voltage, so that one can select which cluster (which material) feeds the signal to the pixel 201 or adjust the strength of the optical signal received relative to each reaction site 206. Above each of the one or more layers 252 is a filter layer 541 acting as an emission filter.

[0065] The one or more layers 252 are situated in signals paths from the respective reaction sites 206 to the pixel sensors 202. As aforementioned, each pixel 201 and pixel sensor 202 receives signals from two reaction sites 206. Thus, for each pixel 201, a first reaction site configured to emit light responsive to a first cluster Cl at a first reaction site 206 (for a given pixel 201) being exposed to the excitation light (from the light energy exciter 10). The emitted light from the first reaction site is propagated through the detector 200 (e.g., biosensor) on a signal path 257 to a pixel 201 of the plurality of pixels as an optical signal. A second reaction site 206 (or a given pixel 201) is configured to emit light responsive to a second cluster C2 at the second reaction site 206 being exposed to the excitation light(from the light energy exciter 10). The emitted light from the second reaction site 206 is propagated through the detector 200 (e.g., biosensor) on a second signal path 257 to a pixel 201 of the plurality of pixels as a second optical signal. Of the one or more layers 252, a first layer associated to the pixel 201 can situated in the first signal path. The first layer (of the one or more layers 252) can be configured to selectively open the signal path 257 of the first optical signal when a first voltage is applied to the first layer. A second layer (of the one or more layers 252) can be situated between the second reaction site 206 and the pixel in a signal path of the second optical signal. The second layer can be configured to selectively open the signal path of the second optical signal when a second voltage is applied to the second layer.

[0066] Hence, electrochromic materials can be utilized in the examples herein (in the one or more layers 252) in a manner similar to how they are utilized in smart windows, which is a more common use of these materials. For a smart window to operate, one can apply a high voltage (approximated 4 V or more) to push lithium-ions into the electrochromic layer, activating the electrochromic material. When this layer is activated, it is non-transparent. For example, when the layer is activated via the introduction of lithium-ions, its oxidation state changes and hence its color changes to dark blue. By applying a lower voltage (approximately 2.5 V for example) the concentration of ions in the electrochromic layer decreases, thus deactivating the electrochromic material (e.g., WO3). When the electrochromic material is deactivated, it is transparent or suitably transparent. Depending on the electrochromic material used, different parts of the spectrum can be blocked, this way UV, visible and IR light can be independently reflected. In the flow cell 282, when the electrochromic layer is opaque, rather than transmitting an optical signal to the pixel 201, it blocks/reflects emissive light (the filter layer 541 blocks excitation light) from the light that originates from the light energy exciter 10. In FIG. 1, the filter layer 541 is situated between the one or more layers 252 and each pixel 101, but in some examples, the filter layer 541 can be situated below the one or more layers 252, enroute to the pixel 201 (in the signal path 257).

[0067] In the flow cell 282 of FIG. 1, the transmission of an optical signal to a common pixel sensor 202 (and pixel 201) by the two reaction sites 206 associated with the pixel sensor 202 can be controlled (at least) by: 1) applying two different voltages to the one or more layers 252, where the one or more layers 252 can be comprised of the same electrochromic material; 2) applying the same voltage to each of the two reaction sites 206 associated with the pixel sensor 202, but not at the same time such that one reaction site of the two reaction sites 206 will transmit a signal to the shared pixel sensor 202 when the other will not, where the one or more layers can be comprised of the same electrochromic material; and/or 3) applying the same voltage to each of the two reaction sites 206 associated with the pixel sensor 202 at the same time, where the one or more layers 252 are comprised of different electrochromic materials such that the opacity of the one or more layers 252 varies at the applied voltage.

[0068] Although the example where the shared pixel sensor 202 receives an optical signal (e.g., photons) from one reaction site 206 assigned to the shared pixel sensor 202 at a given time is easier to understand and illustrate, in some examples, one can expose a portion of one or more layers 252 to a given voltage to change the strength of the optical signal (e.g., number of photons) transmitted to the shared pixel sensor 202. Controlling the signal from a given reaction site 206 can be useful because depending on the processes being completed in the flow cell 282, this voltage switching aspect can be utilized to differentiate two optical signals from the two reaction sites 206 associated with the common pixel sensor 202 from each other. As with examples that include effectively turning on and off the respective reaction sites 206 associated with a common pixel sensor 202 (so only one optical signal is received by a pixel at a given time), in some examples herein, when the common pixel sensor 202 receives two optical signals but can differentiate the optical signals from the two reaction sites 206 associated with the pixel sensor 202, the flow cell 282 can be configured in a similar manner. Utilizing the apparatus 100 such that a pixel 201 and pixel sensor 202 receive two optical signals (which it can differentiate from each other) from the two reaction sites 206 associated with this pixel sensor 202 can include: 1) applying two different voltages to the one or more layers 252, where the one or more layers 252 can be comprised of the same electrochromic material; 2) applying the same voltage to each of the two reaction sites 206 associated with the pixel sensor 202, but not at the same time such that one reaction site of the two reaction sites 206 will transmit a signal to the shared pixel sensor 202 when the other will not, where the one or more layers can be comprised of the same electrochromic material; and/or 3) applying the same voltage to each of the two reaction sites 206 associated with the pixel sensor 202 at the same time, where the one or more layers 252 are comprised of different electrochromic materials such that the opacity of the one or more layers 252 varies at the applied voltage.

[0069] FIGS. 3-4 are cross-sectional views 300400 of the flow cell 282 of FIGS. 1 and 5 taken at an elevation of the one or more layers 252. In this example, the flow cell 282 comprises two layers A B, comprised of one or more electrochromic materials. The layers A B are patterned on a surface of the flow cell 282 such that each pixel 201 is covered (to some extent) by each of the layers A B. The layers A B are positioned between each reaction site 206 and its respective pixel 201 (and hence, pixel sensor 202). These layers can be comprised of the same electrochromic material (WO3) provided that each layer A B is exposed to a voltage at a different time (e.g., thus only one layer is activated at once, meaning that the signal received by a pixel sensor 202 from one layer A is different from the signal received at the same time from a second layer B, including only receiving an optical signal from one of the layers and not the other at the given time). These layers A B can also each be comprised of different electrochromic materials so that when exposed to the same voltage, the opacity of the layers varies so the optical signals can be differentiated by a pixel sensor 202 (e.g., depending on the opacity, one of the layers A B may not transmit a signal at all).

[0070] For ease of understanding, a configuration where reaction sites 206 are turned on an off based on exposing layers A B associated with these sites to one or more voltage, is discussed in more detail than the configuration where both reaction sites 206 are still active but the optical signals transmitted at a given time by both can be differentiated based on exposing layers A B associated with these sites to one or more voltage. However, as one or more voltages are utilized to control the opacity of the layers A B, both types of transmission results can be produced depending on the configuration of the flow cell 282.

[0071] In FIG. 3, the layers A B are configured in a zebra striped pattern, while in FIG. 4, the pattern of the layers A B is angled such that rather than the layers A B appearing horizonal in a cross- sectional view 300, the layers A B appear at a diagonal orientation from on a cross-sectional view 400. FIGS. 5A and 5B (collectively referred to as “FIG. 5”) are cross-sectional views 500 of a portion of a detector 500 that includes various aspects of the detector 200 in the apparatus 100 of FIG. 1. The difference between these detectors 200 is the placement of the filter layer 541a-541b and the layers A B. In FIG. 5A, as will be discussed below, emissions from the reaction sites (e.g., a first reaction site 206a and a second reaction site 206b) are filtered by a filter layer 541a-541b before progressing to a voltage switching layer (e.g., layer A or layer B). In FIG. 5B, the emissions from the reaction sites (e.g., a first reaction site 206a and a second reaction site 206b) can be blocked, transmitted, or partially transmitted through a voltage switching layer (e.g., layer A or layer B) and if transmitted or partially transmitted through the voltage switching layer, will then reach a filter layer 541a-541b.

[0072] Referring to FIG. 5 (both 5A and 5B), in some examples, the first layer A does not transmit the signal to the pixel sensor 202 of a pixel 201 (in FIG. 5B, via a filter layer) when the second voltage (a voltage compatible with the second layer B) is applied to the first layer A. Alternatively, in some examples, the second layer B does not transmit a signal to the pixel sensor 202 of the pixel 201 (in FIG. 5B, via a filter layer) when the first voltage (a voltage compatible with the first layer A) is applied to the second layer B. Depending on the placement of the cluster Cl, C2, depending on the voltage applied, the flow cell 282 can generate results for one of the two clusters at the reaction sites 206 that transmits an optical signal to a given pixel sensor 202 of a pixel 201 by utilizing the advantages of the voltage switching materials comprising the two layers A B. In some examples, the layers A B can transmit at the same voltage but only one layer is exposed to a given voltage at one time. Thus, only one layer (and the reaction site 206 this layer is patterned below) is active. Thus, a given pixel sensor 202 of a pixel 20 can sense either cluster (e.g., either Cl or C2), alternatively (and as aforementioned, in some examples, because of the voltage-switching mechanism described herein, the given pixel sensor 202 of a pixel 201 can sense both clusters, but can differentiate them). In some examples, an electrical contact 207 (e.g., a bond pad) at an edge of the apparatus controls the voltage that is applied to the apparatus (e.g., the voltage switching). In FIG. 1, the electrical contact 207 is illustrated at only one possible orientation, proximate to sidewalls 284 which assist in defining and delimiting the flow channel 283. However, the orientation of the electrical contact 207 can vary provided that is in electrical contact (either directly or indirectly via other connections) with the one or more layers 252.

[0073] The larger broken lined frames in FIGS. 3-4 frame the plurality of pixels 201 (including the pixel sensors 202) and reaction structure 260, and the inner rectangular frames a portion of the layers A B above a respective pixel 201 and pixel sensor 202. The alternating layers can enable each layer A B to provide optical signals (via the voltage switching aspects described herein) to a common pixel 201 and pixel sensor 202. When portions of one or more layers 252 are exposed to a voltage, the opacity of the layers A B is regulated by the voltage.

[0074] FIG. 5 illustrates the one or more layers 252 (e.g., layer A, layer B). In FIG. 5, each pixel 201 (including each pixel sensor 202 in a semiconductor layer 212) can accommodate two clusters Cl C2 (FIG. 1), each at a different reaction site 206 as there are two reaction sites from which a common pixel sensor 202 (and pixel 201) receives optical signals (either not at the same time or if any the same time, at varying strengths such that the signals can be differentiated by the pixel sensor 202). Unlike the detector 200 in the apparatus 100 of FIG. 1, this detector 200 does not include light guides 214 or other cross-talk mitigation structures. In general, aspects in the examples described herein, including the use of a voltage switchable layer and an addition of a reaction site for each pixel sensor in a semiconductor layer, can be integrated into flow cells of various types. Those provided in this disclosure are non-limiting examples provided for illustrative purposes. However, these aspects, which increase throughput in matter analysis, can be integrated into both front side illuminated sensors (FSIs) and back-side illuminated sensors (BSIs).

[0075] In the detector 500 of FIG. 5, a first reaction site 206a is above a first layer, e.g., layer A, while a second reaction site 206b is above a second layer, e.g., layer B. It is within these reaction sites that clusters can be deposited while the flow cell 282 is in use. (The configuration of these layers (e.g., layers A B) at reaction sites 206 of flow cells 282 was illustrated in FIGS. 3-4). FIG. 5 illustrates a pair of given reaction sites 206a-206b, which in this non-limiting example each include nanowells 208a- 208b. In FIG. 5A, below (vertically) each nanowells 208a-208b (on a plane parallel to the bottom of each nanowell 208a-208b), is a filter layer 541a-541b (e.g., an emission filter), for filtering certain wavelengths of light. Meanwhile, in FIG. 5B, below (vertically) each of the layers A B (on a plane parallel to the bottom of each nanowell 208a-208b), is a filter layer 541a-541b (e.g., an emission filter), for filtering certain wavelengths of light. In some examples, the filter is elected to filter red light. In FIG. 5A, oriented (vertically) below each filter layer 541a-541b is one or more (e.g., electrochromic) layers A B, each of which is oriented above a semiconductor layer 212 comprising pixel sensors 202 (see, e.g., FIG. 1). In FIG. 5B, each filter layer 541a-541b is oriented between the one or more (e.g., electrochromic) layers A B and the semiconductor layer 212 comprising pixel sensors 202 (see, e.g., FIG. 1). In FIGS. 5A and 5B, both reaction sites 206a-206b are oriented above (and optically coupled) to a common pixel sensor 202. The reaction sites 206a-206b above a common pixel sensor 202 each accommodate a cluster (see, FIG. 1, Cl, C2). Reaction sites 206a-206b above a common pixel 201 (which includes a pixel sensor 202), can be referred to as frames and thus, a portion of the apparatus 100 inclusive of a first layer (layer A) forms a first frame (e.g., Frame 1) and a portion of the apparatus 100 inclusive of a second layer (layer B) forms a second frame (Frame 2). The first reaction site 206a is optically coupled to layer A and the second reaction site 206b is optically coupled to layer B; both layers A B are formed from one or more voltage-switching (e.g., electrochromic) material and hence, when the aforementioned frames (Frame 1 and Frame 2, e.g., areas comprising each layer electrically coupled to a given reaction site 206) are “switched” on and off (via exposure to voltage impacting the opacity of each layer), each reaction site 206a-206b electrically coupled to a given pixel sensor 202 is separately turned on or off. Table 1 illustrates how the layers A B and their respective reaction sites 206a and 206b are turned on and off (e.g., voltage switched) when at least a portion of the one or more layers 252 is exposed to a voltage (that can impact the opacity of the material comprising the layers 252).

Table 1

[0076] FIGS. 6A-6F illustrate various states in a workflow for forming the detector 500 of FIG. 5 A. FIG. 5 A is used as an example but the configuration of FIG. 5B can be formed using similar means.

[0077] As illustrated in FIG. 6A, one commences with a semiconductor layer 212 comprising pixel sensors 202a-202c. In this illustration, there are three individual pixels sensors 202a-202c representing a plurality of pixels sensors, but this number is provided for illustrative purposes only and as a nonlimiting example. FIG. 6B illustrates that one can form one or more layers 252 of one or more voltage- switchable (e.g., electrochromic) materials over an upper surface 226 of the semiconductor layer 212 (and hence, over the pixels sensors 202a-202c). To form these layers 252, one can deposit the voltage switchable materials over the semiconductor layer 212 using various techniques, including but not limited to, Plasma Enhanced Chemical Vapor Deposition (PECVD), sputter, e-beam evaporation, crystalline growth and etching (e.g., transfer wafer bonding or direct wafer bonding), and radical activation bonding in vacuum. In some examples, the one or more layers 252 can be spluttered using D.C. magnetron spluttering, which is a physical vapor deposition technique for depositing thin layers of fdms onto substrates. In some examples, the one or more layers 252 have an elevation (thickness) of approximately 600 nm to approximately 750 nm (e.g., as measured along a vertical axis extending upwards from the semiconductor layer 212, which is aligned on a horizontal axis). Other examples provide for one or more layers 252 that have a thickness of less than 600 nm.

[0078] In the examples herein, the one or more layers 252 can be comprised of a combination of electrochromic materials and materials that are not electrochromic. But the layer itself will have electrochromic or voltage-switching properties. Table 2 below provides examples of materials that can comprise the one or more layers 252 in the examples herein. This configuration is just one example of a possible configuration for the one or more layers 252. If this (non-limiting) configuration is utilized, the one or more layers 252 can be formed using D.C. magnetron spluttering. The thicknesses of the layers are also non-limiting examples.

Table 2

[0079] In the configuration of Table 2, the resultant one or more layers 252 will have a total thickness of approximately 600 nm, a swing voltage of approximately 2.5 V, a contrast of approximately 10% to approximately 80%, a transition time ranging between approximately 1 second and 13 seconds and a voltage drop of approximately 0.1 uV/cm.

[0080] Depending on the materials that comprise the one or more layers 252, the techniques used to deposit these materials over the semiconductor layer 212 can vary. For example, different materials that comprise the one or more layers 252 can be deposited using different techniques. The materials that comprise the example in Table 2 can all be deposited using D.C. magnetron sputtering, but in some cases, different techniques are combined to apply the one or more layers 252. Table 3 provides another (non-limiting) example for materials (e.g., individual layers) that can be combined to form the one or more layers 252. In this example, certain of the materials (ITO and WO3) can be applied using D.C. magnetron spluttering while others (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT;PSS), SPE) can be applied using spin coating.

Table 3

[0081] Table 4 provides another (non-limiting) example for materials (e.g., individual layers) that can be combined to form the one or more layers 252. In this example, certain of the materials can be applied using D.C. magnetron spluttering.

Table 4

[0082] Once the one or more layers 252 are deposited onto the semiconductor layer 212, as illustrated in FIG. 6C, one can deposit a filter layer 541 onto the one or more (voltage switching) layers 252. Various techniques can be used to deposit this layer as well, including but not limited to Plasma Enhanced Chemical Vapor Deposition (PECVD), sputter, e-beam evaporation, crystalline growth and etching (e.g., transfer wafer bonding or direct wafer bonding), and radical activation bonding in vacuum. In some examples, this filter is about 300 nm thick (e.g., as measured along a vertical axis extending upwards from the semiconductor layer 212, which is aligned on a horizontal axis).

[0083] As illustrated in FIG. 6D, to separate portions of the one or more layers 252 and the filter layer 541 into portions at what will become reaction sites, one can etch trenches 253a-253g through both the one or more layers 252 and the filter layer 541 (which can act as an emission filter) (e.g., utilizing an oxide and metal etching process). To create what will be two reaction sites above each pixel sensor (so that voltage can be applied to change the opacity of portions of the one or more layers 252 so that the processes commenced at these sites will not generate signals that interfere with each other when being transmitted to a common pixel sensor 202a-202c), the trenches are positioned such that the trenches 253a-253g are perpendicular to a horizontal plane comprising the semiconductor layer 212. For each pixel sensor 202a-202c, one forms a trench beyond each edge of the pixel sensor 202a- 202c and a trench on a vertical axis that would divide the pixel sensor 202a-202c into two parts if the trench were continued through the pixel sensor 202a-202c (which it is not). The resulting structure formed above each pixel sensor 202a-202c of the semiconductor layer 212, are two stacks (of the stacks 267a-267f) that are formed based on forming the trenches 253a-253g. The stacks 267a-267f rise from each pixel sensor 202a-202c along a vertical, perpendicular to the pixel sensors 202a-202c, where the pixel sensors 202a-202c are along a horizonal plane. These stacks are each comprised of a portion of the one or more layers 252 and a portion of the fdter layer 541 and are separated by trenches 253a- 253g where portions of the one or more layers 252 and the filter layer 541 have been removed (e.g., by etching).

[0084] Referring to FIG. 6E, once the trenches 253a-253g are etched through the one or more layers 252 and the filter layer 541, one fills the trenches and can planarize the resultant surface. CMP can be utilized to perform the planarization. For example, once the etching is complete (e.g., the trenches 253a-253g have been formed) one can deposit a substance 642, which includes filling the one or more trenches 253a-253g. The substance 642, which can include one or more materials, can be selected to accomplish one or more of the following purposes: 1) reduce crosstalk between the signals from the reaction sites 206a-206f; and 2) prevent adverse atom diffusion into the electrochromic material of the one or more layers 252. Thus, the one or more materials comprising the substance 642 can be selected to act as one or more of a diffusion layer and/or a dielectric layer. In some examples, the material 642 can act as a diffusion layer and/or a light shield. In some examples a material included in the substance 642 can be selected from materials that are electronic conductors, including but not limited to W, Al (aluminum), Cu, Cr (chromium), Ti (titanium), Ta (tantalum), TiN (titanium nitride), various alloys thereof, highly doped Si (Silicon) (or other semiconductor), certain types of perovskites, and generally, materials that can absorb emission wavelengths. Because certain metals, e.g., Cu atoms, can diffuse into the electrochromic layer (e.g., WO3) and affect its electrochromic behavior, certain examples can include a diffusion layer (e.g., TiN, which is often utilized in integrated circuits). The diffusion layers (e.g., TiN) acts as a light barrier and prevent adverse atom diffusion into the electrochromic material (one or more layers 252). In some examples, the substance 642 deposited can include both the material(s) comprising a diffusion layers and material(s) comprising a dielectric layer. In some examples, in addition to a diffusion layer, a dielectric layer (e.g., oxide or nitride layer) can be added between the light shield (e.g., TiN) and electrochromic layers (one or more layers 252). The dielectric layer is added because a possible complication from using metals as light shields is electrical shorts or electrical interference with the electrochromic mechanism. This complication is avoided with the addition of the dielectric layer.

[0085] In some examples, one can planarize the substance 642 such that a portion of the substance 642 forms a contiguous surface 674 with a top surface of the portions of the fdter layer 541. In some examples, the type of CMP utilized to planarize the surface of the fdler material (in this example, tungsten) is advanced tungsten chemical mechanical polishing (W. CMP). This technique includes the use of a slurry for high planarity.

[0086] FIG. 6F depicts forming reaction sites 206a-206f, which in this case, include nanowells 208a-208f atop the stacks 267a-267f. FIG. 7 illustrates the formation of the nanowells 208a-208f in more detail (FIG. 6F shows a general view of the completed formation of the nanowells). As illustrated in FIG. 7, one can form a first conductive layer 780 (e.g., metal) over the contiguous surface 674 (e.g., using a technique including but not limited to, metal sputtering) (728). This example of the method can then include forming a low index layer 783 (e.g., an oxide layer) over a top surface of this first conductive layer 780 (738). Atop the low index layer 783, one can form a second conductive layer 791 (748). One can deposit photoresist 711 on a first portion of a top surface of the second conductive layer 790 (758) (e.g., using photolithography). Portions of the surfaces upon which photoresist 711 is deposited are preserved during a subsequent etching process. One can then etch the portions of the second conductive 790 layer that are not covered by the photoresist 711 (e.g., utilizing an oxide and metal etching process) to form trenches 773 in both conductive layers 780, 790, and then remove the photoresist (e.g., utilizing resist strips, chemical cleaning, and/or etching) (768). In some examples, various chemistries can then be applied to the trenches 773. A passivation layer 797 can be deposited atop the top surface of the structure, which can be a silicon oxide layer (778).

[0087] The examples herein include detectors and flow cells that can be integrated into existing systems and are compatible with components utilized to form various devices, including the apparatus 100 of FIG. 1. In comparing the flow cells and fabrications methods for these cells, certain criteria that one can evaluate can include: signal, additional SNR penalty, CMOS (or CCD) fabrication (the aspects (including new perceived complexities) in forming the elements of the flow cells atop a sensor, which can be a CMOS), clustering chemistry, library preparation, software, and instrument changes. The examples herein are largely consistent and compatible with existing systems. In some examples, an additional SNR penalty of 2-3 dB can exist (during usage) and the CMOS fabrication processes include (as illustrated in FIGS. 6A-6F) the formation of a new layer, an electrochromic layer(s) (in the stack), and the addition of a second (new) nanowell assigned to a given pixel (of the CMOS). However, in these examples, the signal, clustering chemistry, library preparation, and software are consistent with existing approaches and there is no change to instruments.

[0088] The one or more layers 252 (e.g., FIG. 1, FIG. 5) which are voltage switching or electrochromic layers, in the examples herein can be comprised of various materials to achieve the electrochromic mechanism (change in opacity) when various voltages are applied or not applied to them. FIG. 8 illustrates the functionality of an electrochromic layer (EC layer 898). In this example, the EC layer 898 is comprised of WO3. Conducting electrodes (+/-) (e.g., indium tin oxide (IOT)) that are both positive electrodes 892 and negative electrodes 899 form the sides of the example. The opposingly charged electrodes apply the voltage, which in turn will push (or pull) the ions. Central to the configuration is an ion conductor (electrolyte) 896 (e.g., tantalum pentoxide (Ta2Os) or zirconium dioxide (ZrCh)). On the side of the ion conductor (electrolyte) 896 closest to the positive electrodes 892 is an ion storage layer 894 (e.g., Prussian blue, NiO, CeO2). And on the side of the ion conductor (electrolyte) 896 closest to the negative electrodes 899 is the EC layer 898. In this example, which is included to demonstrate electromagnetic mechanisms, generally, the WO3 forms octahedral structure which allows ions to pass when exposed to a volage. The EC layer 898 absorbs ions under the voltage. The excessive ions attract electrons from the electrode. The redox reaction of the tungsten absorbs light. In other words, the redox reaction changes the oxidation state of the electrochromic material and thus its light absorbance.

[0089] As discussed earlier, the one or more layers 252 can be comprised of different materials, some electrochromic and others, not. The opacity of the one or more layers 252 configured according to the materials Table 2 and Table 3 varies in accordance with voltages applied to these layers. FIGS. 9-10 illustrate the transmittance behavior of these configurations; FIG. 9 illustrates the Table 2 configuration while FIG. 10 illustrates the Table 3 configuration and compares the transmittance behavior with the inclusion or exclusion of a PEDOT;PSS layer.

[0090] Each apparatus 100 described herein can be utilized as a biosensor. FIG. 11 provides an illustration of aspects of various workflows 1100 that include utilizing various examples of the apparatuses described herein. Thus, for each apparatus described herein, one can obtain the apparatus (1110). The apparatus, as discussed and illustrated in the accompanying figures, includes a plurality of pixels on a sensor, where each pixel is associated with two reaction sites, a first reaction site and a second reaction site. In an optical signal transmission path from the first reaction site to the pixel is a first layer and in an optical signal transmission path from the second reaction site to the pixel is a second layer. The first layer is configured to selectively transmit a first optical signal to the pixel based when a first voltage is applied to the first layer. The second layer is configured to selectively transmit a second optical signal to the pixel when a second voltage is applied to the second layer. One can place one or more nucleic acid on the first reaction site and on the second reaction site (1120), expose the first reaction site and the second reaction site to light from a light source (the light from the light source comprises excitation light) (1130). One can selectively apply the first voltage to the first layer and the second voltage to the second layer (1140). Based on the first voltage being applied to the first layer, the first layer transmits the first optical signal to the pixel (1150). Based on the second voltage being applied to the second layer, the second layer transmits the second optical signal to the pixel (1160). In some examples, these voltages are the same and applied at different times. Upon receipt of either (or both) optical signals, the pixel provides signals that are used to identify, based on the emitted light, a composition of the nucleic acids (1170). In obtaining this emitted light, in these examples, the biosensor structures propagate the emitted light through the one or more of the first layer on the second layer to reach the pixel. In some examples, the reaction sites comprise fluorophores. In these examples, based on exposing the reaction sites to light from a light source, the excitation light causes the fluorophores to emit the emitted light.

[0091] Like FIG. 11, FIG. 12 also provides an illustration of aspects of various workflows 1200 that include utilizing various examples of the apparatuses described herein. As illustrated in FIG. 12, one detects, using apixel sensor of aplurality of pixels sensors, a cluster signal (1210). In this example, the cluster signal being dependent on a first cluster signal emitted from a first reaction site associated to a pixel sensor and the cluster signal passes from the first reaction site through a first layer based on a first voltage being applied to the first layer. One then detects, using a pixel sensor of a plurality of pixels sensors, a second cluster signal (1220). In this example, the second cluster signal is dependent on a second cluster signal emitted from a second reaction site associated to the pixel sensor. The cluster signal passes from the second reaction site through a second layer based on a second voltage being applied to the second layer. One determines an identity of a first analyte of interest in the first reaction site in dependence on the cluster signal detected using the pixel sensor (1230). One determines an identity of a second analyte of interest in the second reaction site in dependence on the second cluster signal detected using the pixel sensor (1240).

[0092] The examples herein include apparatuses, methods of forming these apparatuses, and methods of using these apparatuses.

[0093] The examples herein include an apparatus that includes a biosensor comprising a plurality of pixels. This example of an apparatus includes a first reaction site configured to emit light responsive to a first cluster at the first reaction site being exposed to excitation light, wherein the emitted light from the first reaction site is propagated through the biosensor on a first signal path to a pixel of the plurality of pixels as a first optical signal. The apparatus example includes a second reaction site configured to emit light responsive to a second cluster at the second reaction site being exposed to the excitation light, where the emitted light from the second reaction site is propagated through the biosensor on a second signal path to the pixel of the plurality of pixels as a second optical signal. The apparatus can include a first layer associated to the pixel, the first layer situated in the first signal path, wherein the first layer is configured to selectively open the signal path of the first optical signal when a first voltage is applied to the first layer. The apparatus can include a second layer associated to the pixel, the second layer situated in the second signal path, wherein the second layer is configured to selectively open the second signal path when a second voltage is applied to the second layer.

[0094] In some examples of the apparatus, the first reaction site and the second reaction site each comprise a separate nanowell.

[0095] In some examples of the apparatus, the first layer is configured to selectively block a portion of the signal path of the first optical signal when the first voltage is not applied to the first layer.

[0096] In some examples of the apparatus, the first layer is configured to selectively block a portion of the signal path of the first optical signal when a voltage different from the first voltage is applied to the first layer.

[0097] In some examples of the apparatus, selectively blocking a portion of the signal path of the first optical signal comprises: enabling fewer photons from the emitted light from the first reaction to reach the pixel or enabling no photons to reach the from the emitted light from the first reaction to reach the pixel.

[0098] In some examples of the apparatus, the second layer is configured to selectively block a portion of the signal path of the second optical signal when the second voltage is not applied to the first layer.

[0099] In some examples of the apparatus, the second layer is configured to selectively block a portion of the signal path of the second optical signal when a voltage different from the second voltage is applied to the second layer.

[0100] In some examples of the apparatus, selectively blocking a portion of the signal path of the second optical signal comprises: enabling fewer photons from the emitted light from the second reaction to reach the pixel or enabling no photons to reach the from the emitted light from the second reaction to reach the pixel.

[0101] In some examples of the apparatus, the first layer is comprised of a first electrochromic material.

[0102] In some examples of the apparatus, the second layer is comprised of a second electrochromic material.

[0103] In some examples of the apparatus, a first electrochromic layer comprises the first layer, and wherein a second electrochromic layer comprises the second layer, and wherein each pixel of the plurality of pixels is communicatively coupled with a portion of the first electrochromic layer and the second electrochromic layer.

[0104] In some examples of the apparatus, a complementary metal-oxide-semi conductor (CMOS) comprises the plurality of pixels.

[0105] In some examples of the apparatus, the first layer and the second layer are patterned over the CMOS in a zebra pattern.

[0106] In some examples of the apparatus, the apparatus further comprises: a pad at an edge of the apparatus, wherein the pad controls when the first voltage and the second voltage are applied to the apparatus.

[0107] In some examples of the apparatus, the apparatus further comprises: a pad at an edge of the apparatus, wherein the pad controls where the first voltage and the second voltage are applied to the apparatus.

[0108] In some examples of the apparatus, the first voltage and the second voltage are a common voltage.

[0109] In some examples of the apparatus, the first voltage and the second voltage are not a common voltage.

[0110] In some examples of the apparatus, the apparatus further comprises: a first filter layer situated between the first reaction site and the first layer; and a second filter layer situated between the second reaction site and the second layer.

[OHl] In some examples of the apparatus, the apparatus further comprises: a first filter layer situated between the first layer and the pixel; and a second filter layer situated between the second layer and the pixel.

[0112] In some examples of the apparatus, the first filter layer and the second filter layer each comprise an emission filter.

[0113] In some examples of the apparatus, the first layer and the second layer each comprise a material selected from the group consisting of: tungsten trioxide, molybdenum, titanium, and niobium oxide.

[0114] In some examples of the apparatus, the first layer and the second layer are coplanar.

[0115] The examples herein include a method that comprises forming one or more layers over a sensor, the sensor comprising a plurality of pixels, the one or more layers comprising at least one material that changes in opacity when exposed to a given range of voltages. In this example, the method includes forming a filter layer over the one or more layers, wherein the one or more layers are situated between the sensor and the filter layer. The method can also include forming trenches through the one or more layers and the filter layer, where the forming creates stacks separated from each other by the trenches, each stack comprising a portion of the filter layer and a portion of the one or more layers, wherein based on forming the trenches, two stacks of the stacks are situated on each pixel of the plurality of pixels. The method can include filling the trenches with a material that reduces crosstalk between the pixels. The method can include planarizing the filled trenches to define a contiguous surface with a top surface of the stacks. The method can include forming reaction sites atop each stack such that each pixel is associated with a first reaction site and a second reaction site.

[0116] In some examples of the method, forming the one or more layers comprises utilizing a technique selected from the group consisting of: Plasma Enhanced Chemical Vapor Deposition (PECVD), sputter, e-beam evaporation, crystalline growth and etching (e.g., transfer wafer bonding or direct wafer bonding), and radical activation bonding in vacuum.

[0117] In some examples of the method, forming the one or more layers comprises performing one or more of: D.C. magnetron spluttering or spin coating.

[0118] In some examples of the method, forming the filter layer comprises utilizing a technique selected from the group consisting of: Plasma Enhanced Chemical Vapor Deposition (PECVD), sputter, e-beam evaporation, crystalline growth and etching, and radical activation bonding in vacuum.

[0119] In some examples of the method, the filter layer comprises an emission filter.

[0120] In some examples of the method, forming the trenches through the one or more layers and the filter layer comprises etching.

[0121] In some examples of the method, the material selected to reduce crosstalk is selected from the group consisting of: oxide, nitride, silicon, and a rare metal.

[0122] In some examples of the method, the material selected to reduce crosstalk is tungsten and the planarizing comprises performing tungsten chemical mechanical polishing.

[0123] In some examples of the method, forming reaction sites atop each stack comprises: forming a first conductive layer over the contiguous surface; forming an oxide layer over a top surface of the first conductive layer; forming a second conductive layer over a top surface of the oxide layer; depositing photoresist on a first portion of a top surface of the second conductive layer; and etching through a second portion of the top surface of the second conductive layer, wherein the photoresist is not deposited on the second portion of the top surface of the second conductive layer, a portion of the oxide layer, and a portion of the first conductive layer, wherein the etching forms one or more trenches nanowell trenches, wherein each stack comprises a nanowell trench based on the etching.

[0124] In some examples of the method, the method includes depositing a passivation layer over a top surface of the apparatus comprising the one or more nanowell trenches.

[0125] In some examples of the method, the method includes applying various chemistries to a top surface of a portion of the passivation layer, wherein the portion of the passivation later is situated in the one or more nanowell trenches such that the trenches become active detection areas.

[0126] In some examples of the method, forming the first conductive layer comprises utilizing metal sputtering.

[0127] In some examples of the method, depositing the photoresist comprises utilizing photolithography.

[0128] In some examples of the method, the method includes: based on completing the etching, removing the photoresist, wherein the removing utilizes a technique selected from the group consisting of: utilizing resist strips, chemical cleaning, and etching.

[0129] In some examples of methods of using examples of the apparatus described herein, the method includes detecting, using a pixel sensor of a plurality of pixels sensors, a cluster signal, the cluster signal being dependent on a first cluster signal emitted from a first reaction site associated to the pixel sensor, wherein the cluster signal passes from the first reaction site through a first layer based on a first voltage being applied to the first layer. The method can include detecting, using a pixel sensor of a plurality of pixels sensors, a second cluster signal, the second cluster signal being dependent on a second cluster signal emitted from a second reaction site associated to the pixel sensor, wherein the cluster signal passes from the second reaction site through a second layer based on a second voltage being applied to the second layer. The method can include determining an identity of a first analyte of interest in the first reaction site in dependence on the cluster signal detected using the pixel sensor. The method can include determining an identity of a second analyte of interest in the second reaction site in dependence on the second cluster signal detected using the pixel sensor.

[0130] In some examples of the method, the first reaction site includes a first nanowell, and the second reaction site includes a second nanowell. [0131] In some examples of the method, the second layer is configured to selectively block a portion of the second cluster signal when the second voltage is not applied to the second layer.

[0132] In some examples of the method, the second layer is configured to selectively block a portion of the first cluster signal when the first voltage is not applied to the first layer.

[0133] In some examples of methods of using examples of the apparatus described herein, the method includes obtaining an apparatus, the apparatus comprising: a biosensor comprising a plurality of pixels; a first reaction site configured to emit light responsive to a first cluster at the first reaction site being exposed to excitation light, wherein the emitted light from the first reaction site is propagated through the biosensor on a first signal path to a pixel of the plurality of pixels as a first optical signal; a second reaction site configured to emit light responsive to a second cluster at the second reaction site being exposed to the excitation light, wherein the emitted light from the second reaction site is propagated through the biosensor on a second signal path to the pixel of the plurality of pixels as a second optical signal; a first layer associated to the pixel, the first layer situated in the first signal path, wherein the first layer is configured to selectively open the signal path of the first optical signal when a first voltage is applied to the first layer; and a second layer associated to the pixel, the second layer situated between the second reaction site and the pixel in a signal path of the second optical signal, wherein the second layer is configured to selectively open the signal path of the second optical signal when a second voltage is applied to the second layer. The method can include exposing the first reaction site and the second reaction site to light from a light source. The method can include selectively applying the first voltage to the first layer and the second voltage to the second layer. The method can include based on the first voltage being applied to the first layer, receiving the first optical signal at the pixel. The method can include based on the second voltage being applied to the second layer, receiving the second the second optical signal at the pixel.

[0134] In some examples of the method, the first voltage and the second voltage are a same voltage.

[0135] In some examples of the method, the selectively applying comprises applying the first voltage to the first layer at a first time and applying the second voltage to the second layer at a different time.

[0136] In some examples of the method, the method includes based on the pixel receiving the first optical signal or the second optical signal, providing one or more signals identifying a composition of the nucleic acids at one or more of the first reaction site or the second reaction site.

[0137] In some examples of the method, the first reaction site and the second reaction site comprise fluorophores.

[0138] In some examples of the method, the excitation light causes the fluorophores to emit the emitted light. [0139] Some examples herein include a biosensor that includes a plurality of pixels. The biosensor can include a first reaction site, where light emitted from the first reaction site propagates on a first signal path to a pixel of the plurality of pixels as a first optical signal. The biosensor can include a second reaction site, where light emitted from the second reaction site propagates on a second signal path to the pixel of the plurality of pixels as a second optical signal. The biosensor can include a first layer situated in the first signal path, where the first layer is comprised of a first electrochromic material, where the opacity of the first electrochromic material changes in response to a first voltage. The biosensor can include a second layer situated in the second signal path, where the second layer is comprised of a second electrochromic material, where the opacity of the second electrochromic material changes in response to a second voltage.

[0140] In some examples of the biosensor, the first reaction site and the second reaction site each comprise a separate nanowell.

[0141] In some examples of the biosensor, the first layer is configured to selectively block a portion of the signal path of the first optical signal when the first voltage is not applied to the first layer.

[0142] In some examples of the biosensor, the first layer is configured to selectively block a portion of the signal path of the first optical signal when a voltage different from the first voltage is applied to the first layer.

[0143] In some examples of the biosensor, selectively blocking a portion of the signal path of the first optical signal comprises: enabling fewer photons from the emitted light from the first reaction to reach the pixel or enabling no photons to reach the from the emitted light from the first reaction to reach the pixel.

[0144] In some examples of the biosensor, the second layer is configured to selectively block a portion of the signal path of the second optical signal when the second voltage is not applied to the first layer.

[0145] In some examples of the biosensor, the second layer is configured to selectively block a portion of the signal path of the second optical signal when a voltage different from the second voltage is applied to the second layer.

[0146] In some examples of the biosensor, selectively blocking a portion of the signal path of the second optical signal comprises: enabling fewer photons from the emitted light from the second reaction to reach the pixel or enabling no photons to reach the from the emitted light from the second reaction to reach the pixel.

[0147] In some examples of the biosensor, the first electrochromic material is the same as the second electrochromic material.

[0148] In some examples of the biosensor, a complementary metal-oxide-semi conductor (CMOS) comprises the plurality of pixels. [0149] In some examples of the biosensor, the first layer and the second layer are patterned over the plurality of pixels in a zebra pattern.

[0150] In some examples of the biosensor, the biosensor includes a pad at an edge of the biosensor, wherein the pad controls when the first voltage and the second voltage are applied to the apparatus.

[0151] In some examples of the biosensor, the biosensor includes a pad at an edge of the apparatus, wherein the pad electrically couples a voltage source to the first layer.

[0152] In some examples of the biosensor, the biosensor includes a second pad at an edge of the apparatus, wherein the second pad electrically couples a second voltage source to the second layer.

[0153] In some examples of the biosensor, the first voltage and the second voltage are a common voltage.

[0154] In some examples of the biosensor, the biosensor includes a filter layer situated between the first reaction site and the first layer; and the filter layer situated between the second reaction site and the second layer.

[0155] In some examples of the biosensor, the filter layer comprises an emission filter.

[0156] In some examples of the biosensor, the first layer and the second layer each comprise a material selected from the group consisting of: tungsten trioxide, molybdenum, titanium, and niobium oxide.

[0157] In some examples of the biosensor, the biosensor includes a crosstalk mitigating structure between the first layer and the second layer.

[0158] In some examples of the biosensor, the crosstalk mitigating structure extends from a layer comprising the first reaction site and the second reaction site to a layer comprising the plurality of pixels.

[0159] In some examples of the biosensor, the crosstalk mitigating structure comprises a diffusion layer.

[0160] In some examples of the biosensor, the crosstalk mitigating structure comprises a dielectric layer.

[0161] The flowchart and block diagrams in the figures illustrate the architecture, functionality, and operation of possible implementations of systems, methods, and computer program products according to various examples of the present implementation. In this regard, each block in the flowchart or block diagrams can represent a module, segment, or portion of instructions, which comprises one or more executable instructions for implementing the specified logical function(s). In some alternative implementations, the functions noted in the blocks can occur out of the order noted in the figures. For example, two blocks shown in succession can, in fact, be executed substantially concurrently, or the blocks can sometimes be executed in the reverse order, depending upon the functionality involved. It will also be noted that each block of the block diagrams and/or flowchart illustration, and combinations of blocks in the block diagrams and/or flowchart illustration, can be implemented by special purpose hardware-based systems that perform the specified functions or acts or carry out combinations of special purpose hardware and computer instructions.

[0162] The terminology used herein is for the purpose of describing particular examples only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising”, when used in this specification, specify the presence of stated features, integers, steps, processes, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, processes, operations, elements, components, and/or groups thereof.

[0163] The corresponding structures, materials, acts, and equivalents of all means or step plus function elements in the claims below, if any, are intended to include any structure, material, or act for performing the function in combination with other claimed elements as specifically claimed. The description of one or more examples has been presented for purposes of illustration and description, but is not intended to be exhaustive or limited to in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art. Any example was chosen and described to best explain various aspects and the practical application, and to enable others of ordinary skill in the art to understand various examples with various modifications as are suited to the particular use contemplated.

[0164] It should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the subject matter disclosed herein at least to achieve the benefits as described herein. In particular, all combinations of claims subject matter appearing at the end of this disclosure are contemplated as being part of the subject matter disclosed herein. It should also be appreciated that terminology explicitly employed herein that also may appear in any disclosure incorporated by reference should be accorded a meaning most consistent with the particular concepts disclosed herein.

[0165] This written description uses examples to disclose the subject matter, and also to enable any person skilled in the art to practice the subject matter, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the subject matter is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

[0166] It is to be understood that the above description is intended to be illustrative, and not restrictive. For example, the above-described examples (and/or aspects thereof) may be used in combination with each other. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the various examples without departing from their scope. While the dimensions and types of materials described herein are intended to define the parameters of the various examples, they are by no means limiting and are merely provided by way of example. Many other examples will be apparent to those of skill in the art upon reviewing the above description. The scope of the various examples should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. In the appended claims, the terms “including” and “in which” are used as the plain-English equivalents of the respective terms “comprising” and “wherein.” Moreover, in the following claims, the terms “first,” “second,” and “third,” etc. are used merely as labels, and are not intended to impose numerical requirements on their objects. Forms of term “based on” herein encompass relationships where an element is partially based on as well as relationships where an element is entirely based on. Forms of the term “defined” encompass relationships where an element is partially defined as well as relationships where an element is entirely defined. Further, the limitations of the following claims are not written in means-plus- function format and are not intended to be interpreted based on 35 U.S.C. § 112, sixth paragraph (35 U.S.C. § 112(f)), unless and until such claim limitations expressly use the phrase “means for” followed by a statement of function void of further structure. It is to be understood that not necessarily all such objects or advantages described above may be achieved in accordance with any particular example. Thus, for example, those skilled in the art will recognize that the systems and techniques described herein may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objects or advantages as may be taught or suggested herein.

[0167] While the subject matter has been described in detail in connection with only a limited number of examples, it should be readily understood that the subject matter is not limited to such disclosed examples. Rather, the subject matter can be modified to incorporate any number of variations, alterations, substitutions or equivalent arrangements not heretofore described, but which are commensurate with the spirit and scope of the subject matter. Additionally, while various examples of the subject matter have been described, it is to be understood that aspects of the disclosure may include only some of the described examples. Also, while some examples are described as having a certain number of elements it will be understood that the subject matter can be practiced with less than or greater than the certain number of elements. Accordingly, the subject matter is not to be seen as limited by the foregoing description, but is only limited by the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An apparatus comprising: a biosensor comprising a plurality of pixels; a first reaction site configured to emit light responsive to a first cluster at the first reaction site being exposed to excitation light, wherein the emitted light from the first reaction site is propagated through the biosensor on a first signal path to a pixel of the plurality of pixels as a first optical signal; a second reaction site configured to emit light responsive to a second cluster at the second reaction site being exposed to the excitation light, wherein the emitted light from the second reaction site is propagated through the biosensor on a second signal path to the pixel of the plurality of pixels as a second optical signal; a first layer associated to the pixel, the first layer situated in the first signal path, wherein the first layer is configured to selectively open the first signal path to the first optical signal when a first voltage is applied to the first layer; and a second layer associated to the pixel, the second layer situated in the second signal path, wherein the second layer is configured to selectively open the second signal path to the second optical signal when a second voltage is applied to the second layer.

2. The apparatus of claim 1, wherein the first reaction site and the second reaction site each comprise a separate nanowell.

3. The apparatus of claim 1 or claim 2, wherein the first layer is configured to selectively block a portion of the signal path of the first optical signal when the first voltage is not applied to the first layer.

4. The apparatus of claim 1 or claim 2, wherein the first layer is configured to selectively block a portion of the signal path of the first optical signal when a voltage different from the first voltage is applied to the first layer.

5. The apparatus of claim 3 or claim 4, wherein the first layer is configured to selectively block the portion of the signal path of the first optical signal by: enabling fewer photons from the emitted light from the first reaction site to reach the pixel or enabling no photons from the emitted light from the first reaction site to reach the pixel.

6. The apparatus of any one of claims 1 through 5, wherein the second layer is configured to selectively block a portion of the signal path of the second optical signal when the second voltage is not applied to the second layer. 7. The apparatus of claim 1 through 5, wherein the second layer is configured to selectively block a portion of the signal path of the second optical signal when a voltage different from the second voltage is applied to the second layer.

8. The apparatus of claim 6 or claim 7, wherein the second layer is configured to selectively block the portion of the signal path of the second optical signal by: enabling fewer photons from the emitted light from the second reaction site to reach the pixel or enabling no photons from the emitted light from the second reaction site to reach the pixel.

9. The apparatus of any one of claims 1 through 8, wherein the first layer is comprised of a first electrochromic material.

10. The apparatus of any one of claims 1 through 9, wherein the second layer is comprised of a second electrochromic material.

11. The apparatus of any one of claims 1 through 10, wherein a first electrochromic layer comprises the first layer, wherein a second electrochromic layer comprises the second layer, and wherein each pixel of the plurality of pixels is communicatively coupled with a portion of the first electrochromic layer and a portion of the second electrochromic layer.

12. The apparatus of any one of claims 1 through 11, wherein a complementary metal - oxide-semi conductor (CMOS) comprises the plurality of pixels.

13. The apparatus of claim 12, wherein the first layer and the second layer are patterned over the CMOS in a zebra pattern.

14. The apparatus of any one of claims 1 through 13, further comprising: a pad at an edge of the apparatus, wherein the pad is configured to control when the first voltage and the second voltage are applied to the apparatus.

15. The apparatus of any one of claims 1 through 14, further comprising: a pad at an edge of the apparatus, wherein the pad is configured to control where the first voltage and the second voltage are applied to the apparatus.

16. The apparatus of any one of claims 1 through 15, wherein the first voltage and the second voltage are a common voltage.

17. The apparatus of any one of claims 1 through 15, wherein the first voltage and the second voltage are not a common voltage.

18. The apparatus of any one of claims 1 through 17, further comprising: a first filter layer situated between the first reaction site and the first layer; and a second filter layer situated between the second reaction site and the second layer.

19. The apparatus of any one of claims 1 through 17, further comprising: a first filter layer situated between the first layer and the pixel; and a second filter layer situated between the second layer and the pixel.

20. The apparatus of claim 18, wherein the first filter layer and the second filter layer each comprise an emission filter.

21. The apparatus of any one of claims 1 through 20, wherein the first layer and the second layer each comprise a material selected from the group consisting of: tungsten trioxide, molybdenum, titanium, and niobium oxide.

22. The apparatus of any one of claims 1 through 21, where the first layer and the second layer are coplanar.

23. A method, comprising: forming one or more layers over a sensor, the sensor comprising a plurality of pixels, the one or more layers comprising at least one material that changes in opacity when exposed to a given range of voltages; forming a filter layer over the one or more layers, wherein the one or more layers are situated between the sensor and the filter layer; forming trenches through the one or more layers and the filter layer, wherein forming the trenches creates stacks separated from each other by the trenches, each stack comprising a portion of the filter layer and a portion of the one or more layers, wherein, based on forming the trenches, two stacks of the stacks are situated on each pixel of the plurality of pixels; filling the trenches with a material that reduces crosstalk between the pixels; planarizing the filled trenches to define a contiguous surface with a top surface of the stacks; and forming reaction sites atop each stack such that each pixel is associated with a first reaction site and a second reaction site.

24. The method of claim 23, wherein forming the one or more layers comprises utilizing a technique selected from the group consisting of: Plasma Enhanced Chemical Vapor Deposition (PECVD), sputter, e-beam evaporation, crystalline growth and etching, and radical activation bonding in vacuum.

25. The method of claim 23, wherein forming the one or more layers comprises performing one or more of: D.C. magnetron sputtering or spin coating.

26. The method of any one of claims 23 through 25, wherein forming the filter layer comprises utilizing a technique selected from the group consisting of: Plasma Enhanced Chemical Vapor Deposition (PECVD), sputter, e-beam evaporation, crystalline growth and etching (e.g. , transfer wafer bonding or direct wafer bonding), and radical activation bonding in vacuum.

27. The method of any one of claims 23 through 26, wherein the filter layer comprises an emission filter.

28. The method of any one of claims 23 through 27, wherein forming the trenches through the one or more layers and the filter layer comprises etching.

29. The method of any one of claims 23 through 28, wherein the material selected to reduce crosstalk is selected from the group consisting of: oxide, nitride, silicon, and a rare metal.

30. The method of any one of claims 23 through 28, wherein the material selected to reduce crosstalk is tungsten, and wherein planarizing the filled trenches comprises performing tungsten chemical mechanical polishing. 1 . The method of any one of claims 23 through 29, wherein forming the reaction sites atop each stack comprises: forming a first conductive layer over the contiguous surface; forming an oxide layer over a top surface of the first conductive layer; forming a second conductive layer over a top surface of the oxide layer; depositing photoresist on a first portion of a top surface of the second conductive layer; and etching through a second portion of the top surface of the second conductive layer, wherein the photoresist is not deposited on the second portion of the top surface of the second conductive layer, a portion of the oxide layer, and a portion of the first conductive layer, wherein the etching forms one or more nanowell trenches, and wherein each stack comprises a nanowell trench based on the etching.

32. The method of claim 31, further comprising: depositing a passivation layer over a top surface of an apparatus comprising the one or more nanowell trenches.

33. The method of claim 32, further comprising: applying various chemistries to a top surface of a portion of the passivation layer, wherein the portion of the passivation later is situated in the one or more nanowell trenches such that the trenches become active detection areas.

34. The method of any one of claims 31 through 33, wherein forming the first conductive layer comprises utilizing metal sputtering.

35. The method of any one of claims 31 through 34, wherein depositing the photoresist comprises utilizing photolithography.

36. The method of any one of claims 31 through 35, further comprising: based on completing the etching, removing the photoresist, wherein removing the photoresist utilizes a technique selected from the group consisting of: utilizing resist strips, chemical cleaning, and etching.

37. A method comprising: detecting, using a pixel sensor of a plurality of pixels sensors, a first cluster signal, the first cluster signal emitted from a first reaction site associated to the pixel sensor, wherein the first cluster signal passes from the first reaction site through a first layer based on a first voltage being applied to the first layer; detecting, using a pixel sensor of the plurality of pixels sensors, a second cluster signal, the second cluster signal emitted from a second reaction site associated to the pixel sensor, wherein the second cluster signal passes from the second reaction site through a second layer based on a second voltage being applied to the second layer; determining an identity of a first analyte of interest in the first reaction site in dependence on the first cluster signal detected using the pixel sensor; and determining an identity of a second analyte of interest in the second reaction site in dependence on the second cluster signal detected using the pixel sensor.

38. The method of claim 37, wherein the first reaction site includes a first nanowell, and wherein the second reaction site includes a second nanowell.

39. The method of claim 37 or 38, wherein the second layer is configured to selectively block a portion of the second cluster signal when the second voltage is not applied to the second layer.

40. The method of any one of claims 37 through 39, wherein the first layer is configured to selectively block a portion of the first cluster signal when the first voltage is not applied to the first layer.

41. A method comprising: obtaining an apparatus, the apparatus comprising: a biosensor comprising a plurality of pixels; a first reaction site configured to emit light responsive to a first cluster at the first reaction site being exposed to excitation light, wherein the emitted light from the first reaction site is propagated through the biosensor on a first signal path to a pixel of the plurality of pixels as a first optical signal; a second reaction site configured to emit light responsive to a second cluster at the second reaction site being exposed to the excitation light, wherein the emitted light from the second reaction site is propagated through the biosensor on a second signal path to the pixel of the plurality of pixels as a second optical signal; a first layer associated to the pixel, the first layer situated in the first signal path, wherein the first layer is configured to selectively open the first signal path to the first optical signal when a first voltage is applied to the first layer; and a second layer associated to the pixel, the second layer situated in the second signal path, wherein the second layer is configured to selectively open the second signal path to the second optical signal when a second voltage is applied to the second layer; exposing the first reaction site and the second reaction site to light from a light source; selectively applying the first voltage to the first layer and the second voltage to the second layer; based on the first voltage being applied to the first layer, receiving the first optical signal at the pixel; and based on the second voltage being applied to the second layer, receiving the second optical signal at the pixel. 42. The method of claim 41, wherein the first voltage and the second voltage are a same voltage.

43. The method of claim 41, wherein selectively applying the first voltage to the first layer and the second voltage to the second layer comprises applying the first voltage to the first layer at a first time and applying the second voltage to the second layer at a different second time.

44. The method of claim 41, further comprising: based on the pixel receiving the first optical signal or the second optical signal, providing one or more signals identifying a composition of nucleic acids at one or more of the first reaction site or the second reaction site.

45. The method of any one of claims 41 through 44, wherein the first reaction site and the second reaction site comprise fluorophores.

46. The method of claim 45, wherein the excitation light causes the fluorophores to emit the emitted light.

47. A biosensor comprising: a plurality of pixels; a first reaction site, wherein light emitted from the first reaction site propagates on a first signal path to a pixel of the plurality of pixels as a first optical signal; a second reaction site, wherein light emitted from the second reaction site propagates on a second signal path to the pixel of the plurality of pixels as a second optical signal; a first layer situated in the first signal path, wherein the first layer is comprised of a first electrochromic material, and wherein an opacity of the first electrochromic material changes in response to a first voltage; and a second layer situated in the second signal path, wherein the second layer is comprised of a second electrochromic material, and wherein an opacity of the second electrochromic material changes in response to a second voltage.

48. The biosensor of claim 47, wherein the first reaction site and the second reaction site each comprise a separate nanowell.

49. The biosensor of claim 47 or claim 48, wherein the first layer is configured to selectively block a portion of the signal path of the first optical signal when the first voltage is not applied to the first layer.

50. The biosensor of claim 47 or claim 48, wherein the first layer is configured to selectively block a portion of the signal path of the first optical signal when a voltage different from the first voltage is applied to the first layer.

51. The biosensor of claim 49 or claim 50, wherein the first layer is configured to selectively block the portion of the signal path of the first optical signal by: enabling fewer photons from the emitted light from the first reaction site to reach the pixel or enabling no photons from the emitted light from the first reaction site to reach the pixel.

52. The biosensor of any one of claims 47 through 51, wherein the second layer is configured to selectively block a portion of the signal path of the second optical signal when the second voltage is not applied to the second layer.

53. The biosensor of any one of claims 47 through 51, wherein the second layer is configured to selectively block a portion of the signal path of the second optical signal when a voltage different from the second voltage is applied to the second layer.

54. The biosensor of claim 52 or claim 53, wherein the second layer is configured to selectively block the portion of the signal path of the second optical signal by: enabling fewer photons from the emitted light from the second reaction site to reach the pixel or enabling no photons from the emitted light from the second reaction site to reach the pixel.

55. The biosensor of any one of claims 47 through 54, wherein the first electrochromic material is the same as the second electrochromic material.

56. The biosensor of any one of claims 47 through 55, wherein a complementary metal- oxide-semi conductor (CMOS) comprises the plurality of pixels.

57. The biosensor of any one of claims 47 through 56, wherein the first layer and the second layer are patterned over the plurality of pixels in a zebra pattern.

58. The biosensor of any one of claims 47 through 57, further comprising: a pad at an edge of the biosensor, wherein the pad is configured to control when the first voltage and the second voltage are applied to the biosensor.

59. The biosensor of any one of claims 47 through 58, further comprising: a pad at an edge of the biosensor, wherein the pad is configured to electrically couple a voltage source to the first layer.

60. The biosensor of any one of claims 47 through 59, further comprising: a pad at an edge of the biosensor, wherein the pad is configured to electrically couple a voltage source to the second layer.

61. The biosensor of any one of claims 47 through 60, wherein the first voltage and the second voltage are a common voltage.

62. The biosensor of any one of claims 47 through 61, further comprising: a filter layer situated between the first reaction site and the first layer and between the second reaction site and the second layer.

63. The biosensor of claim 62, wherein the filter layer comprises an emission filter.

64. The biosensor of any one of claims 47 through 63, wherein the first layer and the second layer each comprise a material selected from the group consisting of: tungsten trioxide, molybdenum, titanium, and niobium oxide.

65. The biosensor of any one of claims 47 through 64, further comprising a crosstalk mitigating structure between the first layer and the second layer.

66. The biosensor of claim 65, wherein the crosstalk mitigating structure extends from a layer comprising the first reaction site and the second reaction site to a layer comprising the plurality of pixels.

67. The biosensor of claim 65 or 66, wherein the crosstalk mitigating structure comprises a diffusion layer.

68. The biosensor of any one of claims 65 through 67, wherein the crosstalk mitigating structure comprises a dielectric layer.