Sensors with multiple reaction sites per pixel

Abstract

In one example, an apparatus is described herein. The apparatus may include, for example, a first reaction site and a second reaction site above a single pixel. In one example, a method is described herein. The method may include, for example, detecting a signal emitted from the first reaction site and the second reaction site, and using the amplitude of the detected signal to determine the identity of a first analyte of interest in the first reaction site, and using the amplitude of the detected signal to determine the identity of a second analyte of interest in the second reaction site.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. patent application Ser. No. 63 / 200,383, entitled “Sensor with Multiple Reaction Sites Per Pixel,” filed March 3, 2021, and incorporated by reference in its entirety herein. [Background technology]

[0002] This application relates generally to sensing, and specifically to optical sensing.

[0003] 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 the properties of the chemicals involved in the reaction. In some multiplex assays, an unknown analyte with an identifiable label (e.g., a fluorescent label) can be exposed to thousands of known probes under controlled conditions. Each known probe can be deposited in a corresponding well of a microplate. Observing any chemical reactions that occur between the known probes and the unknown analyte in the well can help identify or reveal the properties of the analyte. Other examples of such protocols include known deoxyribonucleic acid (DNA) sequencing processes, such as sequencing-by-synthesis (SBS) or circular array sequencing. Summary of the Invention

[0004] In one example, an apparatus is described herein. The apparatus can include, for example, a first reaction site and a second reaction site above a single pixel.

[0005] In one example, a method is described herein that may include, for example, detecting a signal emitted from a first reaction site and a second reaction site, and using the amplitude of the detected signal to determine the identity of a first analyte of interest at the first reaction site, and using the amplitude of the detected signal to determine the identity of a second analyte of interest at the second reaction site.

[0006] In one example, a device is described herein. The device may include, for example, a plurality of pixels, each of the plurality of pixels including a respective pixel sensor, a first reaction site associated with one of the plurality of pixels, and a second reaction site associated with the pixel, the pixel sensor of the pixel detects a read signal, the read signal being dependent on a first cluster signal (or a first cluster signal) emitted from the first reaction site and a second cluster signal (or a second cluster signal) emitted from the second cluster site, the first reaction site and the second reaction site are configured such that the second cluster signal in an "on" state has a larger amplitude than the first cluster signal in an "on" state.

[0007] In one example, a method is described herein that may include, for example, detecting a readout signal using a pixel sensor of a plurality of pixel sensors, where the readout signal depends on a first cluster signal emitted from a first reaction site associated with the pixel sensor and a second cluster signal emitted from a second reaction site associated with the pixel sensor, determining an identity of a first analyte of interest at the first reaction site using an amplitude of the readout signal detected using the pixel sensor, and determining an identity of a second analyte of interest at the second reaction site using an amplitude of the readout signal detected using the pixel sensor.

[0008] By modulating the signals of two different adjacent clusters in two separate nanowells, either through geometric constraints or signal modulation, it may be possible to distinguish signals from adjacent clusters using a single pixel. In other words, the benefits and advantages of the methods and structures disclosed herein include isolating separate signal levels from each unique cluster by a single image pixel. The methods and structures disclosed herein allow for the use of multiple clusters per pixel (e.g., on a CMOS sensor) and distinguishing the on / off signals generated from each pixel.

[0009] Although various examples herein may describe certain methods and structures with respect to CMOS sensors, other sensor types may be utilized as well, including charge coupled device (CCD) sensors. Similarly, the methods and structures disclosed herein may also be applicable detection of analytes on a flow cell, where emitted light is detected via external optics and image sensor(s).

[0010] Increasing the information density on a fixed CMOS-based flow cell has the beneficial side of reducing the cost per gigabyte of sequencing information by a factor proportional to the increased density.

[0011] The first reaction site and the second reaction site are above a single pixel. The first reaction site is a first nanowell and the second reaction site is a second nanowell. The first nanowell may be half the size of the second nanowell. The filter may be between the first reaction site and the single pixel, but not between the second reaction site and the single pixel. The filter attenuates a cluster signal emitted from the first reaction site. The filter may be a thin metallic layer. The filter may include tantalum. The method may include detecting signals emitted from the first reaction site and the second reaction site, using an amplitude of the detected signal to determine an identity of a first analyte of interest in the first reaction site, and using an amplitude of the detected signal to determine an identity of a second analyte of interest in the second reaction site.

[0012] 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 the present disclosure will become apparent from the following detailed description of the various aspects of the disclosure taken in conjunction with the accompanying drawings.

[0013] It is to be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below (provided that such concepts are not mutually inconsistent) are considered to be part of the inventive subject matter disclosed herein and are considered to achieve the advantages and benefits disclosed herein. [Brief description of the drawings]

[0014] One or more aspects are particularly pointed out and distinctly claimed as examples at the conclusion of the specification. The foregoing as well as objects, features, and advantages of one or more aspects will become apparent from the following detailed description taken in conjunction with the accompanying drawings. [Figure 1]FIG. 1 shows a portion of a CMOS sensor with a single nanowell above a single pixel. [Diagram 2] FIG. 1 shows a portion of a CMOS sensor with two nanowells providing substantially similar "on" cluster signal intensities above a single pixel. [Diagram 3] FIG. 1 shows a portion of a CMOS sensor with two nanowells providing substantially different "on" cluster signal intensities above a single pixel. [Figure 4] FIG. 13 is an exemplary scatter plot of two channel signal intensities detected by a single pixel from two nanowells providing substantially different "on" cluster signal intensities. [Diagram 5] FIG. 13 shows an exemplary base calling chart based on two channel signal intensities detected by a single pixel from two nanowells that provide substantially different "on" cluster signal intensities. [Figure 6] 1A and 1B are top and cross-sectional views of a portion of a CMOS sensor with nanowells of substantially similar size. [Figure 7] 1A-1D are top and cross-sectional views of a portion of a CMOS sensor with nanowells of substantially different sizes. [Figure 8] FIG. 1 is a cross-sectional view of a portion of a CMOS sensor in which one of the two nanowells has a filter to attenuate the cluster signal. [Figure 9] FIG. 13 is a cross-sectional view of a portion of another CMOS sensor in which one of the two nanowells has a filter to attenuate the cluster signal. [Figure 10] FIG. 1 is a top view of a portion of a CMOS sensor showing the placement of nanowells over adjacent pixels. [Figure 11] 1A-1C are a top view and corresponding cross-sectional view of a portion of a CMOS sensor showing the placement of nanowells over adjacent pixels. [Figure 12] 1 is a chart showing the number of clusters (or nanowells) above a single pixel along with a scatter plot cloud of the corresponding numbers and signal intensities. [Figure 13] FIG. 1 is a schematic cross-sectional front view of an apparatus for use in the analysis. [Figure 14] FIG. 14 is a top cross-sectional view of the device shown in FIG. 13. [Figure 15] FIG. 13 is a spectral profile diagram showing the alignment between the excitation light, the emission light, and the detection band of the detector. [Figure 16] 1A and 1B are top and cross-sectional views of a portion of a CMOS sensor with nanowells of substantially similar size. [Figure 17] 1A-1D are top and cross-sectional views of a portion of a CMOS sensor with nanowells of substantially different sizes. [Figure 18A] FIG. 1 is a cross-sectional view of a portion of a CMOS sensor in which one of the two nanowells has a filter to attenuate the cluster signal. [Figure 18B] 18B is a diagram of the fabrication stages for making the structure of FIG. 18A. [Figure 18C] 18B is a diagram of the fabrication stages for making the structure of FIG. 18A. [Figure 18D] 18B is a diagram of the fabrication stages for making the structure of FIG. 18A. [Figure 19A] FIG. 13 is a cross-sectional view of a portion of another CMOS sensor in which one of the two nanowells has a filter to attenuate the cluster signal. [Figure 19B] FIG. 19B illustrates the fabrication stages for making the structure of FIG. 19A. [Figure 19C] FIG. 19B illustrates the fabrication stages for making the structure of FIG. 19A. [Figure 20] FIG. 1 is a top view of a portion of a CMOS sensor showing the placement of nanowells over adjacent pixels. [Figure 21] 1A-1C are a top view and corresponding cross-sectional view of a portion of a CMOS sensor showing the placement of nanowells over adjacent pixels. [Figure 22] 11A and 11B are top and cross-sectional views of a portion of an alternative CMOS sensor; [Diagram 23] 11A and 11B are top and cross-sectional views of a portion of an alternative CMOS sensor; [Figure 24]11A and 11B are top and cross-sectional views of a portion of an alternative CMOS sensor; [Diagram 25] 11A and 11B are top and cross-sectional views of a portion of an alternative CMOS sensor; [Figure 26] FIG. 1 is a flow diagram showing a method that can be used to assist DNA sequencing for DNA sequence reconstruction. [Figure 27A] 1 is a chart showing signal to noise considerations in a multiple reaction site per pixel design. [Figure 27B] 1 is a chart showing signal to noise considerations in a multiple reaction site per pixel design. [Figure 28A] 13 is a chart showing the effect of cluster signal amplitude ratio on cloud spacing. [Figure 28B] 13 is a chart showing the effect of cluster signal amplitude ratio on cloud spacing. [Figure 28C] 13 is a chart showing the effect of cluster signal amplitude ratio on cloud spacing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The accompanying drawings, in which like reference numbers refer to the same or functionally similar elements throughout the different views, and which are incorporated in and form a part of this specification, further illustrate the implementation(s) and, together with the detailed description of the implementation(s), serve to explain the principles of the present implementation(s). As will be appreciated by those skilled in the art, the accompanying drawings are provided for ease of understanding and illustrate aspects of certain examples of the present implementation(s). The implementation(s) are not limited to the examples depicted in the drawings.

[0016] The terms "connect," "connected," "contact," "coupled," and the like are broadly defined herein to include a variety of different arrangements and assembly techniques, including, but not limited to, (1) directly joining one component to another without any intervening components between them (i.e., the components are in direct physical contact) and (2) connecting one component to another with one or more components between them, provided that a component that is "connected" or "in contact" or "coupled" to another component is in some operative communication (e.g., electrical, fluid, physical, optical, etc.) with the other component (notwithstanding the presence of one or more additional components between them). It should be understood that some components that are in direct physical contact with each other may or may not be in electrical and / or fluid contact with each other. Furthermore, 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 between them.

[0017] As used herein, the terms "including" and "comprising" mean the same thing.

[0018] The terms "substantially", "approximately", "about", "relatively", or other such similar terms, which may be used throughout this disclosure, including the claims, are used to describe and take into account small variations from a reference or parameter, such as due to variations in processing. Such small variations also include zero-point variations from a reference or parameter. For example, small variations can refer to ±10% or less, such as ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less. As used herein, "substantially", "approximately", "about", "relatively", or other such similar terms can also refer to no variation, i.e., ±0%.

[0019] As used herein, a "flow cell" can include a device having a lid that extends over a reaction structure to form a flow channel between the flow cells that communicates with multiple reaction sites of the reaction structure. In some examples, a detection device, such as an imaging device and / or an optical system, is separate from the flow cell. In other examples, the flow cell can include a detection device that detects a specified reaction occurring at or adjacent to the reaction site. The flow cell can include a solid-state optical detection or "imaging" device, such as a Charge-Coupled Device (CCD) or a Complementary Metal-Oxide Semiconductor (CMOS) (light) detection device. A CMOS detection device or sensor can include multiple detection pixels that detect, for example, incident luminescence signals. In some examples, each detection pixel corresponds to a reaction site. In other examples, there can be more or less pixels than the number of reaction sites. Similarly, a detection pixel in some examples corresponds to a single sensing element to generate an output signal. In other examples, a detection pixel corresponds to multiple sensing elements to generate an output signal. The flow cell may also, or alternatively, include two (or more) opposing sensors without a lid. As a specific example, the flow cell may be fluidically, electrically, or fluidically and electrically coupled to a cartridge, and the cartridge may be fluidically, electrically, or fluidically and electrically coupled to a bioassay system. The cartridge and / or bioassay system may deliver reaction solutions to reaction sites of the flow cell according to a predetermined protocol (e.g., sequencing by synthesis) and perform multiple imaging events. For example, the cartridge and / or bioassay system may direct one or more reaction solutions through a flow channel of the flow cell, and thereby along the reaction sites. At least one of the reaction solutions may include four types of nucleotides with the same or different fluorescent labels. In some examples, the nucleotides are coupled to reaction sites of the flow cell, such as corresponding oligonucleotides of the reaction sites.In some examples, the cartridge, bioassay system, or flow cell itself then illuminates the reaction site using an excitation light source (e.g., a solid-state light source such as a light-emitting diode (LED)). In some examples, the excitation light has a predetermined wavelength or multiple wavelengths, including a range of wavelengths. Fluorescent labels excited by the incident excitation light can provide an emission signal (e.g., light of one or more wavelengths different from the excitation light, and potentially different from each other) that can be detected by a light sensor in the flow cell.

[0020] The flow cells described herein perform a variety of biological or chemical processes and / or analyses. More specifically, the flow cells described herein can be used in a variety of processes and systems in which it is desirable to detect an event, property, quality, or characteristic indicative of a specified reaction. For example, the flow cells described herein can include or be integrated with optical detection devices, sensors, including but not limited to biosensors, and components thereof, as well as bioassay systems that operate in conjunction with sensors, including biosensors.

[0021] The flow cell facilitates multiple designated reactions that can be detected individually or collectively. The flow cell performs multiple cycles in which multiple designated reactions occur in parallel. For example, the flow cell can be used to sequence a high-density array of DNA features through repeated cycles of enzymatic operation and light or image detection / capture. Thus, the flow cell can be in fluid communication with one or more microfluidic channels that deliver reagents or other reaction components in a reaction solution to reaction sites of the flow cell. The reaction sites can be provided or spaced in a predetermined manner, such as a uniform or repeating pattern. Alternatively, the reaction sites can be randomly distributed. Each of the reaction sites can 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, the light guide includes one or more filters to filter out specific wavelengths of light.

[0022] The light guide may be, for example, an absorptive filter (e.g., an organic absorptive filter) such that the filter material absorbs a particular 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 site may be located within a reaction recess or chamber within which a designated reaction may be at least partially compartmentalized. Furthermore, the designated reaction may be involved or more easily detected at temperatures other than ambient temperature, for example, at elevated temperatures.

[0023] As used herein, a "designated reaction" includes a change in at least one of the chemical, electrical, physical, or optical properties (or qualities) of a chemical or biological substance of interest, such as an analyte of interest. In certain flow cells, the designated reaction is a positive binding event, such as, for example, the mixing of a fluorescently labeled biomolecule with the analyte of interest. More generally, the designated reaction may be a chemical conversion, chemical change, or chemical interaction. The designated reaction may also be a change in an electrical property. In certain flow cells, the designated reaction includes mixing a fluorescently labeled molecule with the analyte. The analyte may be an oligonucleotide, and the fluorescently labeled molecule may be a nucleotide. The designated reaction may be detected when excitation light is directed at the oligonucleotide with the labeled nucleotide and the fluorophore emits a detectable fluorescent signal. In another example of a flow cell, the detected fluorescence is the result of chemiluminescence or bioluminescence. The specified reaction can also be, for example, increasing fluorescence (or Forster) resonance energy transfer (FRET) by bringing a donor fluorophore into close proximity with an acceptor fluorophore, decreasing FRET by moving the donor and acceptor fluorophores apart, increasing fluorescence by moving a quencher away from a fluorophore, or decreasing fluorescence by bringing a quencher and a fluorophore into close proximity. The biological or chemical assay can include detecting the specified reaction.

[0024] As used herein, "electrically coupled" and "optically coupled" refer to the transfer of electrical energy and light waves, respectively, between any combination of power sources, electrodes, conductive portions of a substrate, droplets, conductive traces, wires, waveguides, nanostructures, other circuit segments, etc. The terms "electrically coupled" and "optically coupled" may be used in reference to direct or indirect connections, which may pass through various intermediaries, such as fluidic media, voids, etc. Similarly, "fluidically coupled" refers to moving fluid between any combination of sources. The term "fluidically coupled" may be used in reference to direct or indirect connections, which may pass through various intermediaries, such as channels, wells, pools, pumps, etc.

[0025] As used herein, a "reaction solution," "reaction component," or "reactant" includes any material that can be used to obtain at least one specified reaction. For example, potential reaction components include, for example, reagents, enzymes, samples, other biomolecules, and buffers. A reaction component can be delivered in solution to a reaction site in a flow cell disclosed herein and / or immobilized at the reaction site. A reaction component can directly or indirectly interact with another material, such as an analyte of interest, immobilized at the reaction site of the flow cell.

[0026] As used herein, the term "reaction site" is a localized area where at least one designated reaction can occur. A reaction site may include a support surface of a reaction structure or substrate on which a substance can be immobilized. For example, a reaction site may include a surface of a reaction structure (which may be positioned within a channel of a flow cell) having reaction components thereon, such as a colony of nucleic acid thereon. In some flow cells, the nucleic acids in the colony have the same sequence, e.g., are clonal copies of a single-stranded or double-stranded template. However, in some flow cells, a reaction site may only include a single nucleic acid molecule, e.g., in single-stranded or double-stranded form.

[0027] As used herein, the term "transparent" refers to allowing all or substantially all visible and non-visible electromagnetic radiation or light of interest to pass through unobstructed, the term "opaque" refers to reflecting, deflecting, absorbing, or otherwise obstructing all or substantially all visible and non-visible electromagnetic radiation or light of interest to not pass through, and the term "non-transparent" refers to allowing some, but not all, visible and non-visible electromagnetic radiation or light of interest to pass through unobstructed.

[0028] As used herein, the term "waveguide" refers to a structure that guides waves, such as electromagnetic waves, minimizing energy loss by restricting the transmission of energy to a particular direction or range of directions.

[0029] The proposed methods and structures offer many benefits, including higher throughput of sequencing data and lower cost.

[0030] In a particular example, modulating the signals of two different adjacent clusters in two separate nanowells, either through geometric constraints or signal modulation, can increase the information density of a CMOS sensor by more than double, providing an advantage over CMOS-based detection devices with one cluster / well per pixel. Furthermore, increasing the information density on a CMOS-based flow cell can have the benefit of reducing the cost per gigabyte of sequencing information by a factor proportional to the increase in density. The practical impact of such innovations is that CMOS-based sequencing can become competitive with larger platforms in terms of information density. Furthermore, implementing the methods and structures disclosed herein requires only software modifications coupled with modifications to the CMOS fabrication, allowing equipment and reagent consumables to remain substantially, if not completely, unused.

[0031] By placing two or more nanowells on top of a pixel, the sensor accepts a minimum 50% intensity hit for the "bright" cluster and a greater than 50% intensity hit compared to the nominal value for the "attenuated" cluster in the transmission modulation option. Different sized nanowell implementations (e.g., larger "major" sized wells and smaller "lower" sized wells) accept unequal signal sharing between the two (assumed) monoclonal clusters such that the total signal remains approximately the same. Nevertheless, the signal ratio between the major and lower nanowells is large enough to accurately distinguish the signal between the two clusters.

[0032] For ease of understanding, reference is made below to the drawings, which are not drawn to scale and the same reference numbers are used throughout different figures to denote the same or similar components.

[0033] FIG. 1 shows a portion of a CMOS sensor with a single nanowell above a single pixel. One cluster on a pixel will result in two possible different readings on the sensor. For example, the sensor can determine that the cluster is either on or off. Cluster C1 is located within the nanowell NW and resides above a light pipe LP that directs the emission signal (e.g., light) from cluster C1 to the sensor below. If the cluster signal is "on", for example, a particular analyte of interest may be present and there is fluorescence or light emission. Similarly, if the cluster signal is "off", this indicates that a particular analyte of interest may not be present. The table on the right of the portion of the CMOS sensor shows examples of cluster signal strengths and corresponding sensor readout signal strengths, which may assume, for example, that there is no signal loss between the cluster signal and the sensor.

[0034] The nanowells can be boundary structures to the reaction sites that house the DNA clusters. Although the CMOS sensor is shown with nanowells, alternative structures for the reaction sites can be used as well.

[0035] FIG. 2 shows a portion of a CMOS sensor with two nanowells providing substantially similar "on" cluster signal intensities above a single pixel. Two clusters with the same "on" signal level (intensity) result in only three possible different readings on the sensor. In other words, the sensor may not be able to determine which cluster is "on". Clusters C1 and C2 are located in their own respective nanowells NW and above a light pipe LP that directs the emission signals (e.g., light) from clusters C1 and C2 to the sensor below. When the cluster signal is "on", for example, a particular analyte of interest may be present and there is fluorescence or light emission. Similarly, when the cluster signal is "off", this indicates that a particular analyte of interest may not be present. The table on the right of the portion of the CMOS sensor shows examples of cluster signal intensities and corresponding sensor read signal intensities, which may assume, for example, that there is no signal loss between the cluster signal and the sensor.

[0036] FIG. 3 shows a portion of a CMOS sensor with two nanowells providing substantially different "on" cluster signal intensities above a single pixel. Sensor readings from two clusters with 1× (single fold) and 2× (two fold) signal intensities may allow for determining the cluster state, i.e., on or off. Clusters C1 and C2 are located in their own respective nanowells NW and above a light pipe LP that directs the emission signals (e.g., light) from clusters C1 and C2 to the sensor below. When the cluster signal is "on", for example, a particular analyte of interest may be present and there is fluorescence or light emission. Similarly, when the cluster signal is "off", this indicates that a particular analyte of interest may not be present. The table on the right of the portion of the CMOS sensor shows examples of cluster signal intensities and corresponding sensor read signal intensities, which may assume, for example, that there is no signal loss between the cluster signal and the sensor. By modulating the cluster signal intensity between each cluster on the pixel sensor, i.e., by making the signal intensity detected from each cluster by the pixel sensor different, it may be possible to determine the state (on or off) of each cluster (in this example, each cluster within its own nanowell) using a single pixel of the CMOS sensor.

[0037] FIG. 4 shows an example scatter plot of two channel signal intensities detected by a single pixel from two nanowells providing substantially different "on" cluster signal intensities. The scatter plot in FIG. 4 shows 16 separate clouds of cluster signals at exemplary intensities. FIG. 5 shows an exemplary base calling chart based on two channel signal intensities detected by a single pixel from two nanowells providing substantially different "on" cluster signal intensities. By using two channels (e.g., detecting two different wavelengths of emitted light), specific bases in a DNA sequence (e.g., adenine (A), guanine (G), cytosine (C), and thymine (T)) can be determined for each cluster / well. For example, detecting light at a first wavelength but not at a second wavelength may indicate that the base of interest is cytosine (C), detecting light at the second wavelength but not at the first wavelength may indicate that the base of interest is adenine (A), detecting light at both the first and second wavelengths may indicate that the base of interest is thymine (T), and detecting neither the first nor the second wavelength may indicate that the base of interest is guanine (G).

[0038] FIG. 6 shows a top view and a cross-sectional view of a portion of a CMOS sensor with nanowells of substantially similar size. As shown in this figure, each of the two nanowells (which may support clusters of interest, e.g., monoclonal clusters of DNA strands) has the same length and width. However, one nanowell has a filter that attenuates the signal by about 50%, while the other nanowell does not. The filter is shown as a black layer (shown in monochrome with a darker uniform shading) below the top surface of the nanowell. The nanowells in the figure may be shown as rectangular, although other shapes of nanowells are possible, e.g., circular, elliptical, hexagonal, octagonal, etc. The blue material (shown in monochrome with a lighter uniform shading) may also be a metallic material that attenuates the signal. The yellow and gray materials (shown with a hatched pattern) may be dielectric materials.

[0039] FIG. 7 shows a top view and a cross-sectional view of a portion of a CMOS sensor with nanowells of substantially different sizes. As shown in this figure, each of the two nanowells (which may support clusters of interest, e.g., monoclonal clusters of DNA strands) has a different width, with the left nanowell having half the width of the right nanowell. This can limit the size of the clusters of interest and, therefore, the cluster signal emitted by the clusters when the analyte of interest is present. For example, if a first nanowell is twice the size of a second nanowell, the first nanowell may generate twice the cluster signal intensity compared to the second nanowell.

[0040] FIG. 8 shows a cross-sectional view of a portion of a CMOS sensor in which one of the two nanowells has a filter for attenuating the cluster signal. The second nanowell may have a filter layer disposed below, in some instances directly below, the surface of the second nanowell, which is not present in the first nanowell. Other instances may have an additional layer or material present between the surface of the nanowell and the filter layer. The nanowell on the right side of this FIG. 8 is shown with an additional layer. This filter layer may be a thin metal layer. The filter layer should be compatible with the fabrication process of the CMOS sensor. In some instances, the filter layer attenuates approximately 50% (half) of the cluster signal emitted from the cluster when the analyte of interest is present. The filter layer may include or be composed of tantalum.

[0041] FIG. 9 shows a cross-sectional view of a portion of another CMOS sensor in which one of the two nanowells has a filter for attenuating the cluster signal. One of the layers of the CMOS sensor may be a filter layer. However, this filter layer may be removed under the first of the two nanowells above the single pixel, but not under the second nanowell. Thus, this configuration may not attenuate the cluster signal emitted from the first nanowell. In this figure, the blue layer identified by the letter C is the filter layer.

[0042] FIG. 10 shows a top view of a portion of a CMOS sensor showing the placement of nanowells above adjacent pixels. FIG. 11 shows a top view and corresponding cross-sectional view of a portion of a CMOS sensor showing the placement of nanowells above adjacent pixels. Green circles represent attenuated wells (i.e., nanowells with attenuated cluster signals) and blue circles represent unattenuated wells (i.e., nanowells with unattenuated cluster signals). In some examples, it may be preferable to maximize the distance between nanowells, for example, to reduce crosstalk detected by the pixel sensor from adjacent wells. FIG. 10 and FIG. 11 show perpendicular nanowell orientation between adjacent pixels. For example, if a first pixel has a left-to-right nanowell orientation, a second pixel adjacent to the first pixel may have a top-to-bottom nanowell orientation.

[0043] 12 is a chart showing the number of clusters (or nanowells or reaction sites) above a single pixel along with a scatter plot cloud of the corresponding number and signal intensity. As the number of clusters above a single pixel increases, the amplitude of the signal intensity as well as the number of different signal intensities required to determine the on or off state of each cluster may increase.

[0044] With reference to the description accompanying Figures 1-12, a pixel sensor of a pixel herein may alternatively be referred to as a light sensor or light detector, a light sensor of a pixel may alternatively be referred to as a pixel sensor or light detector, and a light detector of a pixel herein may alternatively be referred to as a sensor or light detector. With reference to the description accompanying Figures 1-12, a filter that attenuates light may alternatively be referred to as an attenuator, and a filter layer that attenuates light may alternatively be referred to as an attenuation layer or attenuation material layer.

[0045] Examples herein recognize that an image sensor structure, such as a complementary metal oxide semiconductor (CMOS) sensor, can be combined with a microfluidic device to form a sensor system. The sensor system can be, for example, a biosensor system. Such sensor systems often utilize a high-density array of nanowells disposed on top of a passivation stack of one or more layers (herein "passivation stack") of the image sensor structure to perform a controlled reaction protocol on a polynucleotide strand disposed in the nanowell. The reaction protocol can, for example, determine the order of nucleotides in the strand.

[0046] Examples herein recognize that in examples of such reaction protocols, polynucleotide strands (clusters of DNA fragments, strands of nucleic acid molecules, etc.) disposed in the nanowell array of an image sensor structure can be tagged with distinguishable labels (such as fluorescently labeled nucleotide bases) that are delivered to the strands via fluid flow through a flow cell. One or more excitation lights can then be directed onto the cluster(s) of labeled strands within the nanowells. The labeled strands can then emit photons of emission light indicative of the order of the nucleotide bases within the strands, which can be delivered through a passivation stack to a light guide of the image sensor structure associated with (e.g., located below) each nanowell.

[0047] Examples herein recognize, with reference to the described examples, that the light guide directs the emitted photons to a photodetector disposed within the image sensor structure and associated with the light guide (each light guide and associated photodetector may be referred to as a pixel). The photodetector detects the emitted light photons. Device circuitry within the image sensor structure then uses the detected photons to process and transmit a data signal. The data signal can then be analyzed to reveal the sequence of nucleotide bases within the strand. An example of such a sequencing process is sequencing by synthesis (SBS) or circular array sequencing.

[0048] The examples herein recognize that achieving higher cluster density (and therefore higher detection throughput) on a CMOS sensor is a difficult task. The examples herein recognize that it becomes more difficult to reduce the pixel pitch (i.e., the spacing or periodic distance of each pixel) to less than 0.7 μm. The examples herein recognize that an option to increase cluster density without reducing pixel size is by configuring the CMOS sensor so that multiple clusters per pixel can be detected, i.e., by a single photodetector of the CMOS sensor.

[0049] The examples herein recognize that because each pixel has only one photodetector that measures the number of photons incident on the photodetector, distinguishing between signals resulting from multiple clusters is not possible by simply adding two or more clusters to a pixel. The examples herein recognize that a method and / or structure is needed to distinguish between signals generated from different clusters on a single pixel.

[0050] Herein, with reference to the examples of Figures 1-12, a device is described, where each pixel and pixel sensor of the device has a first and second nanowell associated therewith. The first and second nanowells associated with each pixel and pixel sensor may be configured such that a cluster signal "on" state signal emitted from the second nanowell is greater than a cluster signal "on" state signal emitted from the first nanowell. To configure the device such that the cluster signal "on" state signal of the second nanowell is distinct from the cluster signal "on" state signal of the first nanowell, the second nanowell may be configured differently from the first nanowell. In the examples of Figures 6, 8-11, the first and second nanowells may be configured such that the attenuation of emitted light from the first nanowell is increased relative to the attenuation of emitted light from the second nanowell. In the examples of Figures 6, 8-11, the device may be configured such that the attenuation of emitted light from the first nanowell is increased relative to the attenuation of emitted light from the second nanowell using an attenuating material. In the examples of Figures 6, 8-11, the device may be configured such that the first nanowell includes an attenuator and the second nanowell does not include an attenuator. In the examples of Figures 6, 8-11, the device may be configured such that the first nanowell includes an attenuator that attenuates emitted light radiating vertically downward from the bottom surface of the first nanowell and the second nanowell does not include an attenuator that attenuates emitted light radiating vertically downward from the bottom surface of the second nanowell. In the examples of Figures 6, 8-11, the device may be configured such that the first nanowell includes an attenuator in a region below the bottom surface of the first nanowell and the second nanowell does not include an attenuator in a region below the bottom surface of the second nanowell. In the examples of Figures 7 and 11, the device may be configured such that the attenuating material layer extends below the bottom surface of the first nanowell into the region defining the first nanowell but not below the bottom surface of the second nanowell into the region defining the second nanowell. In the examples of Figures 6 and 8-11, the device may be configured such that a first nanowell includes an attenuator in the region immediately below the bottom surface of the first nanowell, and a second nanowell in the region immediately below the bottom surface is free of an attenuator.In the examples of Figures 6, 8-11, the device may be configured such that the first nanowell includes an attenuator in a region below the bottom surface of the first nanowell that is aligned with the bottom surface of the first nanowell, and the second nanowell does not include an attenuator in a region below the bottom surface of the second nanowell that is aligned with the bottom surface of the second nanowell (or the second nanowell does not include an attenuator in a region below the bottom surface of the second nanowell that is aligned with the bottom surface of the second nanowell). In the example of Figure 7, the second nanowell may include a size larger than the first nanowell to configure the second nanowell such that the cluster signal "on" state signal emitted from the second nanowell is larger than the "on" state signal emitted from the second nanowell. In one example, the second nanowell having a size larger than the first nanowell may have a width larger than the first nanowell. In one example, the second nanowell having a size larger than the first nanowell may have a bottom width larger than the first nanowell. In one example, the second nanowell having a larger size than the first nanowell may have a larger bottom surface area than the first nanowell. As described with reference to Figures 1-12, to configure the device such that the cluster signal "on" state signals emitted from the first and second nanowells are differentiated, the first nanowell may include an attenuator and the second nanowell may be attenuator-free, and additionally or alternatively, the first and second attenuators have different sizes. As described with reference to Figures 10 and 11, adjacent pixels and pixel sensors of the device may be associated with the first and second nanowells, respectively, and the orientations of the first and second nanowells may be differentiated between the adjacent pixels and pixel sensors. The first and second nanowells associated with the first pixel and pixel sensor may have a first orientation, and the first and second nanowells associated with the second pixel and pixel sensor adjacent to the first pixel and pixel sensor may have a second orientation.In Figures 10 and 11, a first orientation can be defined by first and second nanowells having a back-to-front spacing from a top view, and a second orientation can be defined by first and second nanowells having a side-to-side spacing from a top view. Changing the orientation between adjacent pixels and pixel sensors of the device can increase the spacing between nanowells associated with adjacent pixels relative to the spacing between nanowells associated with adjacent pixels in the absence of the orientation change. With reference to Figures 10-12, pixels 2-N of the plurality of pixels can have associated first and second reaction sites respectively configured according to the first and second reaction sites associated with the first pixel, the first and second reaction sites associated with adjacent pixels of the plurality of pixels having first and second different respective orientations with respect to the respective pixels of the reaction sites, the first and second different respective orientations increasing the spacing between the reaction sites from adjacent pixel locations relative to the spacing between the reaction sites from adjacent pixel locations in the absence of the respective orientations.

[0051] A few examples of combinations described above in the "Description of Embodiments" with reference to Figures 1-12 include: (A1) an apparatus comprising a first reaction site and a second reaction site above a single pixel, the pixel comprising a photodetector; (A2) an apparatus as described in A1, wherein the first reaction site is a first nanowell and the second reaction site is a second nanowell; (A3) an apparatus as described in A2, wherein the first nanowell is half the size of the second nanowell; (A4) an apparatus as described in A1 or A2, wherein a filter is present between the first reaction site and the single pixel; (A5) an apparatus as described in A4, wherein a filter is not present between the second reaction site and the single pixel; (A6) an apparatus as described in A4 or A5, wherein the filter attenuates the cluster signal emitted from the first reaction site by about 50%; (A7) an apparatus as described in any of A4-A6, wherein the filter is a thin metal layer; (A8) an apparatus as described in any of A4-A7, wherein the filter comprises tantalum. A few examples of combinations described above in the Detailed Description with reference to Figures 1-12 include: a method comprising detecting signals emitted from a first reaction site and a second reaction site, and using the amplitude of the detected signals to determine an identity of a first analyte of interest at the first reaction site, and using the amplitude of the detected signals to determine an identity of a second analyte of interest at the second reaction site. A few examples of combinations described above in the Detailed Description with reference to Figures 1-12 include: (C1) an apparatus comprising a plurality of pixels, a first reaction site associated with a pixel of the plurality of pixels, and a second reaction site associated with the pixel, wherein a pixel sensor of the pixel detects a readout signal, the readout signal being dependent on a first cluster signal emitted from the first reaction site and a second cluster signal emitted from the second cluster site, the first reaction site and the second reaction site being configured such that the second cluster signal in an "on" state has a greater amplitude than the first cluster signal in an "on" state;(C2) The apparatus of C1, wherein the apparatus determines the identity of each analyte in the first and second reaction sites using a calling chart that maps signal amplitudes of the read signal under different illumination conditions to the identity of each analyte in the first and second reaction sites; (C3) The apparatus of C1, wherein the apparatus determines the identity of each analyte in the first and second reaction sites using 16 cloud calling charts that map signal amplitudes of the read signal under different illumination conditions to the identity of each analyte in the first and second reaction sites; (C4) the apparatus of any of C1-C3, wherein the ratio of the second cluster signal in the "on" state to the first cluster signal in the "on" state is at least about 1.4; (C5) the apparatus of any of C1-C3, wherein the ratio of the second cluster signal in the "on" state to the first cluster signal in the "on" state is from about 1.9 to about 2.0; (C6) the apparatus of any of C1-C5, wherein the second reaction site has a size larger than the first reaction site, such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (C7) the apparatus of any of C1-C5, wherein the second cluster in the "on" state has a size larger than the first reaction site, such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (C8) the apparatus of any of C1-C7, wherein the second reaction site is devoid of an attenuator and the first reaction site includes an attenuator such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (C9) the apparatus of any of C1-C8, wherein the arrangement of the attenuating material provides a greater attenuation of the emission beam emanating from the first reaction site than the attenuation of the emission beam emanating from the second reaction site such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (C10) the apparatus of C9, wherein the attenuating material comprises a metal; (C11) the apparatus of C9, wherein the attenuating material comprises tantalum;(C12) The apparatus according to any one of C1 to C11, wherein the second reaction site does not have an attenuator aligned with and located below the bottom surface of the second reaction site (or the attenuator aligned with and located below the bottom surface of the second reaction site) and the first reaction site includes an attenuator aligned with and located below the bottom surface of the first reaction site such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (C13) The apparatus according to any one of C1 to C11, wherein the second reaction site includes an attenuator aligned with and located below the bottom surface of the first reaction site such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; An apparatus according to any one of C1 to C12, wherein the first reaction site includes an attenuator intersecting a central axis extending in a vertical direction of the first reaction site, such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (C14) the second reaction site does not have an attenuator for attenuating an emission beam radiating vertically downward from a bottom surface of the second reaction site, and the first reaction site includes an attenuator intersecting a central axis extending in a vertical direction of the first reaction site, such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state. (C15) the apparatus includes an attenuator for attenuating emitted light emanating vertically downward from a bottom surface of the first reaction site such that the attenuator for the first reaction site is attenuated by a layer of attenuating material aligned with the bottom surface of the first reaction site and extending through a region below the bottom surface of the first reaction site, the layer of attenuating material having no section extending through a region below the bottom surface of the second reaction site; (C16) the apparatus includes a first nanowell defined by a first nanowell and a second nanowell defined by a second nanowell, the first reaction site being attenuated by a layer of attenuating material aligned with the bottom surface of the first reaction site and extending through a region below the bottom surface of the second reaction site to define the attenuator for the first reaction site; (C17) the device of C16, wherein the second nanowell has a larger size than the first nanowell, such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (C18) the device of C16 or C17, wherein the second nanowell has a larger width than the first nanowell, such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state;(C19) An apparatus according to any of C16-C18, wherein the second nanowell does not have an attenuator and the first nanowell includes an attenuator such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (C20) An apparatus according to any of C16-C19, wherein the attenuator of the first nanowell has an upwardly extending section; (C21) An apparatus according to any of C16-C19, wherein the attenuator of the first nanowell runs parallel to the circumferential sidewall of the first nanowell. (C22) the device of any of C16 to C21, wherein the attenuation of the emitted light beam emanating from the first nanowell is greater than the attenuation of the emitted light beam emanating from the second nanowell, such that the second cluster signal in the "on" state has a greater amplitude than the first cluster signal in the "on" state, due to the placement of the attenuating material; (C23) the device of C22, wherein the attenuating material comprises a metal; (C24) the device of C22, wherein the attenuating material comprises tantalum; (C25) the second nanowell includes an attenuator aligned with a bottom surface of the second nanowell, the attenuator being located at the bottom surface of the second nanowell. (C26) an apparatus according to any one of C16 to C25, wherein the second nanowell is not below an attenuator and the first nanowell includes an attenuator aligned with the bottom surface of the first nanowell such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (C27) an apparatus according to any one of C16 to C25, wherein the second nanowell is not below an attenuator and the first nanowell includes an attenuator intersected by a central axis extending in the vertical direction of the first nanowell such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state;(C27) the apparatus of any of C16-C16, wherein the second nanowell is devoid of an attenuator for attenuating an emission beam emanating vertically downward from a bottom surface of the second nanowell, and the first nanowell includes an attenuator for attenuating an emission beam emanating vertically downward from a bottom surface of the first nanowell such that the second cluster signal in the "on" state has a greater amplitude than the first cluster signal in the "on" state; (C28) the apparatus of any of C16-C27, wherein the apparatus includes an attenuator material layer aligned with and extending through a region underlying the bottom surface of the first nanowell to define an attenuator for the first nanowell, the attenuator material layer being devoid of a section extending through a region underlying the bottom surface aligned with the second reaction site; (C29) the second through Nth pixels of the plurality of pixels have associated first and second reaction sites respectively configured according to the first and second reaction sites associated with the pixels, and the first reaction sites associated with adjacent pixels of the plurality of pixels being devoid of a section extending through a region underlying the bottom surface of the first nanowell. (C30) the device of any of C16 to C29, wherein a first pixel of the plurality of pixels has associated first and second reaction sites configured according to a first and second reaction site associated with the pixel, and the first and second reaction sites associated with adjacent pixels of the plurality of pixels have respective first and second orientations that differ with respect to their respective pixels of the reaction sites, the respective different first orientations being characterized by a back-to-front spacing between the reaction sites and the respective different second orientations being characterized by a side-to-side spacing between the reaction sites;(C31) The apparatus of C29 or C30, wherein the second through Nth pixels are the second through one millionth pixels. A few examples of combinations described above in the Detailed Description with reference to Figures 1-12 include: (D1) a method, comprising: detecting a read signal using a pixel sensor of a plurality of pixel sensors, the read signal being dependent on a first cluster signal emitted from a first reaction site associated with the pixel sensor and a second cluster signal emitted from a second reaction site associated with the pixel sensor, determining an identity of a first analyte of interest at the first reaction site using an amplitude of the read signal detected using the pixel sensor, and determining an identity of a second analyte of interest at the second reaction site using an amplitude of the read signal detected using the pixel sensor; (D2) determining an identity of a first analyte of interest at the first reaction site using an amplitude of the read signal detected using the pixel sensor, and determining an identity of a second analyte of interest at the second reaction site using an amplitude of the read signal detected using the pixel sensor; (D3) the method of D1, wherein determining the identity of the first analyte of interest at the first reaction site using the amplitude of the read signal detected using the pixel sensor and determining the identity of the second analyte of interest at the second reaction site using the amplitude of the detected read signal comprises using a calling chart having 16 signal clouds mapping the signal amplitude of the read signal under different illumination conditions to the identity of the respective analytes at the first reaction site and the second reaction site; (D4) the first reaction site and the second reaction site are in an "on" state and the second reaction site is ... (D5) a method according to any one of D1 to D4, wherein the second reaction site has a larger size than the first reaction site, such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (D6) a method according to any one of D1 to D5, wherein a ratio of the second cluster signal in the "on" state to the first cluster signal in the "on" state is at least about 1.4; (D7) a method according to any one of D1 to D6, wherein a ratio of the second cluster signal in the "on" state to the first cluster signal in the "on" state is from about 1.9 to about 2.0; (D8) a method according to any one of D1 to D7, wherein the second reaction site has a larger width than the first reaction site, such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state. (D9) The method according to any one of D1 to D8, wherein the second reaction site is devoid of an attenuator, and the first reaction site includes an attenuator such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (D10) By disposing the attenuating material,A method according to any one of D1-D9, wherein the attenuation of the emitted light beam emanating from the first reaction site is greater than the attenuation of the emitted light beam emanating from the second reaction site, such that the second cluster signal in the "on" state has a greater amplitude than the first cluster signal in the "on" state; (D11) a method according to D10, wherein the attenuating material comprises a metal; (D12) a method according to D10, wherein the attenuating material comprises tantalum; (D13) a method according to D10, wherein the second reaction site is free of an attenuator aligned with and located below a bottom surface of the second reaction site (or wherein the attenuator is aligned with and below a bottom surface of the second reaction site). (C14) the second reaction site has no attenuator intersected by a vertically extending central axis of the first reaction site, and the first reaction site includes an attenuator below its bottom surface that is aligned with the bottom surface of the first reaction site such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (C15) the second reaction site has no attenuator intersected by a vertically extending central axis of the first reaction site, and the first reaction site includes an attenuator below its bottom surface that is aligned with the bottom surface of the first reaction site such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (C15) the second reaction site does not have an attenuator for attenuating an emission beam emanating vertically downward from a bottom surface of the second reaction site, and the first reaction site includes an attenuator for attenuating an emission beam emanating vertically downward from a bottom surface of the first reaction site such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (D16) the method of any of D1 to D14, wherein the second reaction site does not have an attenuator for attenuating an emission beam emanating vertically downward from a bottom surface of the first reaction site such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (C17) a method according to any of C1-C16, wherein the attenuating material layer extending through the region underlying the bottom surface defines an attenuator of the first reaction site, and the attenuating material layer has no section extending through the region underlying the bottom surface that is aligned with the second reaction site; (C18) a method according to any of D1-D16, wherein the first reaction site is defined by a nanowell and the second reaction site is defined by a nanowell; (D19) the second nanowell is configured to have a larger amplitude than the first cluster signal in the "on" state such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state;(D19) the method of D17 or D18, wherein the second nanowell has a larger width than the first nanowell, such that the second cluster in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (D20) the method of any of C17-C19, wherein the second nanowell is devoid of an attenuator and the first nanowell includes an attenuator, such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (D22) the method of any of D17-D20, wherein the attenuator of the first nanowell has an upwardly extending section; (D23) the attenuator of the first nanowell is disposed on the peripheral sidewall (or circumferential sidewall, (D24) a method according to any of D17-D22, wherein the attenuation of the emitted light beam emanating from the first nanowell is greater than the attenuation of the emitted light beam emanating from the second nanowell, such that the second cluster signal in the "on" state has a greater amplitude than the first cluster signal in the "on" state, by the placement of an attenuating material; (D25) a method according to D24, wherein the attenuating material comprises a metal; (D26) a method according to D24, wherein the attenuating material comprises tantalum; (D27) a method according to D24, wherein the second nanowell is free of an attenuator aligned with and located below a bottom surface of the second nanowell (or wherein the attenuating material is located at a bottom surface of the second nanowell). (D28) a method according to any of D17 to D27, wherein the second nanowell has no attenuator aligned with the bottom surface of the first nanowell and the first nanowell includes an attenuator aligned with the bottom surface of the first nanowell such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state; (D29) a method according to any of D17 to D27, wherein the second nanowell has no attenuator aligned with the bottom surface of the first nanowell and the first nanowell includes an attenuator aligned with the bottom surface of the first nanowell and the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state;A method according to any of D17-D28, wherein the first nanowell includes an attenuator for attenuating emitted light emanating vertically downward from a bottom surface of the first nanowell such that the second cluster signal in an "on" state has a greater amplitude than the first cluster signal in an "on" state; (D30) a method according to any of D17-D28, wherein the first nanowell includes an attenuator for attenuating emitted light emanating vertically downward from a bottom surface of the first nanowell such that the second cluster signal in an "on" state has a greater amplitude than the first cluster signal in an "on" state; (D31) an apparatus including a layer of attenuating material aligned with the bottom surface of the first nanowell and extending through a region underlying said bottom surface to define an attenuator of the first nanowell, the attenuating material (D31) a method according to any of D17 to D29, wherein the material layer has no section extending through an area underlying its bottom surface that is aligned with the second reaction site; (D32) a plurality of pixel sensors are each associated with a plurality of pixels, the pixel sensor defining one pixel of the plurality of pixels, the first reaction site and the second reaction site are associated with the pixel, and pixels 2 through N of the plurality of pixels have associated first and second reaction sites that are respectively configured according to the first reaction site and the second reaction site associated with the pixel; (D32) a plurality of pixel sensors are each associated with the plurality of pixels, the pixel sensor defining one pixel of the plurality of pixels, the first and second reaction sites being associated with the pixel, and a second through Nth pixels of the plurality of pixels having associated first and second reaction sites respectively configured according to the first and second reaction sites associated with the pixel, and the first and second reaction sites associated with adjacent pixels of the plurality of pixels having different respective first and second orientations with respect to their respective pixels of the reaction sites, the different respective first orientations increasing a separation distance between the reaction sites from adjacent pixel locations relative to a separation distance between the reaction sites from the adjacent pixel locations in the absence of the respective orientations;(D33) The method of any of D1-D32, wherein the first nanowell has a first thickness and the second nanowell has a second thickness, the first thickness being less than the second thickness, and the second orientation is characterized by a back-to-front spacing between the reaction sites and each different second orientation is characterized by a side-to-side spacing between the reaction sites. (D33) The method of any of D1-D32, wherein the apparatus includes an attenuating material layer including a first section aligned with and extending through an area underlying a bottom surface of the first nanowell to define an attenuator of the nanowell, and a second section extending adjacent and laterally from the first section, the first section having a first thickness and the second section having a second thickness, the first thickness being less than the second thickness. A few examples of combinations described above in the "Forms for Implementing the Invention" with reference to Figures 1-12 include the following: (E1) An apparatus comprising a plurality of pixels, a first reaction site associated with one pixel of the plurality of pixels, and a second reaction site associated with the pixel, wherein a pixel sensor of the pixel detects a read signal, the read signal being dependent on a first cluster signal emitted from the first reaction site and a second cluster signal emitted from the second cluster site, the second reaction site having a size larger than the first reaction site such that the second cluster signal in an "on" state has a larger amplitude than the first cluster signal in an "on" state. A few examples of combinations described above in the "Description of the Invention" with reference to Figures 1-12 include the following: (F1) An apparatus comprising a plurality of pixels, a first reaction site associated with a pixel of the plurality of pixels, and a second reaction site associated with the pixel, wherein a pixel sensor of the pixel detects a readout signal, the readout signal being dependent on a first cluster signal emitted from the first reaction site and a second cluster signal emitted from the second cluster site, the second reaction site having a size larger than the first reaction site such that the second cluster signal in an "on" state has a larger amplitude than the first cluster signal in an "on" state. A few examples of combinations described above in the "Description of the Invention" with reference to Figures 1-12 include:The following are included: (G1) An apparatus comprising a plurality of pixels, a first reaction site associated with a pixel of the plurality of pixels, and a second reaction site associated with the pixel, wherein a pixel sensor of the pixel detects a read signal, the read signal being dependent on a first cluster signal emitted from the first reaction site and a second cluster signal emitted from the second cluster site, and a second, a first reaction site is devoid of an attenuator, and a first reaction site includes an attenuator such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state. A few examples of combinations described above in the "Description of Embodiments" with reference to Figures 1-12 include the following: (H1) An apparatus comprising a plurality of pixels, a first reaction site associated with one pixel of the plurality of pixels, and a second reaction site associated with the pixel, a pixel sensor of the pixel detecting a read signal, the read signal being dependent on a first cluster signal emitted from the first reaction site and a second cluster signal emitted from the second cluster site, and the arrangement of attenuating material causes an attenuation of an emission light beam emanating from the first reaction site to be greater than an attenuation of an emission light beam emanating from the second reaction site, such that the second cluster signal in the "on" state has a larger amplitude than the first cluster signal in the "on" state.

[0052] 13 shows an apparatus 100 for use in an analysis, such as a biological or chemical analysis. The apparatus 100 may include a light energy excitation device 10 and a flow cell 282. The flow cell 282 may include a detector 200 and an area above the detector 200. The detector 200 may include a plurality of pixels 201 and a detector surface 208 for supporting clusters C1, C2, such as biological or chemical samples to be tested. The detector 200, having side walls 284 and a flow cover 288, and the detector surface 208, may define and demarcate a flow channel 283. The detector surface 208 may have an associated detector surface plane 130. Each pixel 201 may include a light guide 214 and a pixel sensor 202.

[0053] In a further aspect, detector surface 208 may be configured to define reaction sites 206, which may be provided by, in one example, nanowells 207. According to one example, each reaction site 206 may be associated with a particular pixel 201 and a particular pixel sensor 202 of the particular pixel 201. Each of clusters C1 and C2 may be supported on a respective reaction site 206, which may be provided by, in one example, nanowells 207. Detector surface 208 may be defined by surfaces that define nanowells and surfaces intermediate the nanowells as shown by FIG.

[0054] The detector 200 may, according to one example, include a dielectric stack 213, a semiconductor layer 212, a light guide 214 disposed in an optical path between the detector surface 208 and the pixel sensors 202, and an isolation structure 218 that defines and demarcates pixel areas on each of the pixel sensors 202. The dielectric stack 213 may, in one example, include metallization layers that define various circuits, such as circuits for readout, digitization, storage, and signal processing of signals from the sensing pixels. Metallization layers that define such circuits may additionally or alternatively be incorporated into the isolation structure 218.

[0055] The pixel sensor 202 may be provided by, in one example, a sensing photodiode. The sensing photodiode may be defined by, in one example, a doped region of the semiconductor layer 212. Examples herein recognize that the "region" referred to herein may refer to a volumetric space (in other words, is not limited to a two-dimensional space).

[0056] According to one example, the detector 200 may be provided by a solid-state integrated circuit detector, such as a complementary metal oxide semiconductor (CMOS) integrated circuit detector or a charge coupled device (CCD) integrated circuit detector. The pixel sensor 202 may be provided in one example with a two-dimensional pixel array having rows and columns of pixels arranged in a grid pattern as shown in the cross-sectional top view of Figure 14 taken along the height of the pixel sensor 202. In one example, such a pixel array may include at least 1M pixels, or may include fewer pixels.

[0057] In one aspect, a pixel 201 herein may include a respective pixel sensor 202 and a light guide 214. The light guide 214 may be disposed in a region above each pixel sensor 202 and may be bounded by an isolation structure 218 and a reactive structure 260.

[0058] According to an example, the device 100 can be used to perform biological or chemical tests using analytes provided by fluorophores. For example, a fluid having one or more fluorophores can be passed through the inlet port into and out of the flow cell 282 using the inlet port 289 and the outlet port 290. The analytes provided by the fluorophores can be attracted to the various clusters C1, C2, and thus, upon their detection, the analytes provided by the fluorophores can act as markers for the clusters C1, C2, e.g., biological or chemical analytes that the clusters attract.

[0059] To detect the presence of an analyte provided by a fluorophore in the flow cell 282, the optical energy excitation device 10 can be energized such that excitation light 101 in an excitation wavelength range is emitted by the optical energy excitation device 10. Upon receiving the excitation light 101, the fluorophores attached to the samples C1, C2 can emit emission light 501 that is a signal of interest for detection by the pixel sensor 202. The emission light 501 due to fluorescence of the fluorophores attached to the clusters C1, C2 can have a wavelength range that is red-shifted relative to the wavelength range of the excitation light 101.

[0060] The optical energy excitation device 10 may include at least one light source and at least one optical component for illuminating the samples C1, C2. Examples of light sources include lasers, arc lamps, LEDs, or laser diodes. Optical components may include, for example, reflectors, It may include dichroics, beam splitters, collimators, lenses, filters, wedges, prisms, mirrors, detectors, etc. In an example using an illumination system, the light energy excitation device 10 may be configured to direct excitation light 101 to the reaction site 206. As an example, a fluorophore may be excited by light in the green wavelength range, e.g., using excitation light 101 having a central (peak) wavelength of about 523 nm.

[0061] The examples herein recognize that the signal-to-noise ratio of the device 100 can be expressed as set forth in Equation (1) below.

[0062] The examples herein recognize that the signal-to-noise ratio of the device 100 can be expressed as set forth in Equation (1) below.

[0063]

number

[0064] FIG. 15 is an example of a spectral profile correspondence diagram showing target adjustments between the wavelength range of the excitation light, the wavelength range of the signal light, and the detection wavelength range. In the spectral profile correspondence diagram of FIG. 15, the spectral profile 1101 shown as a green light spectral profile is the spectral profile of the excitation light 101 emitted by the light energy excitation device 10. The spectral profile 1501 is the spectral profile of the emission light 501 caused by the fluorescence of the fluorophore excited by the excitation light 101. The spectral profile 1220 is the transmission profile (detection band) of the pixel sensor 202 according to one example. The spectral profile correspondence diagram of FIG. 15 is intended to represent general features common to several examples, but it will be understood that the changes in the illustrated spectral profiles are common. In one embodiment, the excitation light 101 may generally include a blue light spectral profile (not shown) in addition to the green light spectral profile, and the device 100 is switchable between (a) a mode in which the green light spectral profile is active and the blue light spectral profile is inactive, and (b) a mode in which the blue light spectral profile is active and the green light spectral profile is inactive. In other examples, there may be different combinations of excitation light 101 and emission light 501. In one example, the spectral profile 1101 of the excitation light 101 may be characterized by a central wavelength of a blue light wavelength range, and the spectral profile of the emission light 501 may be characterized by a central wavelength of a green wavelength range.

[0065] The detector 200 may be configured to detect light in a wavelength range indicated by the spectral profile 1220. The spectral profile 1220 specifies a detection wavelength range, and the amplitude of the spectral profile 1220 indicates a level of sensitivity. Thus, referring to the spectral profile correspondence diagram of Figure 15, the detector 200 may detect the emitted light 501 in a range of wavelengths where the spectral profile 1501 of the emitted light 501 and the detection band spectral profile 1220 of the pixel sensor 202 intersect.

[0066] As used herein, and with further reference to the exemplary diagram of FIG. 13, a "flow cell" 282 may include a device having a lid 288 extending above a reaction structure to form a flow channel 283 therebetween that communicates with a plurality of reaction sites 206 of the reaction structure 260. In some examples, a detection device, such as an imaging device and / or an optical system, is separate from the flow cell 282. In other examples, as shown in FIG. 13, the flow cell 282 may include a detection device, e.g., a detector 200, that detects a specified reaction occurring at or adjacent to a reaction site. The flow cell 282 may include a solid-state optical detection device or "imaging" device, such as a charge-coupled device (CCD) or a complementary metal oxide semiconductor (CMOS) (photo) detection device. The CMOS detection device or sensor may include a plurality of detection pixels 201 (pixels) that detect, for example, incident luminescence signals. In some examples, each detection pixel 201 corresponds to a reaction site. In other examples, there may be more or less pixels 201 than the number of reaction sites. Similarly, the detection pixel 201 generates an output signal corresponding to a single sensing element in some examples. In other examples, the pixel 201 generates an output signal corresponding to multiple sensing elements. The flow cell 282 may also or alternatively include two (or more) opposing sensors without a lid. As a specific example, the flow cell 282 may be fluidically, electrically, or fluidically and electrically coupled to a cartridge, which may be fluidically, electrically, or fluidically and electrically coupled to a bioassay system. The cartridge and / or bioassay system may deliver reaction solutions to the reaction sites 206 of the flow cell 282 according to a predetermined protocol (e.g., sequencing by synthesis) and perform multiple imaging events. For example, the cartridge and / or bioassay system may direct one or more reaction solutions through the flow channel of the flow cell, and thereby along the reaction sites. At least one of the reaction solutions may include four types of nucleotides with the same or different fluorescent labels. In some examples, the nucleotides bind to the reaction sites 206 of the flow cell 282, such as corresponding oligonucleotides of the reaction sites.In some examples, the cartridge, bioassay system, or flow cell 282 itself then illuminates the reaction site 206 using an excitation light source (e.g., a solid-state light source such as a light emitting diode (LED)). In some examples, the excitation light has a predetermined wavelength or multiple wavelengths, including a range of wavelengths. Fluorescent labels excited by the incident excitation light can provide an emission signal (e.g., light of one or more wavelengths different from the excitation light, and potentially different from each other) that can be detected by a photosensor in the flow cell 282.

[0067] The flow cells 282 described herein may perform a variety of biological or chemical processes and / or analyses. More specifically, the flow cells 282 described herein may be used in a variety of processes and systems in which it is desirable to detect an event, property, quality, or characteristic indicative of a specified reaction. For example, the flow cells 282 described herein may include or be integrated with optical detection devices, sensors including, but not limited to, biosensors, and components thereof, as well as bioassay systems that operate in conjunction with sensors including biosensors.

[0068] The flow cell 282 facilitates multiple designated reactions that can be detected individually or collectively. The flow cell 282 executes multiple cycles in which multiple designated reactions occur in parallel. For example, the flow cell 282 can be used to sequence a high-density array of DNA features through repeated cycles of enzymatic operation and light or image detection / capture. Thus, the flow cell 282 can be in fluid communication with one or more microfluidic channels that deliver reagents or other reaction components in a reaction solution to the reaction sites 206 of the flow cell. The reaction sites 206 can be provided or spaced in a predetermined manner, such as a uniform or repeating pattern. Alternatively, the reaction sites 206 can be randomly distributed. Each of the reaction sites 206 can be associated with one or more light guides 214 and one or more light sensors that detect light from the associated reaction site 206. In one example, the light guide 214 includes one or more filters for filtering out specific wavelengths of light. The light guide 214 may be, for example, an absorptive filter (e.g., an organic absorptive filter) such that the filter material absorbs a particular 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 site 206 may be located within a reaction recess or chamber within which a designated reaction may be at least partially compartmentalized. Furthermore, the designated reaction may take place or be more easily detected at temperatures other than ambient temperature, for example at elevated temperatures.

[0069] As used herein, a "designated reaction" includes a change in at least one of the chemical, electrical, physical, or optical properties (or qualities) of a chemical or biological substance of interest, such as an analyte of interest. In certain flow cells, the designated reaction is a positive binding event, such as, for example, the mixing of a fluorescently labeled biomolecule with the analyte of interest. More generally, the designated reaction may be a chemical conversion, chemical change, or chemical interaction. The designated reaction may also be a change in an electrical property. In certain flow cells, such as flow cell 282, the designated reaction includes mixing a fluorescently labeled molecule with the analyte. The analyte may be an oligonucleotide, and the fluorescently labeled molecule may be a nucleotide. The designated reaction may be detected when excitation light is directed at the oligonucleotide with the labeled nucleotide and the fluorophore emits a detectable fluorescent signal. In another example of a flow cell, the detected fluorescence is the result of chemiluminescence or bioluminescence. The desired reaction can also be, for example, increasing fluorescence (or Forster) resonance energy transfer (FRET) by bringing a donor fluorophore into close proximity with an acceptor fluorophore, decreasing FRET by moving the donor and acceptor fluorophores apart, increasing fluorescence by moving a quencher away from a fluorophore, or decreasing fluorescence by bringing a quencher and a fluorophore into close proximity. Biological or chemical assays can include detecting a specified reaction.

[0070] As used herein, "electrically coupled" and "optically coupled" refer to the transfer of electrical energy and light waves, respectively, between any combination of power sources, electrodes, conductive portions of a substrate, droplets, conductive traces, wires, waveguides, nanostructures, other circuit segments, etc. The terms "electrically coupled" and "optically coupled" may be used in reference to direct or indirect connections, which may pass through various intermediaries, such as fluidic media, voids, etc. Similarly, "fluidically coupled" refers to moving fluid between any combination of sources. The term "fluidically coupled" may be used in reference to direct or indirect connections, which may pass through various intermediaries, such as channels, wells, pools, pumps, etc.

[0071] As used herein, a "reaction solution," "reaction component," or "reactant" includes any substance that can be used to obtain at least one specified reaction. Potential reaction components include, for example, reagents, enzymes, samples, other biomolecules, and buffers. A reaction component can be delivered in solution to a reaction site 206 in a flow cell 282 disclosed herein and / or immobilized at a reaction site. A reaction component can directly or indirectly interact with another substance, such as an analyte of interest, immobilized at the reaction site 206 of the flow cell 282.

[0072] As used herein, the term "reaction site" is a localized area where at least one designated reaction can occur. The reaction site 206 may include a support surface provided by a reaction structure 260 or a substrate onto which a substance can be immobilized. For example, the reaction site 206 may include a surface of a reaction structure (which may be positioned within a channel of a flow cell) having reaction components thereon, e.g., a colony of nucleic acid thereon. In some flow cells, such as flow cell 282, the nucleic acids in the colony have the same sequence, e.g., are clonal copies of a single-stranded or double-stranded template. However, in some flow cells 282, the reaction site 206 may include only a single nucleic acid molecule, e.g., in single-stranded or double-stranded form.

[0073] As used herein, the term "transparent" refers to allowing all or substantially all visible and non-visible electromagnetic radiation or light of interest to pass through unobstructed, the term "opaque" refers to reflecting, deflecting, absorbing, or otherwise obstructing all or substantially all visible and non-visible electromagnetic radiation or light of interest to not pass through, and the term "non-transparent" refers to allowing some, but not all, visible and non-visible electromagnetic radiation or light of interest to pass through unobstructed.

[0074] As used herein, the term "waveguide" refers to a structure that guides waves, such as electromagnetic waves, minimizing energy loss by restricting the transmission of energy to a particular direction or range of directions.

[0075] As used herein, the term "associated" refers to something being directly or indirectly connected to something else. For example, a first element associated with a second element may refer to the first element being located above or on the second element.

[0076] The proposed methods and structures offer many benefits, including higher throughput of sequencing data and lower cost.

[0077] In a particular example, modulating the signals of two different adjacent clusters C1, C2 in two separate nanowells 207, either through geometric constraints and / or signal modulation, can increase the information density of the CMOS sensor by more than double, providing an advantage over CMOS-based detection devices with one cluster / well per pixel. Furthermore, increasing the information density on a CMOS-based flow cell can have the benefit of reducing the cost per gigabyte of sequencing information by a factor proportional to the increase in density. The practical impact of such innovations is that CMOS-based sequencing can become competitive with larger platforms in terms of information density. Furthermore, implementing the methods and structures disclosed herein requires only software modifications coupled with modifications to the CMOS fabrication, leaving the equipment and reagent consumables substantially, if not completely, unchanged.

[0078] By placing two or more nanowells 207 on top of a pixel, the sensor accepts a minimum 50% intensity hit for the "bright" clusters C1, C2 and a greater than 50% intensity hit compared to the nominal value for the "attenuated" clusters C1, C2 in the transmission modulation option. Different size nanowell 207 implementations (e.g., larger "major" sized wells and smaller "lower" sized wells) accept unequal signal sharing between the two (assumed) monoclonal clusters C1, C2 such that the total signal remains approximately the same. Nevertheless, the signal ratio between the major and lower nanowells 207 is large enough to accurately distinguish the signals between the two clusters.

[0079] For ease of understanding, reference is made below to the drawings, which are not drawn to scale and the same reference numbers are used throughout different figures to denote the same or similar components.

[0080] 16 shows a top view and a cross-sectional view of a portion of a CMOS sensor having a reaction site 206 defined by substantially similar sized nanowells 207A and 207B. As shown in this figure, each of the two nanowells 207 (which may support clusters of interest C1, C2, e.g., monoclonal clusters C1, C2 of DNA strands) has the same length and width.

[0081] However, one nanowell 207B has an attenuation filter (attenuator) that attenuates the signal by approximately 50%, while the other nanowell 207A does not. The attenuator of nanowell 207B is shown as being defined by a layer of attenuating material 1204 below the surface that defines the bottom of nanowell 207B. Although the nanowells 207, 207A-207D in the figures may be shown as rectangular, other shapes of nanowells 207, 207A-207D are possible, e.g., circular, elliptical, hexagonal, octagonal, etc.

[0082] 16, reactive structure 260 may include a dielectric stack featuring multiple dielectric layers including dielectric layer 1202, dielectric layer 1206, and dielectric layer 1210. The dielectric stack defined by reactive structure 260 may further include first and second attenuating material layers integrally formed therein, including attenuating material layer 1204, attenuating material layer 1205, and attenuating material layer 1208. Attenuating material layers 1204, 1205, and 1208 may be provided by metal in one example. Attenuating material layer 1204, attenuating material layer 1205, and attenuating material layer 1208 may include, for example, tantalum, aluminum, gold, or copper, etc.

[0083] 16, it can be seen that reaction site 206B defined by nanowell 207B may include an attenuator, and nanowell 207A may be devoid of an attenuator. In the example of FIG. 16, reaction site 206B defined by nanowell 207B may include an attenuator defined by attenuating material layer 1205, and nanowell 207A may be devoid of attenuating material layer 1205 defining an attenuator. As shown in FIG. 16, attenuating material layer 1205 includes a first upwardly extending section that extends parallel to the sidewall of nanowell 207B along the perimeter of nanowell 207B, and a second horizontally extending section that extends parallel to the bottom surface of nanowell 207B.

[0084] In one embodiment, reaction site 206B defined by nanowell 207B and reaction site 206A defined by nanowell 207A can include an associated vertically extending central axis 1216.

[0085] 16, device 100 can be configured such that the vertically extending central axis 1216 of nanowell 207B extends through the attenuator and further such that the vertically extending central axis 1216 of nanowell 207A does not extend through the attenuator. Referring to the example of FIG. 16, device 100 can be configured such that the vertically extending central axis 1216 of nanowell 207B extends through the attenuator defined by attenuating material layer 1205 and further such that the vertically extending central axis 1216 of nanowell 207A does not extend through the attenuator defined by attenuating material layer 1205. In another embodiment, the vertically extending central axis 1216 of reaction site 206B defined by nanowell 207B extends through attenuating material layer 1205, and the vertically extending central axis 1216 of reaction site 206A defined by nanowell 207A does not extend through attenuating material layer 1205. In another embodiment, the vertically extending central axis 1216 of reaction site 206B defined by nanowell 207B extends through an attenuator that is not present in reaction site 206A defined by nanowell 207A.

[0086] 16, nanowell 207B in a region aligned with and below the bottom surface of nanowell 207B may include an attenuator defined by attenuating material layer 1205, and nanowell 207A in a region aligned with and below the bottom surface of nanowell 207A may be devoid of an attenuator. The region of nanowell 207B aligned with and below the bottom surface of nanowell 207B may include a region below the bottom surface of nanowell 207B, which is bounded by a depth dimension extending through vertically extending plane 1217 that intersects a bottom corner of nanowell 207B, and by a width dimension extending through vertically extending plane 1218 that intersects a bottom corner of nanowell 207B, as shown in FIG. 16, which may be concluded as a rectangular base. 16 of nanowell 207B may be configured to include a section vertically below the bottom surface of nanowell 207B and may be configured to attenuate an emission ray of emitted light 501 that radiates vertically downward from the bottom of nanowell 207B. In another embodiment, nanowell 207A may not have an attenuator that attenuates an emission ray of emitted light that radiates vertically downward from the bottom of nanowell 207A.

[0087] FIG. 17 shows a top view and a cross-sectional view of a portion of a CMOS sensor with nanowells 207A and 207B of substantially different sizes. As shown in this figure, each of the two nanowells 207A and 207B (which may support clusters of interest C1, C2, e.g., monoclonal clusters C1, C2 of DNA strands) has a different width, with the left nanowell 207A having half the width of the right nanowell. This allows the size of the clusters of interest C1, C2 to be limited, and therefore the cluster signals emitted by the clusters C1, C2 when the analyte of interest is present. For example, if the nanowell 207B is twice the size of the second nanowell 207A, the nanowell 207B may generate twice the cluster signal intensity compared to the nanowell 207A.

[0088] In the example of FIG. 17, reaction site 206B defined by nanowell 207B may include a greater width and nanowell bottom surface area than reaction site 206A defined by nanowell 207A. As shown in the example of FIG. 17, an increase in the surface area of ​​the nanowell bottom may increase the number of strands that define a cluster. Thus, as a result of nanowell 207B having an increased surface area relative to nanowell 207A, cluster C2 may have a greater number of strands than cluster C1. In one example, the width of nanowell 207B relative to nanowell 207A may be selected such that the number of strands in cluster C2 is about 2.0 times the number of strands in cluster C1. In one example, the width of nanowell 207B may be configured such that the number of strands in cluster C2 is about 1.9 times the number of strands in cluster C1.

[0089] Examples herein recognize that the total volume of nanowells 207A and 207B may control the number of strands. Examples herein recognize that the relationship between volume and number of strands may not be linear, but may be determined empirically. In one example, the bottom surface area of ​​each nanowell 207A and 207B may be configured such that nanowell 207B has twice the bottom surface area of ​​nanowell 207A to produce a chain number difference between the nanowells of about 2.0 or about 1.9 and a corresponding "on" state signal amplitude difference. In another example, experiments may be performed to derive the relationship between nanowell bottom surface area and number of strands for the particular conditions used, and the surface area difference between the nanowells may be designed accordingly.

[0090] FIG. 18A shows a cross-sectional view of a portion of a CMOS sensor in which one of the two nanowells 207A and 207B has an attenuation filter for attenuating the cluster signal. The second nanowell 207B may have an attenuation filter layer disposed below, in some instances directly below, the illustrated bottom surface of the second nanowell 207B, which is not present in the first nanowell 207A. Other examples may have an additional layer or material present between the surface of the nanowell 207 and the attenuation filter layer. In the example of FIG. 18A, the attenuation filter layer may be provided by an attenuating material layer 1205. The right nanowell 207 in this FIG. 18A is shown with an additional layer. This filter layer may be a thin metal layer. The filter layer should be compatible with the fabrication process of the CMOS sensor. In some examples, the filter layer attenuates about 50% (half) of the cluster signal emitted from cluster C2 when the analyte of interest is present. The attenuation filter layer may include or be made of tantalum.

[0091] 18A, reactive structure 260 may include a dielectric stack featuring multiple dielectric layers including dielectric layer 1202, dielectric layer 1206, and dielectric layer 1210. The dielectric stack defined by reactive structure 260 may further include first and second attenuating material layers integrally formed therein, including attenuating material layer 1204, attenuating material layer 1205, and attenuating material layer 1208. Attenuating material layers 1204, 1205, and 1208 may be provided by metal in one example. Attenuating material layer 1204, attenuating material layer 1205, and attenuating material layer 1208 may include, for example, tantalum, aluminum, gold, or copper, etc.

[0092] 18A, it can be seen that reaction site 206B defined by nanowell 207B may include an attenuator, and nanowell 207A may be devoid of an attenuator. In the example of FIG. 18A, reaction site 206B defined by nanowell 207B may include an attenuator defined by attenuating material layer 1205, and nanowell 207A may be devoid of attenuating material layer 1205 defining an attenuator. As shown in FIG. 18A, attenuating material layer 1205 may include a first upwardly extending section that extends parallel to the sidewall of nanowell 207B along the perimeter of nanowell 207B, and a second horizontally extending section that extends parallel to the bottom surface of nanowell 207B.

[0093] In one embodiment, reaction site 206B defined by nanowell 207B and reaction site 206A defined by nanowell 207A can include associated vertically extending central axes 1216. Referring to the example of FIG. 18A, device 100 can be configured such that vertically extending central axis 1216 of nanowell 207B extends through an attenuator and further such that vertically extending central axis 1216 of nanowell 207A does not extend through an attenuator. Referring to the example of FIG. 18A, device 100 can be configured such that vertically extending central axis 1216 of nanowell 207B extends through an attenuator defined by attenuator material layer 1205 and further such that vertically extending central axis 1216 of nanowell 207A does not extend through an attenuator defined by attenuator material layer 1205. In another embodiment, the vertically extending central axis 1216 of reaction site 206B defined by nanowell 207B extends through attenuating material layer 1205, and the vertically extending central axis 1216 of reaction site 206A defined by nanowell 207A does not extend through attenuating material layer 1205. In another embodiment, the vertically extending central axis 1216 of reaction site 206B defined by nanowell 207B extends through an attenuator that is not present in reaction site 206A defined by nanowell 207A.

[0094] 18A , nanowell 207B in a region aligned with and below the bottom surface of nanowell 207B may include an attenuator defined by attenuating material layer 1205, and nanowell 207A in a region aligned with and below the bottom surface of nanowell 207A may be devoid of an attenuator. The region of nanowell 207B aligned with and below the bottom surface of nanowell 207B may include a region below the bottom surface of nanowell 207B, which may include a rectangular base, as shown in FIG. 18A , bounded by a depth dimension extending through vertically extending plane 1217 that intersects a bottom corner of nanowell 207B, and bounded by a width dimension extending through vertically extending plane 1218 that intersects a bottom corner of nanowell 207B. 18A of nanowell 207B may be configured to include a section vertically below the bottom surface of nanowell 207B and may be configured to attenuate an emission ray of emitted light 501 that radiates vertically downward from the bottom of nanowell 207B. In another embodiment, nanowell 207A may not have an attenuator that attenuates an emission ray of emitted light that radiates vertically downward from the bottom of nanowell 207A.

[0095] A method of fabricating device 100 shown in FIG. 18A will be described with reference to the fabrication stage diagrams of FIG. 18B-D. With reference to FIG. 18B, layers 1202, 1204, 1206, and 1208 may be deposited over and aligned to pixel 201, which may be defined, in one example, by pixel sensor 202 and light guide 214. With reference to FIG. 18C, trench 209 may be etched to define reaction site 206B defined by nanowell 207B, shown at an intermediate stage of fabrication in FIG. 18C. As can be seen from FIG. 18C, trench 209 may be fabricated prior to and independent of the trench for fabrication of reaction site 206A defined by nanowell 207A. As shown in Figure 18D, prior to creating trench 219 for providing nanowell 207A, at an intermediate stage of fabrication, attenuating material layer 1205 can be deposited in trench 209 defining reaction site 206B provided by nanowell 207B shown in Figure 18D. At an intermediate stage of fabrication shown in Figure 18D, attenuating material layer 1205 can overfill trench 209 for reaction site 206B provided by nanowell 207B.

[0096] Additional fabrication steps can be performed to complete the fabrication of the structure shown in Figure 18A. These additional fabrication steps can include, for example, planarizing layer 1205 to the height of the top height of layer 1208, depositing a sacrificial layer of dielectric material to overfill trench 209 associated with reaction site 206B defined by nanowell 207B, planarizing the overfilled layer, forming a second trench 209 in the dashed area as shown in the dashed area of ​​Figure 18D for reaction site 206A defined by nanowell 207A, etching away the depicted sacrificial layer, and then depositing a dielectric layer 1210 as shown in Figure 18A to define the top layer of both trench 209 associated with reaction site 206B defined by nanowell 206B and trench 209 associated with reaction site 207A defined by nanowell 207A. According to one example, a method for fabricating a device is described herein, the method including depositing a dielectric stack over a pixel in an area aligned with the pixel, etching a first trench in the dielectric stack to define a first nanowell, etching a second trench in the dielectric stack to define a second nanowell, and depositing a layer of attenuating material in the first trench that is not deposited in the second trench.

[0097] FIG. 19A shows a top view and a cross-sectional view of a portion of another CMOS sensor in which one of the two nanowells 207 has an attenuation filter (attenuator) for attenuating the cluster signal. One of the layers of the CMOS sensor may have an attenuation filter layer. However, this attenuation filter layer may be removed below the bottom surface of the first nanowell 207A of the two nanowells 207A and 207B above the pixel 201 that may be defined by the light guide 214 in combination with the pixel sensor 202, but not below the second nanowell 207B. Therefore, this configuration may not attenuate the cluster signal emitted from the first nanowell 207A. In FIG. 19A, the attenuation filter layer may be provided by the attenuating material layer 1204.

[0098] 19A example, device 100 may include attenuating material layer 1204 positioned such that the section of attenuating material layer 1204 defining nanowell 207B is aligned with and below the bottom surface of nanowell 207B, but the section aligned with and below the bottom surface of nanowell 207A is not below that bottom surface. The illustrated section of attenuating material layer 1204 aligned with and below the bottom surface of nanowell 207B may be disposed in an emission light path between the bottom surface of nanowell 207B and pixel sensor 202 associated with nanowell 207B, but is aligned with and below the bottom surface of nanowell 207A, and is not in an optical path between the bottom surface of nanowell 207A and pixel sensors 202 associated with nanowells 207B and 207A.

[0099] 19A, the light-attenuating material layer 1204 can be configured to provide multiple functions. In a first aspect, the light-attenuating material layer 1204 can suppress and reduce crosstalk between the reaction sites associated with the first pixel 201 (reaction sites 206A and 206B) and the second pixel area 201 (reaction sites 206B and 206C). In another aspect, the light-attenuating material layer 1204 can purposely attenuate the emission from the clusters supported within the reaction sites provided by the nanowells 207A to achieve sequencing using the calling charts described with reference to FIGS. 4 and 5.

[0100] Fabrication stage diagrams for fabricating the device shown in FIG. 19A are described with reference to FIGS. 19B and 19C. With reference to the intermediate fabrication stages of FIG. 19B, a reactive structure 260 shown at an intermediate fabrication stage can be deposited on the illustrated structure defined by the isolation structure 218 and the light guide 214. In the intermediate fabrication stages of FIG. 19B, a dielectric layer 1202, a light attenuating material layer 1204, a dielectric layer 1206, and a light attenuating material layer 1210 can be deposited. With reference to the intermediate fabrication stages of FIG. 19C, trenches 209 can be etched to define reactive sites 206A-206D provided by nanowells 207A-207D shown at an intermediate fabrication stage of FIG. 19C. With reference to the intermediate fabrication stages of FIG. 19C, different trenches 209 can be etched to different heights in different etching stages. In a first etching step, trenches 209 for reaction sites 206A and 206C provided by nanowells 207A and 207C can be etched to a height defined by the top height of dielectric layer 1202. Then, after filling the trenches for reaction sites 206A and 206C described with a sacrificial material layer, trenches 209 for reaction sites 206B and 206D provided by nanowells 207B and 207D can be etched. The etching to define nanowells 207B and 207D can include etching to a height defined by the top height of attenuating material layer 1204 such that nanowells 207B and 207D have bottom surfaces with a higher height than the bottom surfaces of nanowells 207A and 207C, and further such that nanowells 207B and 207D include attenuators defined by attenuating material layer 1204 that are not associated with nanowells 207A or nanowells 207C. For final fabrication to achieve the structure of FIG. 19A, a dielectric layer 1210 defining the top surfaces of nanowells 207A-207D can be deposited within the illustrated trenches 209 shown in FIG. 19C.According to one example, a method for fabricating a device is described herein, the method including: depositing a dielectric stack overlying the pixel in an area aligned with the pixel and having an attenuating material integrally formed therein; etching a first trench in the dielectric stack to define a first nanowell, the first trench being etched to a height terminating at or above a top height of the attenuating material layer such that the attenuating material layer defines an attenuator for the first nanowell; and etching a second trench in the dielectric stack to define a second nanowell, the second trench being etched to a height terminating at or below a bottom height of the attenuating material layer.

[0101] 19A , according to one example, the device 100 may be configured such that in an alternative embodiment, the attenuating material layer 1204 includes a first thickness T1 and a second thickness T2. The attenuating material layer 1204 may include a first thickness T1 in a section of the attenuating material layer 1204 that defines the nanowell 207D below its bottom surface and aligned with the bottom surface of the nanowell 207D in the optical path between the bottom surface of the nanowell 207D and the pixel sensor 202. The thickness T1 may be less than the thickness T2. By providing a configuration as shown in the alternative embodiment shown in FIG. 19A , different levels of attenuation may be facilitated in an area below and aligned with the bottom surface of the nanowell 207 and an area outside of, below and aligned with the bottom surface of the nanowell 207. In one example, attenuating material layer 1204 can provide a first, smaller level of attenuation, e.g., about 50% attenuation in a section of attenuating material layer 1204 aligned with and below the bottom surface of nanowell 207D, while in the region shown outside of the section of attenuating material layer 1204 aligned with and below the bottom surface of nanowell 207D, attenuating material layer 1204 can provide a second level of attenuation, e.g., about 90% attenuation of incident light. The arrangement shown in the exploded view of Figure 19A can be useful when a greater attenuation level is targeted to provide differentiated clusters of signals, e.g., to suppress crosstalk, as described herein.

[0102] In one example, the attenuating material layer 1204 can be fabricated to have multiple heights by first depositing the light-attenuating material layer 1204 to a thickness T2 and then etching away portions of the attenuating material layer 1204 to a defined thickness T1 in areas aligned with and below the nanowell bottom as described. In another example, the attenuating material layer 1204 can be fabricated using first and second deposition stages, where in the first stage a sublayer is deposited to a thickness T1 and then a second sublayer is selectively deposited to define a thickness T2. According to one example, an apparatus is described herein that includes an attenuating material layer including a first section aligned with and extending through a region underlying a bottom surface of a first nanowell to define an attenuator of the nanowell, and a second section extending adjacent and laterally (or horizontally or laterally) from the first section, wherein the first section has a first thickness and the second section has a second thickness, the first thickness being less than the second thickness.

[0103] Figure 20 shows a top view of a portion of a CMOS sensor illustrating the placement of nanowells 207 over adjacent pixels 201. With reference to Figure 20, different sets of reaction sites 206 provided by nanowells 207 associated with different pixels 201 are shown. In Figure 20, it can be seen that the set of reaction sites 206 provided by nanowells 207 associated within a particular rectangle is associated with a particular one of the pixels 201. The different pixels 201 shown in Figure 20 may have different pixel locations A1-D2.

[0104] With reference to FIG. 20, it can be seen that the sets of reaction sites 206 provided by nanowells 207 associated with adjacent pixels 201 defining a pixel array can have different orientations. With reference to FIG. 20, the nanowells 207 associated with a first pixel having pixel location A1 can have a first orientation, and the nanowells 207 associated with a second pixel having pixel location B1 can have a second orientation different from the first orientation. The same pattern between adjacent pixels 201 can be repeated throughout the illustrated pixel array. The described first orientation can be characterized by the first and second reaction sites provided by nanowells being spaced apart in a depth dimension from a top view, i.e., from back to front, and the second set of pixels can be characterized by the first and second reaction sites 206 provided by nanowells being spaced apart in a width dimension (which can also be translated as lateral or side-to-side). The depicted first direction may also be referred to as a direction parallel to the Y-axis of the depicted reference coordinate system, and the depicted second direction may also be referred to as a direction parallel to the X-axis of the depicted reference coordinate system. In another embodiment, a set of reaction sites provided by nanowells associated with pixels 201 at pixel locations A1, C1, B2, D2 may have a first orientation, and pixels in a set of reaction sites 206 provided by nanowells 207 associated with pixels 201 at the depicted pixel locations B1, D1, A2, C2 may have a second orientation that is different from the first orientation.

[0105] FIG. 21 shows a top view and a cross-sectional view of a portion of a CMOS sensor showing the arrangement of a set of nanowells 207 on adjacent pixels 201 according to the example of FIG. 20. The lighter circles represent attenuated wells (i.e., nanowells 207 with attenuated cluster signals) and the darker circles represent unattenuated wells (i.e., nanowells 207 with unattenuated cluster signals). In some examples, it may be preferable to maximize the distance between nanowells, for example, to reduce crosstalk detected by the pixel sensor 202 from adjacent wells. FIG. 20 and FIG. 21 show perpendicular nanowell orientation between adjacent pixels. For example, if a first pixel 201 at, for example, position A1 has a left-to-right nanowell orientation, a second pixel 201 at position B1 adjacent to the first pixel 201 may have a top-to-bottom (back-to-front) nanowell orientation.

[0106] Figure 21 shows an example top view and cross-sectional view according to the example shown in Figure 20. In Figure 21, the darker shaded reaction sites 206A and 206C indicate reaction sites defined by nanowells 207A and 207C, respectively, that do not have attenuators to attenuate emission light rays emanating vertically downward from the bottom of respective nanowells 207A and 207C in a direction parallel to the illustrated Z-axis of the reference coordinate system, and the lighter shaded reaction sites 206B and 206D are reaction sites that have attenuators to attenuate emission light rays of emission light 501 emanating vertically downward from the bottom of nanowell 207D in a direction parallel to the illustrated Z-axis of the reference coordinate system. In the example of FIG. 21, reaction site 206D provided by nanowell 207D can be characterized as having an attenuator defined by a section of attenuating material layer 1204 that attenuates emission rays of emission light 501 emanating vertically downward from detector surface 208 that defines the bottom of nanowell 207D.

[0107] 20 and 21, it can be seen that configuring the reaction sites provided by the nanowells associated with adjacent pixels to have different alternating orientations can increase the spacing distance between the reaction sites associated with adjacent pixels. For example, it can be seen that if the reaction sites 206 associated with pixels in pixel regions B1 and A1 were each spaced apart in the width dimension (lateral or side-to-side, i.e., both oriented as shown for pixel region B1), the minimum spacing distance between the reaction sites in pixel regions B1 and A1 would likely be reduced beyond fabrication capabilities, which could increase crosstalk between the reaction sites associated with a particular pixel 201 and adjacent pixels adjacent to the particular pixel 201 that are not associated with a reaction site.

[0108] Variations on the example of FIG. 21 are shown in FIG. 22-24. In the example of FIG. 22, the illustrated reaction sites provided by nanowells 207A-207D have a defined elliptical shape when viewed from the top, rather than a circular shape when viewed from the top as shown in FIG. 21. In the example of FIG. 23, reaction sites 206A-206D provided by nanowells 207A-207D have first and second different shapes and different sizes. Nanowells 207B and 207D are circular when viewed from the top, while reaction sites 206A and 206C provided by nanowells 207A and 207C have an elliptical shape when viewed from the top, and are larger in terms of nanowell bottom surface area than nanowells 207B and 207D.

[0109] In the example of Figure 23, nanowells 207D and 207B of smaller bottom surface area size have attenuators defined by sections of attenuating material layer 1204 that intersect central axes 1216 of nanowells 207D and 207B, and the associated attenuators associated with nanowells 207D and 207B are positioned to attenuate emission rays of emission light 501 that radiate vertically downward from a surface that defines the bottom of nanowells 207D and 207B. In the example of Figure 23, nanowells 207C and 207A do not have attenuators that attenuate emission rays of emission light 501 that radiate vertically downward from detector surface 208 that defines the bottom of their respective nanowells 207C and 207A.

[0110] The example of FIG. 24 is similar to the example of FIG. 23, except that in the example of FIG. 24, an attenuator defined by attenuating material layer 1204 is associated with the larger nanowells 207C and 207A, but not with the smaller bottom surface area nanowells 207D and 207B. The examples herein recognize that the presence or absence of an attenuator may have a greater effect on the signal intensity of the cluster signal than the nanowell size. In the example of FIG. 24, the presence or absence of an attenuator may be used to provide coarse adjustment of the cluster signal "on" signal amplitude, and the nanowell size may be used to provide fine adjustment of the cluster signal "on" signal amplitude.

[0111] With reference to FIG. 24, the region defining nanowell 207C that is below and aligned with the bottom surface of nanowell 207C may include an attenuator, i.e., an attenuator defined by attenuating material layer 1204, and the region defining nanowell 207D that is below and aligned with the bottom surface of nanowell 207D, nanowell 207D, may be free of an attenuator, as shown in FIG. 24.

[0112] The region defining nanowell 207C that is aligned with and below the bottom surface of nanowell 207C may include a region bounded by a vertically extending plane 1217 that intersects the bottom surface of nanowell 207C at the maximum width of nanowell 207C in the indicated X-axis dimension. The region defining nanowell 207C that is aligned with and below the bottom surface of nanowell 207C may include a region in the optical path between the bottom surface of nanowell 207C and pixel sensor 202 associated with nanowell 207C, which is bounded by a vertically extending plane 1218 that intersects a bottom corner of nanowell 207C at a point of maximum depth of nanowell 207C in a direction extending parallel to the indicated reference Y-axis.

[0113] The region defining nanowell 207D that is below and aligned with the bottom surface of nanowell 207D may include the region in the optical path between the bottom surface of nanowell 207D and the pixel sensor 202 associated with nanowell 207D, which is bounded by a vertically extending plane 1217 that intersects a bottom corner of nanowell 207D at the maximum width of the bottom of nanowell 207D in a direction parallel to the reference X-axis, and by a vertically extending plane 1218 that intersects a corner of the bottom of nanowell 207D at a location that defines the maximum depth of nanowell 207D in a direction extending parallel to the reference Y-axis shown in FIG. 24.

[0114] The same relationship is shown in Figure 24, where the area below and aligned with the bottom surface of a first nanowell in the optical path between the bottom surface of the nanowell and its associated pixel sensor 202 includes an attenuator, and the area below and aligned with the bottom surface of a second nanowell in the optical path between the bottom surface of the second nanowell and the pixel sensor 202 associated with the first nanowell does not have an attenuator, as shown throughout the figures, including those relating to Figures 6, 8, 9, 10, 11, 16, 18, 19, 20, 21, 22, 23, and 24 (Figures 6, 8-11, 16, 18-24).

[0115] In the examples of Figures 6, 8-11, 16, 18-24, the device may be configured to increase the attenuation of emitted light from a first nanowell relative to the attenuation of emitted light from a second nanowell using an attenuating material. In the examples of Figures 6, 8-11, 16, 18-24, the device may be configured such that the first nanowell includes an attenuator and the second nanowell does not have an attenuator. In the examples of Figures 6, 8-11, 16, 18-24, the device may be configured such that the first nanowell includes an attenuator that attenuates emitted light radiating vertically downward from the bottom surface of the first nanowell and the second nanowell does not have an attenuator that attenuates emitted light radiating vertically downward from the bottom surface of the second nanowell. In the examples of Figures 6, 8-11, 16, 18-24, the device may be configured such that the first nanowell includes an attenuator in a region below the bottom surface of the first nanowell and the second nanowell does not include an attenuator in a region below the bottom surface of the second nanowell. In the examples of Figures 6, 8-11, 16, 18-24, the device may be configured such that the first nanowell includes an attenuator in a region directly below the bottom surface of the first nanowell and the second nanowell does not have an attenuator in the region directly below the bottom surface. In the examples of Figures 6, 8-11, 16, 18-24, the device may be configured such that a first nanowell includes an attenuator in a region below its bottom surface that is aligned with the bottom surface of the first nanowell, and a second nanowell does not have an attenuator in a region below its bottom surface that is aligned with the bottom surface of the second nanowell (or the second nanowell does not have an attenuator in a region below its bottom surface that is aligned with the bottom surface of the second nanowell). In one aspect, the region below the bottom surface of the nanowell that is aligned with the bottom surface of the second nanowell may be a region intersected by the vertically extending central axes 1216 of the nanowells. In one aspect, the region below the bottom surface of the nanowell that is aligned with the bottom surface of the nanowell may be a region bounded by vertically extending planes 1217 and 1218, as described herein.

[0116] In the example of Figure 25, each of the smaller sized nanowells 207D and 207B and the larger sized nanowells 207C and 207A does not have an attenuator to attenuate the emission rays of emission light 501 emanating vertically downward from the detector surface 208 that defines the bottom of each nanowell 207A-207D.

[0117] Exemplary dimensions according to the examples described with reference to FIGS. 22-25 may be provided as summarized in Table A.

[0118] [Table 1]

[0119] 16-25, the reactive structure 260 may be defined by a dielectric stack having a plurality of dielectric layers including dielectric layer 1202, dielectric layer 1206, and dielectric layer 1210, which may define the detector surface 208 as shown in FIG. 13. One or more attenuating material layers may be integrally formed within the dielectric stack defining the reactive structure 260, including attenuating material layer 1204, attenuating material layer 1205, and / or attenuating material layer 1208. Exemplary dimensions for the examples shown in FIGS. 16-25 are described with reference to Table B. The materials of the various layers as shown in Table B may be materials summarized in Table A or elsewhere herein.

[0120] [Table 2]

[0121] Referring again to FIG. 13, the device 100 may include a processing circuit 310. The processing circuit 310 may include, according to an example, one or more processors 3101, a memory 3102, and one or more input / output interfaces 3103. The one or more processors 3101, the memory 3102, and the one or more input / output interfaces may be connected via a system bus 3104. The memory 3102 may include a combination of system memory and storage memory. The memory 3102 may store, according to an example, one or more programs for facilitating the processes described herein. The one or more processors 3101 may execute one or more programs stored in the memory 3102 to facilitate the processes described herein. The memory 3102 may define a computer-readable medium.

[0122] The DNA sequencing process facilitated by the light energy excitation device 10 will be described with reference to Figures 13, 15, and 26. Referring to Figure 15, a spectral profile correspondence diagram illustrating an aspect of the operation of the device 100 is shown. According to one example, the light energy excitation device 10 can be configured to emit light of first and second different wavelengths. As shown in Figures 1-12, providing excitation light of first and second different wavelength ranges facilitates a dye chemical DNA sequence reconstruction process in which first and second dyes can be disposed in a fluid within a flow cell 282.

[0123] In the spectral profile correspondence diagram of FIG. 15, the spectral profile 1101 shown as a green light spectral profile is the spectral profile of the excitation light 101 emitted by the light energy excitation device 10. The spectral profile 1501 is the spectral profile of the emission light 501 caused by the fluorescence of the fluorophores excited by the excitation light 101. The spectral profile 1220 is the transmission profile (detection band) of the pixel sensor 202 according to one example. The spectral profile correspondence diagram of FIG. 2 is intended to represent general features common to several examples, but it will be understood that variations in the illustrated spectral profiles are common. In one embodiment, the excitation light 101 may generally include a blue light spectral profile (not shown) in addition to the green light spectral profile, and the device 100 is switchable between (a) a mode in which the green light spectral profile is active and the blue light spectral profile is inactive, and (b) a mode in which the blue light spectral profile is active and the green light spectral profile is inactive. In other examples, there may be different combinations of the excitation light 101 and the emission light 501. In one example, the spectral profile 1101 of the excitation light 101 may be characterized by a central wavelength in a blue light wavelength range, and the spectral profile of the emission light 501 may be characterized by a central wavelength in a green wavelength range.

[0124] Examples herein recognize that, with reference to the spectral profile correspondence diagram of FIG. 15 , the processing circuit 310 can be configured to: (a) determine that a first fluorophore is attached to each cluster C1, C2 based on the fluorescence sensed by the pixel sensor 202 under excitation limited to excitation by one or more green-emitting light sources and the fluorescence not sensed by the pixel sensor 202 under excitation limited to excitation by one or more blue-emitting light sources; (b) determine that a second fluorophore is attached to the clusters C1, C2 based on the fluorescence sensed by the pixel sensor 202 under excitation limited to excitation by one or more blue-emitting light sources and the fluorescence not sensed by the pixel sensor 202 under excitation limited to excitation by one or more green-emitting light sources; and (c) determine that a third fluorophore is attached to the clusters C1, C2 based on the fluorescence sensed by the pixel sensor 202 under excitation limited to excitation by one or more green-emitting light sources and the fluorescence sensed by the pixel sensor 202 also under excitation limited to excitation by one or more blue-emitting light sources.

[0125] The processing circuit 310 can use, for example, a decision logic data structure illustrated by the calling charts of Figures 4 and 5, represented in the decision logic table of Table C, which maps the presence of fluorophores to nucleotide types, to identify which fluorophores are attached to the clusters and determine the nucleotide types, e.g., A, C, T, and G, present in the fragments of the DNA strand providing the clusters C1, C2, and the identified nucleotides Nucleotide-Nucleotide4 are nucleotides of nucleotide types A, C, T, and G (the particular mapping is based on the test setup parameters). Examples herein recognize that the output signal levels sensed under different bands of narrowband illumination can be mapped to a particular cloud based on the closest Euclidean distance.

[0126] [Table 3]

[0127] The processing circuit 310 can perform a process to assist in DNA sequence reconstruction in multiple cycles. In each cycle, a different portion of the DNA fragment can be subjected to a sequencing process to determine the nucleotide type associated with the fragment, e.g., A, C, T, or G, using a determination data structure, such as the determination data structure shown in Figures 4 and 5 and Table C. An embodiment of a process that can be performed by the processing circuit 310 for use in performing DNA sequence reconstruction using the light energy excitation device 10 is described in the flow diagram of Figure 26.

[0128] At block 1802, the processing circuit 310 can clear the flow cell 282, which means that the processing circuit 310 can remove fluid from the flow cell 282 that was used during the previous cycle. At block 1804, the processing circuit 310 can load the flow cell 282 with a fluid having multiple fluorophores, for example a first and a second fluorophore, or a first, second, and third fluorophore.

[0129] In block 1806, the processing circuit 310 can read out a signal from the pixel sensor 202 exposed to a first wavelength range of the active excitation light 101. In block 1806, the processing circuit 310 can control the light energy excitation device 10 such that the light energy excitation device 10 emits excitation light 101 limited to excitation by one or more green light sources during the exposure period of the pixel sensor 202. In block 1806, the processing circuit 310 can energize each of the one or more green light emitting light energy excitation devices 10 during the exposure period of the pixel sensor 202. Once the light energy excitation device 10 is controlled as described above such that the green light source is on and the blue light source is off during the exposure period of the pixel sensor 202, the processing circuit 310 can read out a first signal in block 1806 from the pixel sensor 202 exposed to excitation limited to excitation by one or more green light sources as described herein, including with reference to the spectral profile correspondence diagram of FIG.

[0130] In block 1808, the processing circuit 310 can read out a signal from the pixel sensor 202 exposed to the active second wavelength range excitation. In block 1808, the processing circuit 310 can control the light energy excitation device 10 to emit excitation light limited to excitation by the one or more blue light sources of the light energy excitation device 10 during the exposure period of the pixel sensor 202. In block 1808, the processing circuit 310 can energize each of the one or more blue light emitting light sources of the light energy excitation device 10 while maintaining each of the one or more green light emitting light sources of the light energy excitation device 10 in a non-energized state during the exposure period of the pixel sensor 202. Once the light energy excitation device 10 has been controlled as described above so that the blue light source is on and the green light source is off during the exposure period of the pixel sensor 202, the processing circuit 310 can, in block 1808, read out a second signal from the pixel sensor 202 that has been exposed to excitation limited to excitation by one or more blue light sources of the light energy excitation device 10, as described herein.

[0131] In each of blocks 1806 and 1808, the optical energy excitation device 10 can simultaneously direct the referenced narrowband excitation light 101 (green light in block 1806 and blue light in block 1808) to all reaction sites 206 of the reaction structure 260 such that all clusters C1, C2 supported by the reaction structure 260, including clusters C1 and C2 disposed in adjacent nanowells 207, 207A-207B, 207C-207D commonly associated with a particular pixel 201, are simultaneously excited with the narrowband excitation light 101 described with reference to block 1806. Thus, embodiments herein recognize that the device 100 can facilitate sequencing without precise directional control of the excitation light. In other examples, excitation can be advantageously performed using precise directional control of the excitation light 101 such that a first nanowell 207, 207A-207B, 207C-207D associated with a particular pixel of the pixel sensor 202 is excited without exciting a second nanowell 207, 207A-207B, 207C-207D associated with a particular pixel of the pixel sensor 202.

[0132] In block 1810, the processing circuit 310 for the current cycle may process the first signal read in block 1806 and the second signal read in block 1808 to determine the nucleotide type of the DNA fragment undergoing testing during the current cycle, for example, using a determination data structure as described in Figures 4 and 5 and Table C, according to one example. The processing circuit 310 may perform the described nucleotide identification process described with reference to the flow diagram of Figure 26 for each cycle of the DNA sequencing process until nucleotide identification has been performed for each scheduled cycle.

[0133] 27A and 27B are diagrams illustrating signal-to-noise design considerations in the manufacture of devices herein with multiple reaction sites per pixel. FIG. 27A shows a calling chart for a device with a single reaction site per pixel, where there are four possible clouds defined by the first cluster signal under excitation in a first channel combined with the first cluster signal under excitation by a second channel of excitation light. In the example shown in FIG. 27A, the calling chart can include four signal clouds with a device with a single reaction site per pixel. As shown in FIG. 27A, for a device with a single reaction site per pixel and a 2×2 four cloud calling chart, the signal-to-noise ratio can be expressed as shown in Equation 2.

[0134]

number

[0135] Referring to Equation 2, the signal-to-noise ratio of the four cloud calling chart device is determined by not only the signal amplitude A but also the variables σ , which represent the signal variance of the top left cloud at cloud position X1Y2 and the signal variance of the top right cloud at cloud position X2Y2, respectively. 1 and σ 4 The examples herein recognize that as the signal amplitude of the cloud increases, i.e., for the top right cloud at cloud location X2Y2, the signal variance can be expected to increase.

[0136] 27B is a calling chart for a 16 channel 4×4 calling chart system. The signal to noise ratio in the 16 cloud calling chart system can be expressed as shown in Equation 3.

[0137]

number

[0138] For an apparatus having a first and second reaction site per pixel and an associated 4×4 16 cloud calling chart as shown in the calling chart of FIG. 27B, the signal to noise ratio can be expected to decrease based on the variables shown in Equation 3. First, since three different signal level amplitudes are detected, the numerator of Equation 3 can be expressed as the value A / 3, as opposed to the value A shown in Equation 2, which corresponds to the single reaction site per pixel represented in FIG. 27A. Additionally, the denominator of Equation 3 is the signal dispersion variable σ at cloud location X1Y4. 1 By making it a function of the signal variance variable σ3 at cloud location X3Y4 ​​rather than (Eq. 2), the resulting signal-to-noise ratio can be further reduced.

[0139] Examples herein recognize that the signal-to-noise ratio constraints of multiple reaction sites per pixel device can be managed by appropriate system design, including, for example, design strategies that facilitate minimizing signal variance of the various clouds and also increase separation between the clouds. Examples herein recognize that higher amplitude clouds, e.g., the cloud at cloud location X4Y4, may have greater variance than clouds of relatively smaller signal amplitudes. Thus, in one aspect, examples herein may include design features that facilitate increased separation between higher amplitude clouds, which may be expected to exhibit more pronounced signal variance σn.

[0140] 28A-28C show cloud spacing in a 16 cloud calling chart for different values ​​of R, where R is the ratio between the second cluster emission signal in the "on" state and the first cluster emission signal in the "on" state. With reference to FIG. 28A-28C, it can be seen that by varying the value of R, the spacing between different clouds can be varied. When R=2, i.e., when there is a 2x cluster signal ratio, the various clouds at cloud locations X1Y1-X4Y4 can have equal centroid-to-centroid spacing as shown in FIG. 28A-28C. However, the examples herein recognize that for larger signal amplitude clouds with equal centroid spacing and larger signal variance σn, the edge-to-edge spacing between the larger amplitude clouds can be smaller than the edge-to-edge spacing between the smaller amplitude clouds. With reference to FIG. 28A, the edge-to-edge spacing between clouds at cloud location X3Y4 ​​at cloud location X4Y4 can be smaller than the edge-to-edge spacing between clouds at cloud locations X2Y4 and X1Y4. 28A-C, a 4×4 array of clouds can be represented in terms of their X-axis (horizontal) and Y-axis (vertical) positions from X1Y1 to X4Y4. The X-axis can represent the "on" state luminescence signal amplitude under a first narrowband excitation light, and the Y-axis can represent the "on" state luminescence signal amplitude under a second narrowband excitation light.

[0141] To reduce the overall cloud spacing between the higher amplitude clouds, examples herein recognize with reference to Figures 28A-C that the device 100 can be configured to feature a design exhibiting an "on" state cluster signal emission ratio of about 1.9 to about 2.0. With reference to Figure 28B where R = 1.89, the overall cloud spacing between the clouds at cloud locations X4Y4 and X3Y4 ​​can be increased relative to the overall cloud spacing at cloud locations X4Y4 and X3Y4 ​​shown in Figure 28A where R = 2x. Furthermore, as shown in Figure 28C, if the ratio of R is reduced to a value of, for example, R = 1.4, it can be seen that even with modest cloud dispersion, clouds at various cloud locations may overlap, but if the signal dispersion is small enough, the design represented by Figure 28 will produce adequate cloud spacing. Thus, based on empirical data as shown in Figures 28A-C, a cluster signal "on" state ratio of about R=1.9 promotes edge-to-edge cloud spacing between high signal amplitude clouds while avoiding substantial risk of cloud overlap, as shown in Figure 28C. Referring to Figure 28A, a design featuring an "on" state signal ratio of about R=2.0 can also produce calling charts featuring adequate cloud spacing.

[0142] In the examples described herein, the different reaction sites 206, 206A-206D defined on the reaction structure 260 may be provided by different nanowells 207, 207A-207D. In other examples, the reaction sites 206, 206A-206D may be defined by alternative features that may be defined by the reaction structure 260. The alternative features may include, for example, variations in structural features and / or variations in chemical composition. The structural features defining the variations in structural features may form an array within or on the reaction structure 260. Exemplary structural features may include, but are not limited to, nanowells, posts, ridges, channels, and / or layers of multi-layered materials as described herein. The features may have characteristics such as size (e.g., volume, diameter, and depth), shape (e.g., circular, elliptical, triangular, square, polygonal, star-shaped (having any suitable number of vertices), irregular, or with concentric features separated by dielectric material), and distribution (e.g., spatial location of the features within the dielectric material, e.g., regularly spaced or periodic locations, or irregularly spaced or non-periodic locations). The cross-section of the feature may be, but is not necessarily, uniform along the length of the feature.

[0143] The flow diagrams 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 implementations. In this regard, each block in the flow diagrams or block diagrams represents a module, segment, or portion of instructions, which includes one or more executable instructions for implementing a specified logical function(s). In some alternative implementations, the functions shown in the blocks may occur in an order different from the order shown in the figures. For example, two blocks shown in succession may in fact be executed substantially simultaneously, or the blocks may sometimes be executed in the reverse order, depending on the functionality involved. It should also be noted that each block of the block diagrams and / or flow diagram illustrations, and combinations of blocks in the block diagrams and / or flow diagram illustrations, may be implemented by a dedicated hardware-based system that performs the specified functions or operations, or may be executed by a combination of dedicated hardware and computer instructions.

[0144] The terms used herein are for the purpose of describing particular examples only and are not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly dictates otherwise. It will be further understood that the terms "comprises" and / or "comprising," as used herein, specify the presence of stated features, integers, steps, processes, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, processes, operations, elements, components, and / or groups thereof.

[0145] Where applicable, the corresponding structures, materials, acts, and equivalents of all means or steps in addition to the functional elements of the claims below are intended to include any structures, materials, or acts for performing a 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 the disclosed forms. Numerous modifications and changes will become apparent to those skilled in the art. Each example has been selected and described in order to best explain various aspects and practical applications, and to enable others skilled in the art to understand the various examples with various modifications as suitable for the particular use envisaged.

[0146] It should be understood that all combinations of the above concepts and further concepts discussed in more detail below (provided such concepts are not mutually inconsistent) are contemplated as part of the inventive subject matter disclosed herein to achieve at least the advantages described herein. In particular, all combinations of the subject matter of the claims appearing at the end of this disclosure are contemplated as part of the subject matter disclosed herein. It should also be understood that the terms expressly used in this specification and which may also appear in any disclosures incorporated by reference are to be given the meaning most consistent with the particular concepts disclosed herein.

[0147] This written description uses examples to disclose the subject matter and enables any person skilled in the art to practice the subject matter, including making and using any device or system 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 include structural elements that do not differ from the literal words of the claims, or if they include equivalent structural elements that have insignificant differences from the literal words of the claims.

[0148] It should be understood that the above description is intended to be illustrative and not limiting. For example, the above-described examples (and / or aspects thereof) can be used in combination with each other. In addition, many modifications can 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 in no way limiting and are provided merely as examples. Numerous other examples will become apparent to those skilled in the art upon review of 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. As used herein, the term "based on" encompasses relationships where an element is partially based, as well as relationships where an element is fully based. The term "defined" encompasses relationships where an element is partially defined, as well as relationships where an element is fully defined. Furthermore, the limitations of the following claims are not written in means-plus-function form, and are not intended to be construed under 35 U.S.C. § 112, paragraph 6 (35 U.S.C. § 112(f)), unless such claim limitations expressly use the phrase "means for" followed by a description of a function without further structure. It is to be understood that not all such objects or advantages described above may necessarily be achieved in accordance with any particular embodiment of the present invention.Thus, for example, those skilled in the art will recognize that the systems and techniques described herein can be embodied or performed in a manner that achieves or optimizes one advantage or group of advantages taught herein without necessarily achieving other objectives or advantages that may be taught or suggested herein.

[0149] Although the subject matter has been described in detail with respect to 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 should 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 fewer or more than that certain number of elements. Thus, the subject matter should not be deemed limited by the above description, but only by the scope of the appended claims.

Claims

1. An apparatus comprising: A plurality of pixels; a first reaction site associated with a pixel of the plurality of pixels; a second reaction site associated with the pixel; the first reaction site is defined by a first nanowell and the second reaction site is defined by a second nanowell; the pixel includes a pixel sensor that detects a read signal, the read signal being dependent on a first cluster signal emitted from the first location and a second cluster signal emitted from a second location; An apparatus, wherein the first portion and the second portion are configured such that the second cluster signal in an "on" state has a greater amplitude than the first cluster signal in an "on" state.

2. 2. The device of claim 1, further comprising a processing circuit configured to determine the identity of each analyte in the first reaction site and the second reaction site using 16 cloud calling charts that map signal amplitudes of the read signal under different lighting conditions to the identity of each analyte in the first reaction site and the second reaction site.

3. 2. The apparatus of claim 1, wherein a ratio of the second cluster signal in the "on" state to the first cluster signal in the "on" state is from about 1.9 to about 2.

0.

4. 2. The device of claim 1, wherein the attenuation of emitted light emanating from the first reaction site is greater than the attenuation of emitted light emanating from the second reaction site such that the second cluster signal in the "on" state has a greater amplitude than the first cluster signal in the "on" state due to the arrangement of attenuating material.

5. 2. The device of claim 1, wherein the second reaction site does not have an attenuator located below its bottom surface that is aligned with the bottom surface of the second reaction site, and the first reaction site includes an attenuator below its bottom surface that is aligned with the bottom surface of the first reaction site such that the second cluster signal in an "on" state has a larger amplitude than the first cluster signal in an "on" state.

6. 2. The apparatus of claim 1, wherein the apparatus includes an attenuating material layer aligned with and extending through an area below a bottom surface of the first reaction site to define an attenuator of the first reaction site, the attenuating material layer having no section extending through an area below the bottom surface aligned with the second reaction site.

7. 2. The apparatus of claim 1, wherein pixels 2 through N of the plurality of pixels have associated first and second reaction sites configured, respectively, according to the first and second reaction sites associated with the pixels, and the first and second reaction sites associated with adjacent pixels of the plurality of pixels have different respective first and second orientations with respect to their respective pixels, the different respective first and second orientations increasing a spacing distance between reaction sites from adjacent pixel locations relative to a spacing distance between reaction sites from adjacent pixel locations in the absence of the different respective orientations.

8. 2. The device of claim 1, wherein the second nanowell does not have an attenuator located below its bottom surface that is aligned with the bottom surface of the second nanowell, and the first nanowell includes an attenuator below its bottom surface that is aligned with the bottom surface of the first nanowell such that the second cluster signal in an "on" state has a greater amplitude than the first cluster signal in an "on" state.

9. 2. The device of claim 1, wherein the second nanowell has no attenuator intersected by a vertically extending central axis of the first nanowell, and the first nanowell includes an attenuator intersected by a vertically extending central axis of the first nanowell such that the second cluster signal in an "on" state has a greater amplitude than the first cluster signal in an "on" state.

10. 2. The device of claim 1, further comprising a processing circuit configured to determine the identity of each analyte in the first reaction site and the second reaction site using a calling chart that maps signal amplitudes of the read signal under different lighting conditions to the identity of each analyte in the first reaction site and the second reaction site.

11. The apparatus of claim 1 , wherein a ratio of the second cluster signal in an "on" state to the first cluster signal in an "on" state is at least about 1.

4.

12. 2. The device of claim 1, wherein the second reaction site has a width greater than the first reaction site such that the second cluster signal in an "on" state has a greater amplitude than the first cluster signal in an "on" state.

13. An apparatus comprising: A plurality of pixels; a first reaction site associated with a pixel of the plurality of pixels; a second reaction site associated with the pixel; the pixel includes a pixel sensor that detects a read signal, the read signal being dependent on a first cluster signal emitted from the first location and a second cluster signal emitted from a second location; the first portion and the second portion are configured such that the second cluster signal in an "on" state has a greater amplitude than the first cluster signal in an "on" state; The device, wherein the second reaction site does not have an attenuator positioned below its bottom surface that is aligned with the bottom surface of the second reaction site, and the first reaction site includes an attenuator positioned below its bottom surface that is aligned with the bottom surface of the first reaction site such that the second cluster signal in an "on" state has a greater amplitude than the first cluster signal in an "on" state.

14. The device of claim 13, further comprising a processing circuit configured to determine the identity of each analyte in the first reaction site and the second reaction site using a calling chart that maps signal amplitudes of the read signal under different lighting conditions to the identity of each analyte in the first reaction site and the second reaction site.

15. 14. The apparatus of claim 13, wherein a ratio of the second cluster signal in an "on" state to the first cluster signal in an "on" state is at least about 1.

4.

16. An apparatus comprising: A plurality of pixels; a first reaction site associated with a pixel of the plurality of pixels; a second reaction site associated with the pixel; the pixel includes a pixel sensor that detects a read signal, the read signal being dependent on a first cluster signal emitted from the first location and a second cluster signal emitted from a second location; the first portion and the second portion are configured such that the second cluster signal in an "on" state has a greater amplitude than the first cluster signal in an "on" state; An apparatus, wherein the attenuation of emitted light emanating from the first reaction site is greater than the attenuation of emitted light emanating from the second reaction site, such that the second cluster signal in the "on" state has a greater amplitude than the first cluster signal in the "on" state due to the arrangement of attenuating material.

17. 17. The device of claim 16, wherein the second reaction site does not have an attenuator located below its bottom surface that is aligned with the bottom surface of the second reaction site, and the first reaction site includes an attenuator below its bottom surface that is aligned with the bottom surface of the first reaction site such that the second cluster signal in an "on" state has a larger amplitude than the first cluster signal in an "on" state.

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