Biosensor for biological or chemical analysis and method of manufacturing the same

Abstract

To provide a biosensor improved for a biological and chemical analysis.SOLUTION: According to an embodiment, there is provided a biosensor improved for a biological and chemical analysis. According to the embodiment, a back surface irradiation type (BSI) complementary metal oxide film semiconductor (CMOS) image sensor can be used to analyze and measure fluorescence or chemical emission of a sample effectively. Using the measured value is useful to determine the sample. The embodiment of the present invention also provides a method for manufacturing a biosensor improved for a biological and chemical analysis, and a system and a method for determining a DNA sequence.SELECTED DRAWING: Figure 13

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 416,813, filed November 3, 2016, the contents of which are incorporated by reference in their entirety.

[0002] Field The present invention relates generally to biosensors for biological or chemical analysis, and more particularly to biosensors including backside illuminated (BSI) complementary metal oxide semiconductor (CMOS) image sensors, and methods for fabricating the same. [Background technology]

[0003] Background of the Invention CMOS image sensors are used in electronic imaging devices including digital cameras, medical imaging equipment, radar devices, etc. Using an integrated circuit and an array of photodiodes, CMOS image sensors are able to capture light and convert it into an electrical signal.

[0004] CMOS image sensors are typically implemented on a chip. The chip may have an amplifier for each pixel. Including many amplifiers within a chip can reduce the area available for light capture, but other components can be integrated on the chip to direct more light to the photodiode. For example, a microlens can be placed in front of the photodiode to direct light onto the photodiode. To further increase the amount of light reaching the photodiode, backside illumination (BSI) can be used. BSI effectively places the photodiode closer to the light source, rather than under and between the integrated circuit wiring, reducing destructive interference. BSI CMOS sensors also have other advantages. For example, BSI CMOS sensors may have lower operating voltages, lower power consumption, higher efficiency, and lower noise.

[0005] A BSI CMOS image sensor typically has two functional areas: a light-sensing area and an electronics area. The light-sensing area contains an array of photodiodes coupled to metal-oxide-semiconductor (MOS) transistors that detect light intensity. The electronics area provides connections between the MOS transistors and external connections, for example to other devices for processing data from the MOS transistors.

[0006] In practice, BSI CMOS image sensors use filters that split the incoming light into bands of light with different wavelengths. The light is received by photodiodes on the substrate and converted into electrical signals of different intensities. For example, an incoming beam can be split into red, green, and blue light and received by a respective photodiode for each color. Each photodiode converts the detected light intensity into an electrical signal. This is achieved by the photodiode accumulating an electrical charge. For example, the higher the light intensity, the higher the charge accumulated in the photodiode. The accumulated charge is then correlated to color and brightness.

[0007] In addition to the uses described above, CMOS image sensors can also be used for biological or chemical analysis. For such analysis, a biological or chemical sample can be placed on a photodiode, and light emitted from the biological or chemical sample is directed toward the photodiode. The fluorescence or chemiluminescence of the sample can be detected by the photodiode, and color and brightness can be determined. The color and brightness can be used to identify the biological or chemical sample. Summary of the Invention

[0008] Summary of the Invention Embodiments of the present invention address shortcomings associated with conventional approaches by providing improved biosensors for biological or chemical analysis. According to embodiments of the present invention, a BSI CMOS image sensor can be used to effectively analyze and measure the fluorescence or chemiluminescence of a sample. This measurement can be used to aid in sample identification. Embodiments of the present invention also provide methods for fabricating improved biosensors for biological or chemical analysis. As used herein, the term "biosensor" can be used to refer to devices for measuring luminescent materials within or attached to biological molecules, particularly nucleic acid polymers exemplified by DNA and branched or otherwise derivatized nucleic acids. As used herein, the term "nucleic acid polymer" can refer to, for example, DNA or single-stranded embodiments.

[0009] According to some embodiments of the present invention, a biosensor is provided. The biosensor includes a back-illuminated complementary metal-oxide semiconductor (CMOS) image sensor. The back-illuminated CMOS image sensor includes an electronic circuit layer and a light-sensing layer on the electronic circuit layer. The light-sensing layer includes a substrate layer and a photodiode in contact with the electronic circuit layer. A light-receiving surface is defined by a surface of the photodiode opposite the electronic circuit layer. The biosensor further includes a color filter material on the photodiode. The biosensor further includes spots or wells on the color filter material, the spots or wells being sized and functionalized to receive nucleic acid polymers and either absorb light from the nucleic acid polymers or transmit light from the nucleic acid polymers to the light-receiving surface.

[0010] A manufacturing method according to some embodiments includes providing a back-illuminated complementary metal-oxide semiconductor (CMOS) image sensor. Providing the back-illuminated CMOS image sensor includes providing an electronic circuit layer and providing a light-sensing layer on the electronic circuit layer. The light-sensing layer includes a substrate layer and a photodiode in contact with the electronic circuit layer. A light-receiving surface is defined by a surface of the photodiode opposite the electronic circuit layer. The method further includes depositing a color filter material on the photodiode. The method further includes providing spots or wells on the color filter material, the spots or wells being sized and functionalized to receive nucleic acid polymers and either absorb light from the nucleic acid polymers or transmit light from the nucleic acid polymers to the light-receiving surface.

[0011] A method of DNA sequencing according to some embodiments includes iteratively performing a process that may include labeling a nucleic acid polymer with a fluorescent label that identifies a nucleotide base at a specific position within the nucleic acid polymer. The process further includes detecting the fluorescent label associated with the nucleic acid polymer. Detecting the fluorescent label includes illuminating the nucleic acid polymer with excitation light. The nucleic acid polymer absorbs the excitation light and transmits emitted light through a color filter onto a photodiode of a back-illuminated complementary metal-oxide semiconductor (CMOS) image sensor. Detecting the fluorescent label further includes measuring at least one parameter of the emitted light received by the photodiode. Detecting the fluorescent or chemiluminescent label further includes correlating at least one parameter of the emitted light to the fluorescent label. The process further includes removing the fluorescent label from the nucleic acid polymer. Without limitation, biosensors of embodiments of the present invention can be used to perform sequencing-by-synthesis (SBS), sequencing-by-ligation, cPAL sequencing, pyrosequencing, and combinations thereof.

[0012] The foregoing, together with other features and embodiments, will become more apparent with reference to the following specification, claims, and accompanying drawings.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS Exemplary embodiments of the present invention are described in detail below with reference to the following drawings: [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a cross-sectional view of a back-illuminated CMOS image sensor according to some embodiments. [Figure 2] FIG. 2 is a cross-sectional view of a back-illuminated CMOS image sensor having a first passivation layer according to some embodiments. [Figure 3] FIG. 3 is a cross-sectional view of a back-illuminated CMOS image sensor having a first metal layer according to some embodiments. [Figure 4] FIG. 4 is a cross-sectional view of a back-illuminated CMOS image sensor having an etched first metal layer according to some embodiments. [Figure 5] FIG. 5 is a cross-sectional view of a back-illuminated CMOS image sensor with a dielectric layer according to some embodiments. [Figure 6] FIG. 6 is a cross-sectional view of a back-illuminated CMOS image sensor with a color filter layer according to some embodiments. [Figure 7] FIG. 7 is a cross-sectional view of a back-illuminated CMOS image sensor with a planarized color filter layer according to some embodiments. [Figure 8A] FIG. 8A is a cross-sectional view of a back-illuminated CMOS image sensor having a second passivation layer, a first material layer, and a second metal layer according to some embodiments. [Figure 8B] FIG. 8B is a cross-sectional view of a back-illuminated CMOS image sensor having a second passivation layer and a second metal layer according to some embodiments. [Figure 9]FIG. 9 is a cross-sectional view of a biosensor using a back-illuminated CMOS image sensor, according to some embodiments. [Figure 10] FIG. 10 is a cross-sectional view of a biosensor using a back-illuminated CMOS image sensor and a microlens, according to some embodiments. [Figure 11] FIG. 11 is a cross-sectional view of a biosensor using a back-illuminated CMOS image sensor, a microlens, and a third metal layer according to some embodiments. [Figure 12] FIG. 12 is a cross-sectional view of a biosensor using a back-illuminated CMOS image sensor, a microlens, a third metal layer, and a planarization layer according to some embodiments. [Figure 13] FIG. 13 is a cross-sectional view of a biosensor using a back-illuminated CMOS image sensor, according to some embodiments. [Figure 14A] FIG. 14A is a top view of a two-channel color filter that can be used in a biosensor, according to some embodiments. [Figure 14B] FIG. 14B is a top view of a four-channel color filter that can be used in a biosensor, according to some embodiments. [Figure 15] 15A-15C are photographic images showing signals from DNBs at multiple spots on an array in a BSI CMOS chip at various stages of multi-stage sequencing, according to some embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description of the Invention 1-13 illustrate various stages in the fabrication of a biosensor according to an embodiment of the present invention. Other embodiments of fabrication and construction will be apparent to those skilled in the art from this description. Therefore, the following description is intended to be illustrative and not limiting.

[0016] For ease of reading, the following text is organized into sections, however, it will be understood that discussion of subject matter in one section (e.g., descriptions of polymers, filters, sequencing methods, etc.) may also apply to subject matter in other sections.

[0017] Biosensors according to embodiments of the present invention are not limited to any particular application. In one aspect, biosensors according to embodiments of the present invention find particular application for massively parallel DNA sequencing. Because DNA sequencing techniques are well known (see, e.g., Drmanac et al., 2010, "Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays," Science 327:78-81; Shendure & Ji, (2008, "Next-generation DNA sequencing," Nature Biotechnology 26:1135-45), only general terms are described in the following sections. The following paragraphs provide a brief initial discussion of sequencing and related terminology so that the specific features of the biosensors described below can be more readily understood.

[0018] Various DNA sequencing methods are known. In many techniques, macromolecules (e.g., genomic DNA) are broken down into many smaller fragments, each with a unique DNA sequence. In array-based techniques, fragments are distributed across an array of locations on a substrate, such that each location in the array contains a DNA fragment with a single unique sequence. Sequence information ("reads") is simultaneously obtained from the DNA at each of thousands, or more often millions, of locations and assembled by computer. In most sequencing techniques, fragments are amplified before sequencing. Amplification can occur before placing fragments at each location, after placing fragments at each location, or both before and after placement. The amplification step(s) generates "amplicons" that serve as "templates" in the sequencing process. Thus, by way of example, amplification can be performed using RCA to generate single-stranded concatemers (e.g., DNA nanoballs) at each location on the array, or bridge PCR to generate clonal populations (or clusters) of DNA molecules with the same sequence at each location.

[0019] References to "DNA polymers" and the like will be understood to include DNA nanoballs, branched structures, and clustered clonal populations (i.e., more than a single molecule) or precursors thereof. Furthermore, "DNA polymers" and the like can include auxiliary DNA molecules, such as primers, and growing strands generated by primer extension or other processes. In many sequencing techniques, it is the auxiliary DNA molecules that contain (or are "labeled" with) a detectable (e.g., fluorescent or chemiluminescent) dye that emits light that is detected by a photodiode in a biosensor. Thus, phrases such as "illuminating the nucleic acid polymer with an excitation light source and detecting light emitted from the polymer" will be understood to include "exposing DNA nanoballs or clonal clusters and associated labeled auxiliary molecules with an excitation light source and detecting light emitted from the dye in the labeled auxiliary molecules."

[0020] In array-based sequencing methods and biosensors of the present invention, DNA polymers are disposed on a substrate in wells, or "spots." The wells or spots are capable of receiving and retaining the polymers. Often, the spots (sometimes called "discrete spaced apart regions" or "pads") comprise a substrate functionalized to accept nucleic acid polymers, and the spots are separated by regions that are "inactive," in the sense that DNA polymers do not bind to such regions. See, for example, but not by way of limitation, Drmanac 2010, supra. A "well" is a type of spot that includes a wall that forms a boundary or barrier to the DNA polymers. Unless otherwise clear from the context, references below to "spots" can include wells.

[0021] In biosensors of the present invention, the spots generally have uniform dimensions and are organized in a regular (i.e., non-random) array. The spots in the array are generally organized in a linear pattern, often columns and rows, although other regular patterns may be used (e.g., a spiral). The spots in the array may have a characteristic dimension, pitch, and density. The spots themselves may be circular, square, hexagonal, or other shapes. In the following discussion, the spots are generally assumed to be circular (i.e., may be described as having a diameter). It will be understood that references to "diameter" can also refer to the linear dimension (e.g., diagonal, length, or width) of spots of other shapes. Thus, as used herein, "linear dimension (length dimension)" can refer to the diameter of a circle, the width, diagonal, etc. of a square. In the context of biosensors of the present invention, spot size is meaningful in two ways. First, the spots may be sized and / or functionalized to restrict occupancy to a single target sequence. This can be a single DNA nanoball (concatamer of a single target sequence) or a clonal cluster with a single target sequence. See, e.g., U.S. Pat. No. 8,133,719 and U.S. Patent Application Publication No. 2013 / 0116153, both of which are incorporated by reference in their entirety for all purposes. Second, in general, spots can be sized and positioned relative to the underlying photodiodes such that each photodiode receives emitted light from a single spot. In some embodiments, an array of spots can be positioned on an array of corresponding photodiodes (and / or color filters) in a one-to-one correlation.That is, for example, light emitted from a DNA polymer at an individual spot passes through an underlying filter, and light not blocked by the filter is detected by a single photodiode associated with the filter; alternatively, for example, light emitted from a DNA polymer at an individual spot passes through multiple underlying filters, each associated with a filter (specific to a particular wavelength) and each associated with a single photodiode, and light not blocked by the filter is detected by the associated photodiode. Thus, as discussed below, in some embodiments, light emitted from a single spot can be detected by two or more photodiodes (e.g., two, three, four, etc.). In these embodiments, a group of multiple photodiodes associated with a single spot can be referred to as a photodiode "unit cell." The spots and filters (e.g., a single filter or unit cell) can be arranged in a biosensor such that each photodiode in a unit cell receives light emitted from the same single spot. Furthermore, in some embodiments, the area of ​​the light-receiving surface of a photodiode, or the combined area of ​​the light-receiving surfaces of multiple photodiodes associated with the same spot, is smaller than the area of ​​the spot (from which light is emitted). In other words, the spot may be smaller than the underlying photodiode(s) so that the boundary of the spot, when projected onto the light-receiving surface of the photodiode(s), is contained within the light-receiving surface.

[0022] As is well known, nucleic acid sequencing generally involves an iterative process, in which a fluorescent or chemiluminescent label is bound in a specific manner to the DNA template (amplicon) being sequenced, the binding is detected, and the label is removed, meaning that it no longer emits a signal.See, for example, US Patent Application Publication No. 2016 / 0237488; US Patent Application Publication No. 2012 / 0224050; US Patent Nos. 8,133,719; 7,910,354; 9,222,132; 6,210,891; 6,828,100; 6,833,246; and 6,911,345 (the entirety of which is incorporated herein by reference).Therefore, for example, it will be understood that "labeling a nucleic acid polymer with a fluorescent label" can refer to associating a DNA template immobilized on a spot with labeled auxiliary molecules (multiple).

[0023] Referring now to the drawings, FIG. 1 is a cross-sectional view of a backside illuminated (BSI) CMOS image sensor 100 according to some embodiments. The BSI CMOS image sensor 100 may include a first dielectric layer 110. Although described as a dielectric, it is contemplated that the first dielectric layer 110 may include any suitable electrically insulating material. The first dielectric layer 100 may include metal wiring 113. The metal wiring 113 may include integrated circuit material and external connections. Together, the first dielectric layer 100 and the metal wiring 113 may be collectively referred to herein as the "electronic circuitry layer" of the BSI CMOS image sensor.

[0024] A substrate layer 115 may be provided over the first dielectric layer 110 and the metal wiring 113. The substrate layer 115 may be made of any suitable material, such as silicon, III-V on silicon, graphene-on-silicon, silicon-on-insulator, or combinations thereof. The substrate layer 115 may include an opening in which a light-sensing component (e.g., a photodiode 117) may be disposed. While described herein with respect to a photodiode 117, it is contemplated that any suitable light-sensing component may be used. The photodiode 117 may be configured to convert the measured light into a current. The photodiode 117 may include a source and a drain of a MOS transistor (not shown), which may transfer the current to other components, such as another MOS transistor. The other components may include a reset transistor, a current source follower, or a row selector for converting the current into a digital signal. Together, substrate layer 115 and photodiode 117 are sometimes collectively referred to herein as the "photosensitive layer" of the BSI CMOS image sensor.

[0025] Photodiode 117 is in contact with metal wiring 113 and can transmit digital signals to external connections via metal wiring 113. In BSI CMOS image sensor 100 shown in Figure 1, the light-receiving surface is located at the top of photodiode 117 (i.e., the surface that is not in contact with the electronic circuit layer and is opposite the electronic circuit layer), and incident light is received by photodiode 117 at this light-receiving surface.

[0026] Referring to FIG. 2 , to construct biosensor 200, first passivation layer 120 may be deposited by conventional semiconductor processing techniques (e.g., low-temperature plasma enhanced chemical vapor deposition) on substrate layer 115 and photodiode 117 of BSI CMOS image sensor 100. First passivation layer 120 may comprise any suitable protective material. For example, first passivation layer 120 may comprise a material such as silicon, oxide, metal, combinations thereof, or the like. First passivation layer 120 may act as an etch stop for a subsequent etching step, as described further herein. Alternatively or additionally, first passivation layer 120 may act to protect the active device (i.e., a back-illuminated CMOS sensor). Alternatively or additionally, first passivation layer 120 may act to protect photodiode 117 from wear caused by frequent use. First passivation layer 120 may be transparent. In one example, the first passivation layer 120 may have a thickness of 100 nanometers or less.

[0027] A. Biosensor 200 of FIG. FIG. 2 illustrates a biosensor 200 that can be used for biological or chemical analysis (e.g., to detect the chemiluminescence of a polymer or polymer complex) according to some embodiments. The biosensor 200 includes a back-illuminated CMOS image sensor 100. The back-illuminated CMOS image sensor 100 includes an electronic circuit layer (comprising a first dielectric layer 110 and metal wiring 113) and a light-sensing layer (comprising a substrate layer 115 and a photodiode 117) on the electronic circuit layer. The photodiode 117 may be in contact with the electronic circuit layer so that an electrical signal can be transmitted from the photodiode 117 to the electronic circuit layer and, in some embodiments, to an external device. The light-receiving surface is defined by the surface of the photodiode 117 opposite the electronic circuit layer (i.e., the surface in contact with the first passivation layer 120).

[0028] Biosensor 200 may further include a first passivation layer 120 on back-illuminated CMOS image sensor 100, and spots or wells (not shown) formed on or within first passivation layer 120, onto or over which chemical or biological samples may be placed for analysis. In some embodiments, biosensor 200 may be adapted to detect optical signals (e.g., fluorescence or chemiluminescence) from a corresponding array of biomolecules, where individual biomolecules may be disposed on one or more photodiodes (e.g., in spots or wells), which then receive light from the biomolecules, as discussed in more detail below.

[0029] Various further embodiments for constructing a biosensor using the back-illuminated CMOS sensor 100 can now be described. According to FIG. 3 , the first metal layer 123A can be deposited on the first passivation layer 120 of the biosensor 200 by conventional semiconductor processing techniques (e.g., by metal deposition techniques). The first metal layer 123A can include any suitable metal material. For example, the first metal layer 123A can include materials such as tungsten, aluminum, gold, copper, combinations or alloys thereof, and the like. In some embodiments, the first metal layer 123A can be a thick layer, e.g., a layer thicker than the first passivation layer 120. For example, the first metal layer 123A can be up to 3 micrometers thick.

[0030] Referring to FIG. 4 , the first metal layer 123A may be etched to provide a first opening over the photodiode 117, leaving behind the first metal layer 123B. The first metal layer 123A may be etched by any suitable process, such as, for example, wet etching, dry etching, a combination thereof, or the like. It is contemplated that etching the first metal layer 123A may include, for example, the use of a mask. The etching may be completed using any of a variety of materials, such as, for example, an acid (e.g., hydrochloric acid, hydrofluoric acid, nitric acid, etc.), an alkali with an oxidizing agent, a combination thereof, or the like. It is contemplated that the type of acid required to etch the first metal layer 123A may depend on the material used to form the first metal layer 123A. In some embodiments, the first opening may be aligned with and centered on the photodiode 117 to maximize the efficiency of the photodiode 117 in subsequent use. A mask (not shown) can define openings over the photodiodes 117, leaving the first metal layer 123B, and the first passivation layer 120 can act as an etch stop when etching the openings in the first metal layer 123A. As described herein, the pillars of the first metal layer 123B can separate light received by the different color filters and can reflect light directed to a particular color filter back to that color filter or to the corresponding photodiode 117.

[0031] 5, a second dielectric layer 125 may be deposited over the first metal layer 123B and within the first opening by conventional semiconductor processing techniques. In some embodiments, the second dielectric layer 125 may be formed over all exposed surfaces of the first metal layer 123B. Although described as being a dielectric, it is contemplated that the second dielectric layer 125 may comprise any suitable electrically insulating material, such as silicon nitride, tantalum oxide, combinations thereof, and the like. The second dielectric layer 125 may be formed from the same or a different material as the first dielectric layer 110.

[0032] 6, color filter material 127A may be deposited on second dielectric layer 125. In some embodiments, color filter material 127A may be deposited by spin coating. Color filter material 127A fills the openings formed by second dielectric layer 125. In this embodiment, color filter material 127A is also deposited on portions of second dielectric layer 125 between the openings. Thus, according to FIG. 7, excess color filter material 127A over the openings in second dielectric layer 125 can be removed, for example, by chemical mechanical planarization (CMP), leaving color filter material 127B within the openings in second dielectric layer 125.

[0033] 6 may be omitted. In other words, as in FIG. 7, color filter material 127B may be selectively deposited only in the openings in second dielectric layer 125, such that two or more (e.g., two, three, or four) different color filter materials 127B may be disposed over photodiode 117. In some applications, each different color filter material 127B may be associated with a separate photodiode 117.

[0034] Color filter material 127B may include, for example, pigment-based polymers, pigment-based dyes, dye-based polymers, resins or other organic-based materials, combinations thereof, etc. Color filter material 127B may be necessary for biosensors, for example, because photodiode 117 detects light intensity alone with little or no wavelength specificity and therefore cannot separate color information.

[0035] The color filter material 127B may include blue filter material, red filter material, green filter material, emerald filter material, cyan filter material, yellow filter material, magenta filter material, white filter material, combinations thereof, etc. Thus, the color filter material 127B can filter incident light by wavelength range, such that the separate filtered intensities contain information about the color of the light. For example, the red color filter material 127B can provide information about the intensity of light in the red wavelength region; the blue color filter material 127B can provide information about the intensity of light in the blue wavelength region; the green color filter material 127B can provide information about the intensity of light in the green wavelength region, etc.

[0036] In some embodiments, the color filter material 127B may include a single color material. For example, each of the color filter materials 127B may be red. In some embodiments, the color filter material 127B may include different color materials, with each color filter material 127B corresponding to a different photodiode 117. For example, one color filter material 127B may be red and an adjacent color filter material 127B may be green. FIG. 14A illustrates such an embodiment in which a two-channel color filter is used. In FIG. 14A, a biological or chemical sample (e.g., a DNA polymer) may be placed in a spot or well 1450, such that light emitted from the polymer enters both the red color filter material 1427B and the green color filter material 1427A (e.g., overlaps both the red color filter material 1427B and the green color filter material 1427A), and emission wavelengths of light passing through different colors of color filter materials can be detected. In another example, more than two surrounding color filter materials 127B may include different color materials. 14B shows such an embodiment in which a four-channel color filter is used. The four-channel color filter may include one color filter material 1427B that is red, one color filter material 1427D that is yellow, one color filter material 1427A that is green, and one color filter material 1427C that is blue. In this example, a biological or chemical sample may be placed in a spot or well 1450 at the intersection of the four color filters, so that emission wavelengths of light passing through the four colors of the color filter material can be detected. In some embodiments, the spot or well 1450 may be evenly overlaid on each of the underlying color filter materials (and corresponding photodiodes), i.e., an equal area of ​​each filter underlies the spot.

[0037] FIG. 8A illustrates an embodiment in which a biosensor 800 may be constructed. According to FIG. 8A, a second passivation layer 130 may be deposited on the second dielectric layer 125 and the color filter material 127B according to conventional semiconductor techniques. The second passivation layer 130 may be as described below with respect to FIG. 8B. A first material layer 135 may be deposited on the second passivation layer 130. The first material layer 135 may comprise any suitable material, such as silicon nitride, tantalum oxide, combinations thereof, etc. A second material layer 137 may be deposited on the first material layer 135. The second material layer 137 may comprise any suitable material, such as silicon dioxide, etc. In some embodiments, the first material layer 135 may have a higher refractive index than the second material layer 137. In some embodiments, the first material layer 135 may have a higher refractive index than the second material layer 130. Thus, the embodiment of FIG. 8A can provide efficient delivery of excitation light to the light-receiving surface for fluorescence measurements. For example, first material layer 135 can form the core of an optical waveguide, thus enabling low-loss transmission of excitation light. In some embodiments, biological or chemical samples can be placed on second material layer 137 above photodiode 117 (in some embodiments, within openings or wells formed on second material layer 137), and their fluorescence or chemiluminescence can be measured by photodiode 117, as described further herein. However, when measuring fluorescence in the embodiment shown in FIG. 8A, in some examples, the excitation light may be directed laterally along the surface of biosensor 800.

[0038] B. Biosensor 800 of Figure 8A 8A thus illustrates a biosensor 800 that can be used for biological or chemical analysis, according to some embodiments. The biosensor 800 may include a back-illuminated CMOS image sensor 100. The back-illuminated CMOS image sensor 100 includes an electronic circuit layer (comprising a first dielectric layer 110 and metal wiring 113) and a light-sensing layer (comprising a substrate layer 115 and a photodiode 117) on the electronic circuit layer. The photodiode 117 may be in contact with the electronic circuit layer such that an electrical signal can be transmitted from the photodiode 117 to the electronic circuit layer and, in some embodiments, to an external device. A light-receiving surface is defined by the surface of the photodiode 117 opposite the electronic circuit layer (i.e., the surface in contact with the first passivation layer 120).

[0039] Biosensor 800 may further include a first passivation layer 120 on back-illuminated CMOS image sensor 100 and a first metal layer 123B on first passivation layer 120. First metal layer 123B may also be disposed on substrate layer 115. First metal layer 123B may include a first opening. Biosensor 800 may further include a second dielectric layer 125 on metal layer 123B and first passivation layer 120. Second dielectric layer 125 may also be disposed within the first opening in metal layer 123B.

[0040] Biosensor 800 may further include color filter material 127B over second dielectric layer 125 and in and over the first opening in metal layer 123B, such that the top surface of color filter material 127B may be planar with the top surface of second dielectric layer 125 over metal layer 123B. Biosensor 800 may further include second passivation layer 130 over second dielectric layer 125 and color filter material 127B. Biosensor 800 may further include first material layer 135 and second material layer 137. First material layer 135 may have a higher refractive index than second material layer 137. Biological or chemical samples may be placed in spots or wells (not shown) formed in or on second material layer 137 for analysis, as further described herein.

[0041] FIG. 8B illustrates an alternative embodiment to FIG. 8A. According to FIG. 8B, a second passivation layer 130 can be deposited over the second dielectric layer 125 and the color filter material 127B according to conventional semiconductor techniques. The second passivation layer 130 can include any suitable material, such as, for example, silicon nitride, tantalum oxide, combinations thereof, or the like. In some embodiments, the second passivation layer 130 can include one or more high-k materials. The second passivation layer 130 can include the same or different material as the first passivation layer 120. In some embodiments, the second passivation layer 130 is made of a denser material than the first passivation layer 120. In some embodiments, the second passivation layer 130 can act as a protective material between the sample being analyzed and the color filter material 127B. In some embodiments, the second passivation layer 130 acts as an etch stop for a subsequent etching step. The second passivation layer 130 can be transparent.

[0042] 8B, a second metal layer 133A may be deposited over the second passivation layer 130 according to conventional semiconductor techniques. The second metal layer 133A may comprise any suitable metal material such as, for example, tungsten, aluminum, copper, combinations thereof, etc. The second metal layer 133A may be made of the same or a different material than the first metal layer 123B. The second metal layer 133A may be opaque to incident light or excitation light.

[0043] Next, referring to FIG. 9 , second metal layer 133B may be etched or patterned from second metal layer 133A to form second openings 150A-C within second metal layer 133A. In some embodiments, second openings 150A-C may be aligned with and centered on photodiode 117. In some embodiments, second openings 150A-C may have diameters ranging from 100 nanometers to 1 micrometer. Second openings 150A-C may have a width or diameter smaller than color filter material 127B. In some embodiments, biological or chemical samples may be placed within second openings 150A-C, and light emitted from the samples may be used to measure their fluorescence or chemiluminescence, as described further herein. In embodiments in which second openings 150A-C have a width or diameter smaller than color filter material 127B, there may be increased shading of incident or excitation light, resulting in less noise in detecting the sample's fluorescence or luminescence. The width or diameter of the second openings 150A-C may correspond approximately to the size of the biological or chemical sample being analyzed.

[0044] C. Biosensor 900 of FIG. 9 illustrates a biosensor 900 that can be used for biological or chemical analysis, according to some embodiments. The biosensor 900 includes a back-illuminated CMOS image sensor 100. The back-illuminated CMOS image sensor 100 includes an electronic circuit layer (comprising a first dielectric layer 110 and metal wiring 113) and a light-sensing layer (comprising a substrate layer 115 and a photodiode 117) on the electronic circuit layer. The photodiode 117 may be in contact with the electronic circuit layer so that an electrical signal can be transmitted from the photodiode 117 to the electronic circuit layer and, in some embodiments, to an external device. The light-receiving surface is defined by the surface of the photodiode 117 opposite the electronic circuit layer (i.e., the surface in contact with the first passivation layer 120).

[0045] Biosensor 900 may further include a first passivation layer 120 on back-illuminated CMOS image sensor 100 and a first metal layer 123B on first passivation layer 120. First metal layer 123B may also be disposed on substrate layer 115. First metal layer 123B may include a first opening. Biosensor 900 may further include a second dielectric layer 125 on metal layer 123B and first passivation layer 120. Second dielectric layer 125 may also be disposed within the first opening in metal layer 123B.

[0046] Biosensor 900 may further include a color filter material 127B over second dielectric layer 125 and in and over the first openings in metal layer 123B, such that the top surface of color filter material 127B may be planar with the top surface of second dielectric layer 125 over metal layer 123B. Biosensor 900 may further include a second passivation layer 130 over second dielectric layer 125 and color filter material 127B. Biosensor 900 may further include a second metal layer 133B having second openings 150A-C. Second openings 150A-C may function as spots or wells configured to receive biological or chemical samples, as described further herein.

[0047] Referring again to the embodiment of FIG. 9, various additional fabrication techniques can be implemented for further signal enhancement, as described herein with respect to FIGS. 10-13. According to FIG. 10, a microlens 140A can be grown on the second passivation layer 130 and the second metal layer 133B. In some embodiments, the microlens 140A can be aligned and centered with the photodiode 117. The microlens 140A can comprise various materials, such as glass, polymer, plastic, combinations thereof, etc. A microlens 140A can be included in the device above each of the color filters 127B to focus light emitted onto each of the color filters 127B.

[0048] The microlenses 140A can be grown according to any suitable microlens fabrication process, such as those commonly used for CMOS image sensors. As one example, photolithography can be performed on a photoresist or UV-curable epoxy material, which can then be melted to form an array of microlenses 140A. As another example, a small filament of glass can be melted, and the surface tension of the molten glass can form a smooth spherical surface. The spherical surface glass can then be appropriately attached and polished to form the microlenses 140A. As yet another example, wafer-level optics (WLO) can be used, in which multiple lens wafers are precisely aligned, bonded together, and diced to form a multi-element stack that can be used as the microlenses 140A.

[0049] 11 , third metal layer 143A may be deposited on microlens 140A according to conventional semiconductor processing techniques. Third metal layer 143A may comprise any suitable material, such as tungsten, aluminum, copper, combinations thereof, etc. Third metal layer 143A may be a relatively thin layer, e.g., thinner than second metal layer 123B. Third metal layer 143A may be made of the same or a different material than first metal layer 123B and / or second metal layer 133B.

[0050] 12 , a planarization layer 145A may be deposited over the third metal layer 143A. The planarization layer 145A may comprise any suitable material. The planarization layer 145A may be deposited, for example, by spin coating or by any other suitable method. If the planarization layer 145A exceeds the top exposed surface of the third metal layer 143A, the planarization layer 145A may be planarized, for example, by chemical mechanical planarization (CMP), leaving the planarization layer 145A in the openings between the third metal layer 143A and forming a substantially flat top surface.

[0051] 13, third openings 155A-C can be etched through planarization layer 145A (while leaving planarization layer 145B), third metal layer 143A (while leaving third metal layer 143B), and microlens 140A (while leaving microlens 140B). For example, planarization layer 145B can be spin-coated with photoresist (not shown) to etch third openings 155A-C. In some embodiments, the widths of third openings 155A-C can correspond to the widths of second openings 150A-C, such that further etching of second metal layer 133B is not necessary. Third openings 155A-C can be etched down to second passivation layer 130, with second passivation layer 130 acting as an etch stop. In some examples, the third openings 155A-C may have a diameter between 100 nanometers and 1 micrometer and may be aligned with and centered on the color filter material 127B and / or the photodiode 117. In some embodiments, a biological or chemical sample can be placed within the third openings 155A-C on the second passivation layer 130, and the fluorescence or chemiluminescence of the sample can be measured, as described further herein.

[0052] D. Biosensor 1300 of Figure 13 13 illustrates a biosensor 1300 that can be used for biological or chemical analysis, according to some embodiments. The biosensor 1300 includes a back-illuminated CMOS image sensor 100. The back-illuminated CMOS image sensor 100 includes an electronic circuit layer (comprising a first dielectric layer 110 and metal wiring 113) and a light-sensing layer (comprising a substrate layer 115 and a photodiode 117) on the electronic circuit layer. The photodiode 117 may be in contact with the electronic circuit layer so that an electrical signal can be transmitted from the photodiode 117 to the electronic circuit layer and, in some embodiments, to an external device. The light-receiving surface is defined by the surface of the photodiode 117 opposite the electronic circuit layer (i.e., the surface in contact with the first passivation layer 120).

[0053] The biosensor 1300 may further include a first passivation layer 120 on the back-illuminated CMOS image sensor 100 and a first metal layer 123B on the first passivation layer 120. The first metal layer 123B may also be disposed on the substrate layer 115. The first metal layer 123B may include a first opening. The biosensor 1300 may further include a second dielectric layer 125 on the metal layer 123B and the first passivation layer 120. The second dielectric layer 125 may also be disposed within the first opening in the metal layer 123B.

[0054] Biosensor 1300 may further include a color filter material 127B over second dielectric layer 125 and in and over the first openings in metal layer 123B, such that the top surface of color filter material 127B may be planar with the top surface of second dielectric layer 125 over metal layer 123B. Biosensor 1300 may further include a second passivation layer 130 over second dielectric layer 125 and color filter material 127. Biosensor 1300 may further include a second metal layer 133B over second passivation layer 130 with second openings 150A-C.

[0055] Biosensor 1300 may further include a microlens 140B on second metal layer 133B, a third metal layer 143B on microlens 140B, and a planarization layer 145 on third metal layer 143B. Third metal layer 143B may serve a number of different purposes in biosensor 1300. For example, third metal layer 143B may help block incident light from entering color filter material 127B. Furthermore, because third metal layer 143B is curved, any light emitted from the biological or chemical sample may pass through microlens 140B, reflect off third metal layer 143B, and be directed back toward color filter material 127B and thus toward the light-receiving surface of photodiode 117. In other words, the amount of emitted light that can be measured by photodiode 117 may be maximized.

[0056] The planarization layer 145 may form a planar surface on the third metal layer 143B. The microlenses 140B, the third metal layer 143B, and the planarization layer 145 may have third openings 155A-C formed therein, which may overlap with the second openings 150A-C in some embodiments. For example, the third openings 155A-C may have the same width as the second openings 150A-C. However, it is contemplated that in some embodiments, the third openings 155A-C may have a different width than the second openings 150A-C. Together, the second openings 150A-C and the third openings 155A-C may function as spots or wells configured to receive biological or chemical samples, as described further herein. 13 are deeper than the second openings 150A-C of FIG. 9, excitation light can generally be directed from a light source located directly above the third openings 155A-C in biosensor 1300. Because the second openings 150A-C are not as deep as the third openings 155A-C, biosensor 900 can tolerate greater angular misalignment of the excitation light.

[0057] Nucleic Acid Sequencing Applications As described above with respect to Figures 2, 8A, 9, and 13, a biological or chemical sample can be disposed on each of the described biosensors above the color filter material 127B and photodiode 117. The biological or chemical sample can include any number of components. For example, the sample can include nucleic acid macromolecules (e.g., DNA, RNA, etc.), proteins, etc. The sample can be analyzed to determine gene sequence, DNA-DNA hybridization, single nucleotide polymorphisms, protein interactions, peptide interactions, antigen-antibody interactions, glucose monitoring, cholesterol monitoring, etc.

[0058] As noted above, in some embodiments, the biomolecule is a nucleic acid, e.g., DNA. Without limitation, a DNA biomolecule can be a DNA nanoball (single-stranded concatemer) that is hybridized to a labeled probe (e.g., in DNB sequencing by ligation or cPAL methods) or a complementary growing strand (e.g., in DNB sequencing by synthesis methods), or both; or to a single DNA molecule (e.g., in single-molecule sequencing); or to a clonal population of DNA molecules, such as those generated in bridge PCR-based sequencing. Thus, reference to a "biomolecule," "DNA polymer," or "nucleic acid polymer" can encompass two or more molecules (e.g., a DNB associated with multiple growing complementary strands, or a DNA cluster comprising a clonal population of hundreds or thousands of DNA molecules). See, e.g., U.S. Patent No. 8,133,719; U.S. Patent Application Publication No. 2013 / 0116153, U.S. Patent Application Publication No. 2016 / 0237488; U.S. Patent Application Publication No. 2012 / 0224050; U.S. Patent Nos. 8,133,719; 7,910,354; 9,222,132; 6,210,891; 6,828,100; 6,833,246; and 6,911,345, which are incorporated by reference herein in their entireties.

[0059] In some embodiments, the nucleic acid polymer may be an amplicon of a genomic DNA fragment or a cDNA library. As used herein, an "amplicon" may refer to an amplification product of a nucleic acid molecule, typically an amplification product of a genomic DNA fragment or a cDNA library. Amplification methods include, but are not limited to, rolling circle amplification, as described, for example, in U.S. Pat. No. 8,445,194 (incorporated herein by reference in its entirety), or bridge polymerase chain reaction (PCR), as described, for example, in U.S. Pat. No. 7,972,820 (incorporated herein by reference in its entirety). Amplification may be performed before the nucleic acid contacts the biosensor or in situ, as described, for example, in U.S. Pat. No. 7,910,354 (incorporated herein by reference in its entirety).

[0060] In some embodiments, color filter material 127B may be sized and functionalized to receive a biological or chemical sample (in a spot or well above color filter material 127B) and absorb light emitted from the biological or chemical sample, in some instances. For example, if color filter material 127B is red and the emitted light from the biological or chemical sample is green, color filter material 127B may absorb green emitted light. In some embodiments, color filter material 127B may be sized and functionalized to receive a biological or chemical sample (in a spot or well above color filter material 127B) and pass light emitted from the biological or chemical sample through color filter material 127B and onto the light-receiving surface of photodiode 117. For example, if color filter material 127B is blue and the emitted light from the biological or chemical sample is blue, color filter material 127B may pass blue emitted light onto the light-receiving surface of the corresponding photodiode 117. In other words, in some embodiments, emitted light may be absorbed by color filter material 127B. In some embodiments, the emitted light may be transmitted onto the photodiode 117 through the color filter material 127B.

[0061] For example, a biological sample, such as a DNA polymer, oligonucleotide, or nucleotide, associated with a fluorescent or chemiluminescent dye may be placed on the photodiode 117. In the case of fluorescence, the dye may be illuminated by excitation light from an excitation light source. The excitation light may correspond to any suitable type or intensity of light, including, for example, visible light, infrared (IR), ultraviolet (UV), etc. The excitation light may also originate from any suitable light source, such as a light-emitting diode (LED), lamp, laser, or combinations thereof. When the dye is illuminated with excitation light of a particular wavelength, the biological sample may absorb the light and then emit light of a different wavelength. For example, a biological sample may absorb excitation light having a wavelength of 450 nm but emit light having a wavelength of 550 nm. In other words, when the dye is illuminated with light of a different, characteristic wavelength (i.e., an excitation light source), fluorescence of the characteristic wavelength may be emitted. However, because the excitation light is used to measure fluorescence, it must be filtered to ensure accurate measurements at the photodiode 117.

[0062] In the case of chemiluminescence, to detect the emitted light, no excitation light source is required for the photodiode 117. Instead, the biological sample can emit light due to a chemical or enzymatic reaction that may occur between the biological sample and the chemiluminescent dye (or other solution), causing the emission of light by breaking or forming chemical bonds.

[0063] For both fluorescence and chemiluminescence, the photodiode 117 can detect the intensity of the emitted light and convert it into an electrical signal based on the light intensity, which can be provided to an external device via metal wiring 113. The external device can correlate the electrical signal to a particular wavelength and brightness based on the electrical signal and the color of the color filter material 127B used over that particular photodiode 117.

[0064] To achieve high density and aid in alignment between nucleic acid polymers and the biosensor's photodiode 117, the biosensor surface may be configured with active spots or wells (e.g., openings 150A-C, openings 155A-C, etc.) sized and chemically functionalized to receive nucleic acid polymers and surrounded by areas of the surface to which the nucleic acid polymers cannot bind. Nucleic acid polymers can be immobilized on the active surface aligned with the photodiode 117 using any suitable surface chemistry. This may involve non-covalent interactions (e.g., to positively charged regions) or interactions with capture probes or surface-bound oligonucleotides having sequences complementary to those contained in the nucleic acid polymers. See, e.g., U.S. Pat. No. 8,445,194, incorporated herein by reference in its entirety.

[0065] In some embodiments, the active spots or wells and nucleic acid polymers on the surface of a biosensor can be configured relative to one another so that each spot binds only one nucleic acid polymer. This can be achieved, for example, by contacting the surface with amplicons that correspond in size to the active spots (e.g., amplicons with diameters effectively equal to or larger than the diameter of the active spots). See U.S. Patent No. 8,445,194, incorporated herein by reference in its entirety. Alternatively, the active spots can be chemically adapted to bind a single DNA fragment, which can be amplified to fill a larger area at and around the original binding site.

[0066] Some embodiments of the present invention can be used to determine different labels corresponding to different wavelengths of light. The labels can be, for example, fluorescent, chemiluminescent, or bioluminescent labels. For example, in gene sequencing (or DNA sequencing), embodiments of the present invention can be used to determine the precise order of nucleotide bases within a nucleic acid polymer (e.g., a strand of DNA). The nucleotide bases can be labeled with specific fluorescent labels (e.g., adenine (A), guanine (G), cytosine (C), or thymine (T)). Alternatively, for example, one-, two-, or three-color sequencing methods can be used.

[0067] With regard to fluorescence, each nucleotide base can be determined in turn by sequentially exciting the nucleic acid polymer with excitation light. The nucleic acid polymer can absorb the excitation light and transmit emitted light of different wavelengths onto a biosensor, as described herein (e.g., as shown in Figures 2, 8A, 9, or 13). The biosensor can measure the wavelength of the emitted light and the intensity received by a photodiode. When excited by excitation light of a particular wavelength and / or intensity, each nucleotide can emit a particular wavelength and / or intensity of light to the photodiode (i.e., a "fluorescent label"), allowing identification of the presence of a particular nucleotide base at a particular position in the nucleic acid polymer. Once that particular nucleotide base is determined, it is removed from the nucleic acid polymer, so that the next successive nucleotide base can be determined following a similar process.

[0068] Nucleic acid polymers can be labeled with one or more different fluorescent, chemiluminescent, or bioluminescent labels before or after attachment to a biosensor for any purpose. For example, nucleic acid polymers can be hybridized with labeled oligonucleotide probes or amplification primers. Alternatively, nucleic acid polymers can be hybridized with unlabeled oligonucleotides, which can then be bound to labeled probes or extended with labeled nucleotide analogs. For example, labeling can be performed for the purpose of characterizing the nucleic acid polymer (e.g., the presence of disease-associated single nucleotide polymorphisms (SNPs)) or, as described above, for nucleic acid sequencing of all or part of the nucleic acid polymer. DNA sequencing by probe hybridization is described, for example, in U.S. Pat. No. 8,105,771 (incorporated herein by reference in its entirety). Sequencing by anchor probe ligation is described, for example, in U.S. Pat. No. 8,592,150 (incorporated herein by reference in its entirety). Sequencing by synthesis is described, for example, in U.S. Patent No. 7,883,869 (its entirety is incorporated herein by reference).Generally, sequencing by synthesis is a method in which nucleotides are successively added to the free 3' hydroxyl group provided by the sequencing primer hybridized to the template sequence, resulting in the synthesis of a nucleic acid chain in the 5' to 3' direction.In one method, another exemplary type of SBS, pyrosequencing technology, is used (Ronaghi et al., 1998, Science 281:363).

[0069] In some embodiments, the biosensor shown in FIGS. 2, 8A, 9, and 13 may be coupled to a flow cell (not shown). Nucleic acid polymers may be attached to the biosensor by contacting the biosensor with a liquid sample in the flow cell. The flow cell may include one or more flow channels (e.g., openings 150A-C, openings 155A-C, etc.) that are in fluid communication with the reaction site. In one example, the biosensor may be fluidly and electrically coupled to a bioassay system. The bioassay system may deliver reagents to the reaction site and perform imaging events according to a predetermined protocol. For example, the bioassay system may direct a solution to flow along the reaction site. The solution may contain four types of nucleotides with the same or different fluorescent labels. The bioassay system may then illuminate the reaction site using an excitation light source. The excitation light may have a predetermined wavelength or wavelengths. The excited fluorescent labels may provide an emission signal that can be detected by photodiode 117.

[0070] A user can prepare for sequencing by contacting a biosensor according to the embodiments described (e.g., in Figures 2, 8A, 9, and 13) with a nucleic acid amplicon or a nucleic acid to be subsequently amplified, so that the nucleic acid polymer binds and is retained by the active spot or well, and excess nucleic acid polymer can be washed away. The nucleic acid polymer is contacted with a labeling reagent, either beforehand or in situ. The biosensor can then be operated as described herein to determine light emitted on or around the nucleic acid polymers on the array. The light can be quantified, or it can be sufficient to dually determine which nucleic acid polymers on the surface are labeled with a label that emits light at a particular wavelength. Different probes or different nucleic acid analogs with labels that emit light at different wavelengths can be used simultaneously, for example, to determine different bases at a particular position in a sequence or to sequence multiple positions. [Example]

[0071] Example This example demonstrates that a BSI CIS sensor can be used to detect weak signals from photon-emitting molecules attached to a surface. We constructed a biosensor as described in Figure 9, but without the color filter layer (i.e., lacking elements 120, 123B, 125, and 127B). Furthermore, we made surface 133B hydrophobic and the bottom surfaces of openings 150A / B / C hydrophilic (so that DNB distributes toward the hydrophilic surface and away from the hydrophobic surface).

[0072] A dilute solution of DNA nanoballs (DNBs) was applied to the biosensor array, allowing individual DNBs to precipitate onto the spots on the array. For the purposes of this experiment, all of the DNBs had the same sequence; this is in contrast to sequencing methods, where essentially every DNB on the array will have a different sequence and the sequence / location of a DNB at any particular spot will not be known prior to sequencing.

[0073] Two primers were hybridized to the DNA template (see Figure 15A, top). The "left" primer has a blocked (non-extendable) 3' end and is labeled at the 5' end with a fluorescent dye. The fluorescent dye was used to establish the position of the DNB on the array (not shown). The "right" primer acts as an extendible primer for sequencing by synthesis. Sequencing and detection reagents (DNA polymerase, streptavidin, biotinylated luciferase, ATP, and luciferin) were added along with biotin-tagged dATP via a cleavable linker. In this system, streptavidin associates with biotin conjugated to the incorporated nucleotide and also associates with biotinylated luciferase (biotin represented by a diamond) as shown in Figure 15A. ATP serves as a substrate for light generation via the luciferase-mediated conversion of luciferin to oxyluciferin. The light is received by a photodiode, which generates a signal. The signal correlates with the incorporation of dATP and indicates the presence of thymine at the corresponding position in the template sequence. Figure 15A shows the signal from DNB at multiple spots on the array.

[0074] The cleavable linker was then cleaved using THPP to release the biotin / streptavidin / luciferase complex, and the array was washed to remove all soluble reagents. Figure 15B shows that there was either no signal or a significant decrease in signal from the array after the washing step.

[0075] A second round of incorporation was performed using dTTP-digoxin and DNA polymerase, as shown in Figure 15C. Incorporation of dTTP was detected using biotinylated anti-digoxin antibody, streptavidin, biotinylated luciferase, ATP, and luciferin. The use of biotinylated anti-digoxin antibody amplifies the signal generated by each incorporation event. Figure 15C is an image showing that chemiluminescent light was generated at multiple spots on the array. This example demonstrates that using two different dNTPs and two different detection systems, the BSI CIS sensor of the present invention can be used to detect weak signals from photon-emitting molecules, such as DNB, attached to a surface.

[0076] Although the processes described herein are described with respect to a particular number of steps performed in a particular order, it is contemplated that additional steps not expressly shown and / or described may be included. Further, it is contemplated that fewer steps than shown and described may be included without departing from the scope of the described embodiments (i.e., one or more of the described steps may be optional). Furthermore, it is contemplated that the steps described herein may be performed in an order different from that described.

[0077] In the foregoing description, aspects of the present application are described with reference to specific embodiments thereof, but those skilled in the art will recognize that the present invention is not limited thereto. Accordingly, while exemplary embodiments of the present application have been described in detail herein, it should be understood that the concepts of the present invention can be variously embodied and employed, and that the appended claims are intended to be construed to include such variations except insofar as limited by the prior art. Various features and aspects of the above-described invention can be used individually or together. Moreover, the embodiments can be utilized in any number of environments and applications beyond those described herein without departing from the broader spirit and scope of the present specification. Accordingly, the specification and drawings should be regarded as illustrative rather than restrictive. For illustrative purposes, methods have been described in a particular order. It should be understood that in alternative embodiments, methods may be performed in an order different from that described.

[0078] Other variations are within the spirit and scope of the present disclosure. Thus, while the disclosed technology is susceptible to various modifications and alternative constructions, specific illustrated embodiments thereof have been shown in the drawings and have been described above in detail. It is to be understood, however, that there is no intention to limit the disclosure to the particular forms or forms disclosed, but on the contrary, the intention is to cover all modifications, alternative constructions, and equivalents falling within the spirit and scope of the disclosure, as defined by the appended claims.

Claims

1. 1. A method of manufacturing a biosensor, said method comprising: (a) depositing a passivation layer onto a back-illuminated complementary metal-oxide semiconductor (CMOS) image sensor, wherein the image sensor comprises: (i) an electronic circuit layer; and (ii) a photodetector layer on the electronic circuit layer; the light sensing layer includes a plurality of photodiodes including a light receiving surface opposite the electronic circuit layer, and wherein the passivation layer deposited is on the light receiving surface and opposite the electronic circuit layer; (b) depositing a layer of material over the passivation layer; (c) forming a plurality of openings in the material layer deposited on the passivation layer, wherein each opening is aligned with a single photodiode or with a unit cell containing multiple photodiodes; and (d) functionalizing spots or wells within each of the plurality of openings, each spot or well being sized and chemically functionalized to bind a single nucleic acid polymer; A method comprising:

2. 2. The method of claim 1, wherein forming the plurality of openings and forming the spots or wells in each of the plurality of openings comprises forming a regular array of spots or wells, wherein each spot or well is a discrete positively charged region, and wherein each spot is separated from other spots by regions that are inactive, in the sense that they are configured not to bind to nucleic acid polymers.

3. 10. The method of claim 1, wherein depositing the passivation layer on the back-illuminated complementary metal-oxide-semiconductor (CMOS) image sensor comprises depositing the passivation layer on the light-receiving surface.

4. The method of claim 3 , wherein the passivation layer is an oxide.

5. The method of claim 3 , wherein the passivation layer is a transparent layer.

6. The method of claim 3 , wherein forming a plurality of openings in the material layer deposited on the passivation layer comprises etching the material layer.

7. The method of claim 6 , wherein etching the material layer further comprises using a mask to define a plurality of openings in the material layer.

8. The method of claim 6 , wherein the passivation layer acts as an etch stop during the formation of the plurality of openings.

9. The method of claim 6 , wherein the plurality of openings are aligned and centered with the photodiode or the unit cell.

10. The method of claim 3 , wherein the passivation layer has a thickness of 100 nm or less.

11. The method of claim 1 , wherein depositing the material layer on the passivation layer comprises depositing a metal layer on the passivation layer.

12. 10. The method of claim 1, wherein the photodiode is configured to detect light emitted from a fluorescent or chemiluminescent label on the nucleic acid polymer.

13. 10. The method of claim 1, further comprising forming a color filter layer on the back-illuminated complementary metal-oxide semiconductor (CMOS) image sensor, wherein a passivation layer is deposited on the color filter layer.

14. 14. The method of claim 13, The formation of the color filter layer comprises the steps of: (i) depositing a first passivation layer over the back-illuminated complementary metal oxide semiconductor (CMOS) image sensor; (ii) depositing a first metal layer over the first passivation layer; (iii) etching the first metal layer; and (iv) depositing a color filter material in the openings etched in the first metal layer; and The method, wherein the passivation layer deposited on the color filter layer is a second passivation layer.

15. The method of claim 13 , wherein the color filter layer comprises a single color color filter material.

16. The method of claim 13 , wherein the color filter layers comprise color filter materials of different colors.

17. 17. The method of claim 16, wherein the spots or wells are located at the intersections of different color filter materials.

18. The method of claim 1 , further comprising forming a microlens on the passivation layer.

19. 20. The method of claim 18, wherein forming the microlens comprises growing the microlens on the passivation layer.

20. 20. The method of claim 19, wherein the microlens is aligned centrally with the photodiode or the unit cell.

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