WO2012061818A1

WO2012061818A1 - Flowcells and flowcell reaction chambers

Info

Publication number: WO2012061818A1
Application number: PCT/US2011/059607
Authority: WO
Inventors: Steven Boege; Alan Blanchard; James Ball; Evan Foster; John Bridgham
Original assignee: Life Technologies Corporation
Priority date: 2010-11-05
Filing date: 2011-11-07
Publication date: 2012-05-10

Abstract

Flowcell designs that are suitable for large scale gene sequencing analyses are disclosed. Different layouts of flowcells are presented. Methods of functionalizing the surface where DNA samples are placed for enhanced attachment are discussed. Special treatments of the functionalized surface to achieve desired optical characteristics are also featured.

Description

FLOWCELLS AND FLOWCELL REACTION CHAMBERS

FIELD

[001] The present disclosure relates to various flowcell assemblies designed for next generation gene sequencing technologies.

BACKGROUND

[002] Nucleic acid sequencing techniques are of major importance in a wide variety of fields ranging from basic research to clinical diagnosis. The results available from such technologies can include information of varying degrees of specificity. For example, useful information can consist of determining whether a particular polynucleotide differs in sequence from a reference polynucleotide, confirming the presence of a particular polynucleotide sequence in a sample, determining partial sequence information such as the identity of one or more nucleotides within a polynucleotide, determining the identity and order of nucleotides within a polynucleotide, etc.

SUMMARY

[003] System and methods of large-scale, parallel analysis of polynucleotides are provided herein. More specifically, various embodiments of a flowcell are disclosed herein. In some embodiments, the flowcell can include at least one channel. In some embodiments, portions of the flowcell and/or portions of the channel can include, comprise, be formed of, have layers of, etc. some material capable of improving some performance of the system. For example, in some embodiments, the flowcell can include a material capable of improving binding and/or retention of analytes, samples, polynucleotides, etc. within any one of the channels of the flowcell. In some embodiments, an area or portion of the flowcell or channel can include some material capable of improving reflectance of incident light thereby improving performance of an autofocus system.

[004] These and other embodiments are provided herein. BFIEF DESCRIPTION OF THE DRAWINGS

[005] FIG. 1 is a functional diagram showing a flow cell under a microscope objective lens system and an auto-focus system;

[006] FIG. 2 is an embodiment of a flow cell featuring molded fluid channels;

[007] FIG. 3A is an embodiment of a flow cell chip featuring a top plate, a bonding layer, and a bottom plate;

[008] FIG. 3B is a plane view of a flow cell chip with fluid channels;

[009] FIG. 4 illustrates an adhesion mechanism between a sample surface and a templated bead;

[010] FIG. 5 A illustrates the dependence of reflectance on the thickness of a reflecting surface;

[011] FIG. 5B is a spectrum diagram of the transmittance characteristics of a flow cell imaging surface;

[012] FIG. 6A is a cross-sectional view of a fluid channel of a flow cell;

[013] FIG. 6B shows an optical path for an auto-focus system in the fluid channel of FIG. 6A;

[014] FIG. 7A is a cross-sectional view of another embodiment of a fluid channel; and

[015] FIG. 7B shows an optical path for an auto-focus system in the fluid channel of FIG. 7A.

[016] The drawings are for illustration purposes only. They are not intended to limit the scope of the present disclosure in any way.

DETAILED DESCRIPTION

[017] The present disclosure pertains generally to substrates (e.g., flowcells with a channel or channels, planar substrates such as slides, etc.) used for performing large scale parallel gene sequencing analyses and/or any other type of analysis of biological samples. For example, the various flow cells can be used with the SOLiD DNA Sequencing System (Life Technologies, Carlsbad, CA), as described in PCT

Publication No. WO 2006/084132, entitled "Reagents, Methods, and Libraries for Bead-Based Sequencing", U.S. Patent Application Serial No. 12/873,190, entitled "Low- Volume Sequencing System and Method of Use," filed on August 31, 2010, and U.S. Patent Application Serial No. 12/873,132, entitled "Fast-Indexing Filter Wheel and Method of Use," filed on August 31, 2010, the entirety of each of these applications being incorporated herein by reference thereto.

[018] Various other sequencing systems and/or chemistries are within the spirit and scope of the present disclosure. For example, the presently disclosure flowcells can be utilized with sequencing by synthesis platforms, single-molecule sequencing platforms, pyrosequencing platforms, etc. Additionally, those skilled in the art will appreciate that the presently disclosed substrates can be utilized with virtually any type of biological analysis system or protocol.

[019] Various embodiments of biological analysis systems are provided herein. For example, in some embodiments, the system can include flowcell assembly having a channel, multiple channels, no channels, wells, etc. The channels can include one or more ribs and the surface of the one or more channels (or the surface of the substrate) can be functionalized to receive, bind, attach sample. In some embodiments, the sample can include one or more templated beads, polynucleotides, peptides, proteins, etc. In some embodiments, the substrate or channels with the substrate can be substantially planar (i.e., no ribs) thereby allowing for random arrays. In some embodiments, the flowcell can include one or more covers, and the substrate and covers can be assembled together to form a flowcell assembly.

[020] The flowcell can include various materials and/or features. In some embodiments, various components, layers, portions, etc. of the flowcell can be formed of a polyolefin material. In those embodiments having channels, the one or more channels can be produced in various manners. For example, in some embodiments, the channels can be molded recesses on the substrate. In some embodiments, the one or more channels can further have microfeatures to facilitate sample (e.g., bead) attachment. The microfeatures can be irregularly spaced troughs or regularly spaced grooves. Sample can attach to the top and/or bottom of the flowcell.

[021] In some embodiments, the one or more covers may be a top plate (e.g., glass) and a bottom plate. The substrate can be (or a portion thereof can be) a bonding layer constructed of, for example, polydimethylsiloxane or pressure-sensitive-adhesive and/or the one or more channels on the substrate can be of a depth that equals or is less than about 50 μιη. The top plate may be of a thickness that equals or is less than about 150 μιη. The one or more covers have outlets connected to the one or more channels on the substrate for receiving and draining liquids. The functionalization of the surface of the one or more channels may include coating the surface with a metal oxide. The metal oxide may be zirconium oxide. The surface of the one or more channels may be coated with one single layer of zirconium oxide or multi layers of zirconium oxide. In some embodiments, the layer of zirconium oxide can be optimized so as to facilitate sample (e.g., beads, polynucleotides, etc.) binding while also being optimized to demonstrate enhanced reflectivity in the near-IR spectrum thereby enhancing the effectiveness of an associated auto-focus mechanism.

[022] In some embodiments, the surface of the one or more channels can be coated with sputtered metal oxide to form a rough surface with nano-scale features to facilitate sample (e.g., bead, polynucleotide, etc.) attachment. In some embodiments, the surface of the one or more channels can be coated with one or more selected materials for a desired optical property. The desired optical property can be that the coated layer is reflective within a spectrum range. For example, the spectrum range is the infrared portion of the spectrum. In some embodiments, the one or more selected materials can include a single layer of zirconium oxide of a desired thickness (e.g., have-wave layer). In some embodiments, the one or more selected materials include a layer of zirconium oxide that has a thickness equal to half wavelength of a light beam.

[023] Aspects of the present disclosure can include one or more of the following advantages. The flowcell assemblies can be optimized for large-scale polysequencing analysis. The flowcell assemblies can also include functionalized surfaces for strong sample attachment and/or thinner and slimmer designs that work well with objective lens of high numerical apertures and reduce the volumes of reagent fluids. [024] FIG. 1 shows an embodiment of a flowcell system 200 of a polynucleotide sequencing system. The flow cell 200 can be positioned under an objective lens 204. In the system 200, a flow cell reaction chamber 216 can be on top of a thermal block 202. The flow cell reaction chamber 216 can include a cover 206, an imaging surface 212, a bottom plate 213, and gaskets 208 for sealing flowcell reaction chamber (or channel(s) 216. The cover 206 can be transparent thereby providing an optical window (e.g., glass). Sample can be deposited or grown or amplified within each or at least one reaction chamber or channel 216. The sample, for example, DNA, can be deposited directly on the surface, or the sample and be templated beads 210 having DNA attached thereto. In some embodiments, the sample can be positioned on the imaging surface 212. The thermal block 202 can control the temperature of the reaction chamber 216 and can be made of thermally conductive materials. The thermal block 202 may also have an internal temperature sensor (not shown) imbedded in the center of the block 202 to better control the chamber temperature.

[025] The analysis system can also include an imaging system (not shown). The imaging system can include at least one light source (e.g., a laser, an arc lamp, an LED, etc.) and an objective lens 204. The objective lens 204 can be used for collecting signals (e.g., fluorescent signals) emitted by the nucleotides (or tags coupled thereto) on the templated beads. During data acquisition, signals emitted by the templated beads can be captured by the objective lens 204 and then can be converted into electronic signals by a detector (e.g., a CCD, CMOS, etc.).

[026] To maintain best focus, in some embodiments, the imaging system can further includes an auto focus system 236, which can include, for example, a light detector 218, a first lens 222, a magnifier 224, a beam splitter 226, a filter 228, and/or a light source 230. The light source 230 can be a laser. For example, the laser can include a round TEMOO laser, an astigmatic laser directly from a laser diode, etc. In some embodiments, the light source 230 can be an infrared laser. In some embodiments, the light source 230 can generate an incident laser beam 232 which can pass through a neutral density filter 228. The incident laser beam can be directed by the beam splitter 226 into the primary optical path of the optical lens system 204 and falls upon the imaging surface 212. The imaging surface of the flow cell can reflect the incident light beam. The reflected light beam 234 can pass back through the objective lens 204. If the imaging surface is in focus, the reflected light beam 234 can exit the objective lens 204 parallel to the optical axis. The light beam 234 can pass through a first lens 222 and a magnifier 224 and reach a detector 218 at the end of its path. The detector 218 may be a CCD detector or a CMOS detector or the like.

[027] In some embodiments, the real time auto focus system 236 can detect shifts of the imaging surface and adjust the positions of the flowcell system such that the images of the imaging surface stay on the focal plane 220 of the objective lens system. In some embodiments, when the imaging surface is out of best focus, the laser beam 234 can exit the optical lens at an angle relative to the optical axis, which results in a lateral translation of position of the beam at the detector relative its previous position. Detection of position shifts of the laser beam can allow for re- calibration of the flowcell position before image capturing begins in a next run. In some embodiments, the real time auto focus system 236 can rely on the imaging surface being reflective at the wavelength of the laser beam used by the auto focus system 236. As described below, the surface of the flow cell can be configured so as to optimize this reflectivity.

[028] In some embodiments, the optical lens 204 can influence the resolution of the imaging system. For example, in some embodiments, sufficiently high resolution can be needed to allow the detector to discern fluorescent emissions coming off individual samples. To achieve better resolution, optical lenses of higher numerical apertures can be used. For example, in some embodiments, the optical lens system 204 can include a 0.45nm N.A. objective or a 0.7 N.A. objective. Lenses of higher numerical apertures require shorter working distances, which in turn can utilize a slimmer and/or a more compact flowcell design.

[029] In some embodiments, the imaging light source and the light source 230 can be different and can use light beams of the same or different wavelengths. In some embodiments, while the imaging surface 212 can be reflective with respect to the infrared laser beam in the auto focus system 236, the imaging surface 212 can be of different optical characteristics with respect to the light beam of the imaging light source. For example, in some embodiments, the imaging surface 212 can be required to be transparent with respect to the imaging light beam if the imaging surface 212 is located before the samples on the imaging (primary) optical path. [030] FIG. 2 illustrates an embodiment of a flowcell 300 with a plurality of channels. As shown, multiple flow channels 322 are of approximate dimensions of about 5.25 mm wide x 50 micron (μιη) deep and can be configured to fill and drain with well controlled flow. Multiple channels, for example, four, of such width and with a length of approximately 64mm can be fitted on an existing 25mm x 75mm slide area with little or no sacrifice in imaging footprint. In FIG. 2, the separating ribs between the channels are of a width approximately 0.8 mm or somewhere between about 0.1 mm to 1.5 mm. In such a configuration, each of these channels contains approximately 16.7 microliters of reagent. If the depth of the channels is dropped to approximately 30 microns, the volume of each channel drops to approximately 10 microliters. Those skilled in the art will appreciate that various other dimensions and/or volumes in addition to those provided above are within the spirit and scope of the present disclosure.

[031] The substrate and/or channels can be formed of, include layers of, etc. various types of materials. In some embodiments, the slide with the four chambers as described above can be constructed of injection molded polyolefin material.

Polyolefin materials typically have low fluorescence optical properties and can be incorporated with microfeatures which may be used to create, for example, an ordered array of beads for the high density bead loading assays. Those skilled in the art will appreciate that the number of channels and/or dimensions described above are merely exemplary and flow cells having a wide range of channels (e.g., 4, 5, 6, 7, 8, etc.) and/or dimensions are within the spirit and scope of the present disclosure.

[032] Channels can be provided in various manners. For example, in some embodiments, the cover surface (unfunctionalized) can be bonded to the sample deposition surface (functionalized), prior to deposition. In some embodiments, the sample can then be introduced through a port (not shown) or otherwise in the closed assembly for deposition. In some embodiments, sample can be deposited on the functionalized surface prior to the assembly of the opposing surface. For this method, a very thin (for example a few microns thick) layer of pressure sensitive adhesive (PSA), patterned can be provided on the mating surface.

[033] In various embodiments, sample (e.g., templated beads) can be immobilized by means of attachment to a functionalized surface and the imaging surface of the chamber can be functionalized to accept the sample. The imaging surface can be functionalized prior to assembly or after assembly. In some embodiments, templated beads can be positioned on the top of a flowcell and/or on the bottom of a flowcell. According to some embodiments, the surface functionalization can comprise the deposition of a thin layer of zirconium oxide, for example, a layer that is only a few atoms thick, such as ten or fewer or five or fewer atoms thick. As detailed herein, the thickness of this layer can be optimized to allow for optimized bead binding as well as optimized reflectivity thereby enhancing operation of the autofocus mechanism.

[034] FIG. 3A illustrates one embodiment of a presently disclosed flowcell design 400. In some embodiments, the flowcell design 400 can include three layers: a top plate 402, a bonding layer 404, and a bottom plate 406 which can be assembled together. In some embodiments, the top plate 402 can be made of or include some material (e.g., glass) and has, for example, two alignment holes 408 positioned at, for example, two diagonally opposite corners. The bottom plate 406 and the bonding layer 404 can also include two alignments holes 408 similarly located. These alignment holes can facilitate aligning the three plates during assembly.

[035] In some embodiments, on the bonding layer 404, parallel channels 412 can run lengthwise. In these parallel channels 412, templated beads can be positioned on the top and/or on the bottom. In some embodiments, tubes 414 can be extended at the end of each channel for the purpose of filling and draining reagent and wash fluids. The filling and draining outlets 410 on the bottom layer 406 can be connected with the tubes 414 on the bonding layer 416 when the plates are assembled together.

Alternatively, in some embodiments, the filling and/or draining outlets 410 can be placed on the top plate 402 to allow reagent and wash fluids to be filled and drained from the top.

[036] In some embodiments, the bottom plate 406 can be made of glass and the bonding layer 404 may be made of polydimethylsiloxane (PDMS) or pressure- sensitive adhesive (PSA), both of which are suitable materials for use in microfluidic chips for flow delivery.

[037] The bonding layer can be of various thicknesses. For example, the bonding layer thickness can be about 50 μιη, about 30 μιη, etc. The thickness of the top plate can be about 1.1 mm, about 175 μιη, etc. The thickness of the bottom plate glass can be about 1.1 mm, about 300 μιη, etc. Those skilled in the will appreciate that various such dimensions are within the spirit and scope of the present disclosure.

[038] In FIG. 3B, an enlarged plane view 420 of two channels is presented. In some embodiments, each channel 422 can include a width of about 5 mm, a length of about 87 mm, and a depth of about 30 μιη. The rib between two adjacent channels can be about 8.2 mm wide. In some embodiments, two capillary tubes 424 can extend out at ends of at least some of the channels. In some embodiments, the capillary tubes 424 can be about 1 mm wide and about 6 mm long and are connected to the outlets 410 in FIG. 3A for filling and draining fluids in the channels.

[039] In some embodiments, the flow cell reaction chamber (or channel) can be defined by the top plate, the bottom plate, and the ribs. The surface of the top plate and/or the bottom plate can be an imaging surface. In some embodiments, the imaging surface can be functionalized to facilitate sample attachment.

[040] In use, in some embodiments, as reagent and/or wash fluids are flushed in and out, sample (e.g., templated beads, polynucleotides, etc.) should remain attached to the imaging surface. In some embodiments, sample can be attached to the flowcell (or channel) surface. Attachment may be mechanical or chemical. In some embodiments, the attachment scheme is isothiocyanate chemistry. In this scheme, the imaging surface of the reaction chamber can be modified with an amino siloxane to generate an amino functionalized surface. In some embodiments, the surface can be further modified with phenylene diisothiocyanate, which, in some embodiments, can react with the dUTP attached at the 3' end of the DNA templates anchored on the beads and form a linkage between the surface and the sample.

[041] In some embodiments, the imaging surface of the flowcell reaction chamber can be coated with a metal oxide to effect sample attachment. In some embodiments, zirconium oxide can be used to effect sample attachment. Zirconium treatment of a surface that may be made of, for example, glass or plastic, produces a surface that effective for binding molecules containing phosphate, such as DNA molecules.

Those skilled in the art will appreciate that various other metal oxides are within the spirit and scope of the present disclosure. In some embodiments, the metal or the metal oxide can include any transition metal or some alloy thereof. In some embodiments, the collective binding of many phosphates along the DNA backbone with the zirconium treated surface can result in strong bead attachment. FIG. 4 provides an example of zirconium chemistry that can be used for permanent sample (e.g., templated bead) attachment. In some embodiments, as is shown in FIG. 4, an imaging surface 502 can be coated with a zirconium layer. In some embodiments, a templated bead 504 can be linked to the surface via zirconium chemistry, an example of which is shown in diagram 506. In the diagram 506, two phosphate atoms along the DNA backbone are linked to the surface via chemical bonds.

[042] In some embodiments, the imaging surface can be made of or include glass and the zirconium modified glass surface can exhibit a high degree of nano-scale roughness, which can have a positive effect on bead binding. An atomic force micrograph showing the nano-scale roughness on a zirconium modified glass is presented in the graph 508.

[043] In some embodiments, the imaging surface can be made of plastic and, if desired, can be patterned with grooves as shown in the diagram 510. In some embodiments, the effects of the grooves on bead attachment are at least three-fold. First, the shape of the grooves can restrict movement of the beads significantly during changing of fluids. Second, the contact area between the beads and the grooves is doubled, as compared when the surface is flat, further strengthening the linkage between template DNA molecules and the zirconium coating. Third, use of the grooves can simplify imaging procedures as the system can be configured to only analyze or image along the grooves.

[044] In some embodiments, the coated layer on an imaging surface of a flow cell reaction chamber can include a single layer of zirconium oxide molecules, or multiple layers of zirconium oxide molecules, or a single layer of zirconium oxide molecules plus multiple layers of optically thin materials.

[045] When a single layer of zirconium oxide molecules is coated on the imaging surface, this monolayer coating is transparent or substantially transparent to lights of wavelengths that are in the visible portion of a spectrum. Such optical characteristics can be desirable when the imaging surface is in the optical path of fluorescent signals emitted by the fluorescent nucleotides (see FIG. 5A and discussion thereof).

[046] When multiple layers of zirconium oxide are coated on the imaging surface, the reflectance of the imaging surface varies periodically with the thickness, i.e., the number, of layers. FIG. 5A illustrates the physics behind the phenomenon.

[047] The diagram 610 in FIG. 5A shows a light beam being reflected on a surface 600. In some embodiments, the surface 600 can have a thickness of T. In some embodiments, the incoming light ray 602 can be reflected or substantially reflected off the top layer and the incoming light ray 606 can be reflected off the bottom layer. The phase difference between the two outgoing light rays 604 and 608 depends at least in part on the thickness of the surface 600. When T is such that the phase difference between the two outgoing light rays 604 and 608 is equal or close to about 1/2 λ, half of the wavelength of the light beam, cancellation between the two outgoing light rays 604 and 608 will be significant, which results in weak reflectivity. On the other hand, when T is such that the phase difference between the two outgoing light rays 604 and 608 is λ or near λ, cancellation between the two outgoing light rays 604 and 608 is minimum, which can result in strong reflectivity. When the incoming light beam is perpendicular or close to perpendicular to the surface, the phase difference between the two outgoing light rays 604 and 608 can be approximately represented by 2T.

[048] Therefore, when the thickness of a reflecting surface is one half wavelength of the incoming light beam, the reflectance of the surface is the strongest.

[049] As discussed previously, the real time auto-focus system 236 in FIG. 1 can rely at least in part on a reflective imaging surface to function properly. In some embodiments, the imaging surface of a flowcell reaction chamber is a water-glass interface, the reflectance of which is low, approximately 0.4%. An interface between glass and air or between metal and water typically has a much higher reflectance, usually ten to hundred times higher than that of a water-glass interface. Thus if applied on a glass imaging surface of a flowcell reaction chamber, in some embodiments, a metal oxide coating of an appropriate thickness can yield a high reflectance that is often crucial to the proper operation of the real time auto focus system 236.

[050] Many auto-focus monitor concepts comprise a beam, generally from a laser, which reflects from a surface substantially disposed in the front focal plane of a microscope objective. For example, a bead-laden surface can be substantially disposed in the front focal plane of a microscope objective. As indicated above, that surface constitutes a water-glass interface, the reflectance of which is approximately 0.4%. There are air-glass and metal-water interfaces in close proximity to the water- glass interface that have reflectances 10X - 100X larger than that of the water-glass interface. Focus monitoring apparatus that depend on reflected beams are often unable to discriminate the weak reflection from the water-glass interface in the presence of the air-glass and metal-water interfaces. Thus, in some embodiments, the present disclosure enhances the reflectance of the bead-laden surface by changing the water-glass interface into a water-coating-glass interface while at the same time providing an oxide layer for attachment of beads.

[051 ] In some embodiments, the coating can be a single half- wave layer of zirconium oxide deposited on a glass microscope slide via electron beam evaporation. In some embodiments, in the evaporation process, a block of the material (source) to be deposited can be heated to the point where it starts to boil and evaporate, and it can then be allowed to condense on the substrate. In some embodiments, this process can take place inside a vacuum chamber, enabling the molecules to evaporate freely in the chamber, where they then condense on all surfaces. For e-beam evaporation, in some embodiments, an electron beam can be used to heat the source material and cause evaporation.

[052] In some embodiments, the substrates can include single half-wave layers of other metal oxides. Other embodiments can include multilayer "stacks" of optical thin film materials, the final layer of which is zirconium oxide or another metal oxide. It is the final, metal oxide layer that binds the sample (e.g., templated beads, polynucleotides, etc.). As disclosed herein, by thickening the coating as described in the half-wave of zirconium oxide embodiment of the present disclosure, a substantial increase in reflectance in the near-infrared (NIR) portion of the spectrum can be realized. In some embodiments, this can increase the amount of focus monitor laser beam reflected by the sample-laden interface thereby providing a highly desirable result.

[053] In some embodiments, the use of multilayer "stacks" of optical thin film materials, the final layer of which is zirconium oxide or another metal oxide, can provide a larger increase in NIR reflectance and a smaller decrease in VIS reflectance.

[054] In some embodiments, the coating process can be carried out in a vacuum chamber through an electron beam evaporation process. During the evaporation process, an electron beam can be used to heat the coating material into a vapor which can move freely in the vacuum chamber. In some embodiments, the vapor can then condense on the surface desired to be coated. The duration of the heating process and/or the intensity of the electron beam can determine the amount of coating material evaporated, which in turn can determine the thickness of the coated layer.

[055] In some embodiments, one single layer (monolayer) of zirconium oxide can be applied on the imaging surface(s) of a flowcell reaction chamber. In some embodiments, a layer of zirconium oxide with a thickness equal to one half of the wavelength of the laser beam can be used in the auto-focus system may be coated. In some embodiments, the monolayer of zirconium can be generally much thinner, for example, about thirteen times thinner, than the half-wavelength layer.

[056] In some embodiments, the half-wavelength layer of zirconium oxide can provide an increase of the reflectance of the imaging surface in the visible portion of the spectrum. Because the imaging light source can use light beams of a wavelength that falls in the visible portion, such increase of the reflectance of the imaging surface can cause substantial signal losses. In some embodiments, several layers of optically thin materials can be coated on the imaging surface to achieve the desired thickness. In some embodiments, a final layer of zirconium oxide can be added to provide the adhesive properties for sample attachment. In some embodiments, such multi/hybrid layer embodiments can be utilized to achieve a larger increase in the reflectance in the near-infrared portion of the spectrum and a smaller decrease in the reflectance in the visible portion. FIG. 5B shows the transmittance value of a multi/hybrid layer embodiment at each wavelength in between the range of 400nm to 800nm. The reflectance at each wavelength may be calculated using the following simple relationship:

T = l - R , where T is transmittance and R is reflectance.

[057] In FIG. 5B, the transmittance/reflectance characteristics of the multi/hybrid layer are shown. The transmittance of the multi/hybrid layer remains larger than about 97% and the reflectance stays near zero between wavelengths 400nm to 700nm. This range corresponds to the visible portion of the spectrum. In the near-infrared region (> 750 nm), the transmittance drops significantly and the reflectance increases by a same degree.

[058] Although zirconium is particularly mentioned in the above discussions, other metals/metal oxides may be used as well as suitable coating materials.

[059] In some embodiments, as shown in FIGS. 6A and 7A, the flowcell can include ridges thereby allowing for ordered arrays. In some embodiments, the surfaces can be substantially planar (i.e., no ridges) thereby allowing for random arrays. As for FIGS. 6A and 6B, only flowcell reaction chambers are featured in the two figures. Other details have been omitted for simplicity reasons. In these two examples, the types of materials used and the geometric shapes and dimensions indicated are exemplary and are for illustration purposes only. To those skilled in the art, substitutes with equivalent functionalities for the specific materials mentioned and numerical ranges encompassing the precise dimensions cited are within the scope of the present teachings.

[060] FIG. 6A provides an example of a flowcell reaction chamber 700 in which sample (e.g., beads) 712 are positioned on the top surface of the flowcell. In some embodiments, the top of the flowcell reaction chamber 700 can be constructed out of injection molded poly olefin of a thickness of about 1 mm. In some embodiments, the flowcell 700 can include fluidic channels 702 which are about 50μηι deep and can be configured on the surface of the molded polyolefin during molding. In some embodiments, between the fluid channels 702 are ribs which can be made of various materials, e.g., PDMS, PSA, etc. In some embodiments, the imaging surface 704 can be functionalized and can sit on the top of the fluid channels 702. In some embodiments, a thin layer of zirconium oxide can be coated on the imaging surface 704 for the purposes of immobilizing sample (e.g., the templated beads) 712. In some embodiments, the zirconium coating can be a monolayer of zirconium, a multilayer coating of zirconium, or sputtered zirconium oxide and functions as an attachment layer 706 for sample to attach permanently via chemical bonds. In some

embodiments, the attachment layer can be transparent to allow viewing/imaging of the sample (e.g., beads) 712 from above.

[061] In some embodiments, as shown in FIG. 6A, the attachment layer 706 can be serrated and include regularly spaced grooves. In some embodiments, the attachment layer 706 can also be a rough surface with irregularly spaced troughs and crests.

[062] In some embodiments, the bottom of the flow cell reaction chamber 700 can be constructed with a thermally conductive material, such as, for example, glass, metal, silicone, plastic, or a mixture thereof. In some embodiments, a thermal block (not shown) beneath the flow cell may be used to provide temperature control by heating or cooling the reaction chamber 702 via the thermally conductive material.

[063] As discussed earlier, the real time auto-focus system depends on a reflective imaging surface to function properly. When the coating constitutes a stack of optically thin films topped with a single layer of zirconium oxide, the reflectance of the coating can be as high as 35% in the infrared region (see FIG. 5B). FIG. 6B illustrates the optical path of an incident infrared laser beam being reflected on the imaging surface of the flow cell reaction chamber in accordance with some embodiments. In some embodiments, the laser beam can come from the light source in an auto-focus system used to maintain best focus.

[064] In some embodiments, as illustrated in FIGS. 6A and 6B, the flowcells can include sample (e.g., templated beads) positioned on the top of the reaction chambers. In some embodiments, the samples can be positioned at the top and/or bottom of the reaction chamber(s), as shown in FIG. 7 A.

[065] In some embodiments, as shown in FIG. 7A, the flowcell 800 can have a shorter optical path than, for example, the flowcell 700 for objective lenses used in image acquisition. In the flow cell 800, the top of the flow cell can be a 150μπι glass cover-slip 810. The cover-slip 810 can be considerably thinner than the 1mm polyolefin material used for the top of the flow cell 700. The fluid channels 802 featured in the flow cell 800 can be shallower than the fluid channels 702 featured in the flow cell 700. In some embodiments, the fluid channels 802 can be only 30μπι deep and the ribs 808 in between two fluid channels are of the same height. In some embodiments, the bottom 814 of the flow cell 800 can be constructed with thermally conductive plastic.

[066] While the attachment layer 806 where the sample 812 can be placed on the top and/or at the bottom of the reaction chamber 800, in FIG. 7A, the attachment layer 806 can be at the bottom of the reaction chamber 800. In some embodiments, the attachment layer 806 can be a layer of sputtered zirconium oxide. In some embodiments where the samples 812 can be viewed/imaged from the top/above, the attachment layer 806 can be made selectively opaque to block the optical noises, such as auto-florescent emissions that are coming from the underlying materials, i.e., the bottom 814 of the flowcell reaction chamber and the thermal block (not shown) underneath the flow cell reaction chamber. The attachment layer 806 can also be made thicker to act as a chemical barrier to any undesired chemical

reactions/interactions between the underlying materials and the reagent fluids in the fluid channels 802. Moreover, in some embodiments, the thicker attachment layer 806 can result in more robust sample attachment.

[067] To accommodate the real time auto focus system 236, in some embodiments, the attachment layer 806 can be made reflective at the infrared portion of a spectrum. For example, in some embodiments, a layer of coating that comprises a stack of optically thin films and a single top layer of zirconium oxide can be used. FIG. 7B shows the optical path of an infrared laser beam which can be used by the real time auto focus system 236. In some embodiments, the imaging surface 804 may have a reflectance 35% or higher (see FIG. 5B) and act as a reflecting surface with respect to the infrared laser beam.

[068] A further advantage of the flowcell reaction chamber arrangement as shown in FIG. 7 A is that placing the sample beads 812 at the bottom of the reaction chamber 800 can produce better images. This is because first the side with the most efficient chemistry of the sample beads 812 is facing the objective lens and secondly the thin glass cover-slip 810 causes less optical distortions than the 1mm polyolefin material used in the flow cell reaction chamber 700.

[069] Another advantage of the flow cell reaction chamber arrangement as shown in FIG. 8A is that the overall design is much slimmer than the arrangement shown in FIG. 6A. With a thinner glass cover-slip and shallower fluid channels, the overall height of the flow cell reaction chamber 800 is about five times smaller than that of the flow cell reaction chamber 700, reducing the length of the image acquisition optical length significantly. With a much shorter optical length, the flow cell arrangement 800 is particularly suitable for use with objective lens system of a higher numerical aperture.

[070] A number of embodiments of the disclosure have been described.

Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. For example, some of the steps described above may be order independent, and thus can be performed in an order different from that described.

[071] It is to be understood that the foregoing description is intended to illustrate and not to limit the scope of the disclosure, which is defined by the scope of the appended claims. For example, a number of the function steps described above may be performed in a different order without substantially affecting overall processing. Other embodiments are within the scope of the following claims.

[072] One skilled in the art will appreciate further features and advantages of the present disclosure based on the above-described embodiments. Accordingly, the disclosure is not to be limited by what has been particularly shown and described, except as indicated by the appended claims. All publications and references cited herein are expressly incorporated herein by reference in their entirety.

Claims

What is claimed is:

1. A flowcell, comprising:

a substrate having at least one channel formed therein; and

at least one layer of a metal oxide disposed within the at least one channel.

2. The flowcell of claim 1 , wherein the metal oxide is zirconium oxide.

3. The flowcell of claim 1, further comprising a cover.

4. The flowcell of claim 2, wherein the cover includes a polyolefin material.

5. The flowcell of claim 1, wherein the one or more channels include microfeatures configured to facilitate bead attachment.

6. The flowcell of claim 5, wherein the microfeatures are irregularly spaced troughs.

7. The flowcell of claim 5, wherein the microfeatures are regularly spaced grooves.

8. The flowcell of claim 3, wherein the cover includes at least inlet port and at least one output port.

9. The flowcell assembly in claim 1, wherein a single layer of metal oxide is disposed within the at least one channel.

10. The flowcell of claim 1, wherein multiple layers of metal oxide are disposed within the at least one channel.

11. The flowcell of claim 2, wherein a surface of the one or more channels is coated with sputtered zirconium oxide to form a functionalized surface with nano- scale features to facilitate bead attachment.

12. The flowcell of claim 1 , wherein at least one metal oxide layer is on a top surface of the at least one channel.

13. The flowcell of claim 1, wherein at least one metal oxide layer is on a bottom surface of the at least one channel.

14. The flowcell of claim 1 , wherein at least one metal oxide layer is on a bottom surface and on a top surface of the at least one channel.

15. The flowcell of claim 1, wherein the at least one layer of metal oxide provides a desired optical property.

16. The flowcell of claim 15, wherein the desired optical property is being reflective within a spectrum range.

17. The flowcell of claim 15, wherein the spectrum range is the infrared portion.

18. The flowcell of claim 16, wherein the metal oxide is zirconium oxide.

19. The flowcell of claim 18, wherein the layer of zirconium oxide has a thickness equal to half wavelength of a light beam.