WO2012031234A2

WO2012031234A2 - Methods, systems and apparatus for sequencing

Info

Publication number: WO2012031234A2
Application number: PCT/US2011/050389
Authority: WO
Inventors: Shifeng Li; Michael W. Lafferty; Theo Kotseroglou; Mark Oldham
Original assignee: Life Technologies Corporation
Priority date: 2010-09-03
Filing date: 2011-09-02
Publication date: 2012-03-08
Also published as: WO2012031234A3

Abstract

A device includes a substrate having a refractive index of at least 1.45 and a surface layer disposed over a major surface of the substrate and defining a plurality of reaction volumes. The surface layer has a refractive index in a range of 1.32 to 1.38.

Description

METHODS, SYSTEMS AND APPARATUS FOR SEQUENCING

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Application No.

61/380, 171 filed September 3, 2010, which is herein incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

[0002] This disclosure, in general, relates to sequencing devices, systems incorporating such sequencing devices, methods of making such sequencing devices, and methods of using such sequencing device.

BACKGROUND

[0003] Sequencing of nucleic acid strands, particularly DNA, has become increasingly important in a variety of advancing fields including medicine, agriculture, and biological research. However, conventional gel techniques for sequencing nucleic acid strands are time-consuming and expensive. More recent developments rely on the deposition of nucleic acid containing samples on substrates. Depending upon the sequencing technique, the sequence of the nucleic acid sample can be determined by measuring ionic responses to nucleotide addition or by measuring fluorescent emissions resulting from nucleotide addition.

[0004] However, such sequencing techniques suffer from problems associated with background noise and signal strength. Poor signal strength can lead to misreads in which a nucleic acid addition fails to register. High noise leads to difficulties with instrument calibration. Further, a low signal-to-noise ratio can also lead to deletion resulting from a missed signals associate with nucleic acid addition and insertion associated with an incorrect attribution of noise to a nucleic acid addition.

[0005] As such, an improved sequencing device would be desirable. SUMMARY

[0006] In a first aspect, a device includes a substrate having a refractive index of at least 1.45 and a surface layer disposed over a major surface of the substrate and defining a plurality of reaction volumes. The surface layer has a refractive index in a range of 1.32 to 1.38.

[0007] In a second aspect, a system includes a device comprising a substrate having a refractive index of at least 1.45 and a surface layer disposed over a major surface of the substrate and defining a plurality of reaction volumes. The surface layer has a refractive index in a range of 1.32 to 1.38. The system further includes a chamber defining a volume over the surface layer of the device and a light source to direct excitation light to the substrate.

[0008] In a third aspect, a method of sequencing a nucleic acid strand includes applying the nucleic acid strand into a reaction volume of a device. The device includes a substrate having a refractive index of at least 1.45 and a surface layer disposed over a major surface of the substrate and defining a plurality of reaction volumes. The surface layer has a refractive index in a range of 1.32 to 1.38. The method further includes flowing a reagent solution over the surface layer of the device. The reagent solution includes nucleotides of a set of nucleotide types. Each type of nucleotide is labeled to provide a unique fluorescence signal. The method also includes directing excitation light to the substrate and detecting fluorescence signals associated with nucleotide incorporation onto the nucleic acid strand.

[0009] In a fourth aspect, a device includes a substrate having a major surface and comprising a plurality of detectors and a surface layer disposed over the major surface. The surface layer defines a plurality of reaction volumes. Each reaction volume of the plurality of reaction volumes is uniquely associated with a detector of the plurality of detectors. The surface layer has an index of refraction in a range of 1.32 to 1.38.

[0010] In a fifth aspect, a system includes a device including a substrate having a major surface and comprising a plurality of detectors and a surface layer disposed over the major surface. The surface layer defines a plurality of reaction volumes. Each reaction volume of the plurality of reaction volumes is uniquely associated with a detector of the plurality of detectors. The surface layer has an index of refraction in a range of 1.32 to 1.38. The system also includes a chamber to receive the device and define a volume over the surface layer.

[0011] In a sixth aspect, a method of sequencing a nucleic acid strand includes applying the nucleic acid strand into a reaction volume of a device. The device includes a substrate having a major surface and comprising a plurality of detectors and a surface layer disposed over the major surface. The surface layer defines a plurality of reaction volumes. Each reaction volume of the plurality of reaction volumes is uniquely associated with a detector of the plurality of detectors. The surface layer has an index of refraction in a range of 1.32 to 1.38. The method further includes flowing a reagent solution over the surface layer of the device. The reagent solution includes nucleotides of a set of nucleotide types. Each type of nucleotide is labeled to provide a unique fluorescence signal. The method also includes detecting fluorescence signals associated with nucleotide incorporation onto the nucleic acid strand.

[0012] In a seventh aspect, a method of sequencing a nucleic acid strand includes applying the nucleic acid strand into a reaction volume of a device. The device includes a substrate having a major surface and comprising a plurality of detectors and a surface layer disposed over the major surface. The surface layer defines a plurality of reaction volumes. Each reaction volume of the plurality of reaction volumes is uniquely associated with a detector of the plurality of detectors. The surface layer has an index of refraction in a range of 1.32 to 1.38. The method further includes sequentially flowing reagent solutions over the surface layer of the device. Each of the reagent solutions includes a type of nucleotide of a set of nucleotide types. The method further includes detecting signals associated with nucleotide

incorporation onto the nucleic acid strand.

[0013] In an eighth aspect, a method of forming a device includes coating a substrate with a polymeric coating having a refractive index in a range of 1.32 to 1.38. The substrate is transparent and has a refractive index of at least 1.45. The method further includes etching the polymeric coating to define a plurality of reaction volumes. The plurality of reaction volumes includes at least 100 reaction volumes. [0014] In a ninth aspect, a method of forming a device includes coating a major surface of a substrate with a polymeric coating having a refractive index in a range of 1.30 to 1.40. The substrate includes a plurality of detectors. The method further includes etching the polymeric coating to define a plurality of reaction volumes. Each reaction volume of the plurality of reaction volumes is uniquely associated with a detector of the plurality of detectors.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present disclosure may be better understood, and its numerous features and advantages made apparent to those skilled in the art by referencing the accompanying drawings.

[0016] FIG. 1 includes an illustration of a cross-sectional view of an exemplary sequencing device.

[0017] FIG. 2 includes an illustration of a planar view of an exemplary sequencing device.

[0018] FIG. 3 and FIG. 4 include cross-sectional illustrations of exemplary sequencing devices.

[0019] FIG. 5 includes an illustration of exemplary system incorporating the sequencing device.

[0020] FIG. 6, FIG. 7, and FIG. 8 include block flow diagrams illustrating exemplary methods associated with the sequencing device.

[0021] FIG. 9 and FIG. 10 include illustrations of alternative embodiments of the sequencing device.

[0022] FIG. 1 1, FIG. 12, and FIG. 13 include graph illustrations of electric field intensity relative to distance from a surface of a substrate for configurations of devices.

[0023] FIG. 14 includes a graph illustration of background noise reduction using refractive index matching structures, here in Cytop nano wells. [0024] FIG. 15, FIG. 16, and FIG. 17 include illustrations of exemplary light coupling methods.

[0025] FIG. 18A-E, FIG. 19A-F, and FIG. 20A-F include illustrations of processes for forming surface layers.

[0026] FIG. 21 and FIG. 22 include scanning electron microscopy illustrations of well structures in a surface layer.

[0027] FIG. 23 and FIG. 24 include illustrations of fluorescent intensity for a control sample and a surface layer sample respectively.

[0028] FIG. 25 includes a graph illustration of an exemplary sequencing time trace.

[0029] The use of the same reference symbols in different drawings indicates similar or identical items.

DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

[0030] In an exemplary embodiment, a sequencing device includes a substrate having a major surface and a surface layer disposed over the major surface of the substrate. The surface layer defines a plurality of reaction volumes, such as wells or channels. Further, the surface layer has a refractive index of approximate value relative to the refractive index of a background solution. In an example, when the background solution is water, 1.33, the refractive index of the surface layer can be in a range of 1.3 to 1.4. The substrate can be formed of a material that propagates electromagnetic radiation. For example, the substrate can be a transparent substrate or can include a transparent layer disposed adjacent the surface layer. The substrate can have a greater refractive index than the refractive indices of the background solution and the surface layer. In an example, the transparent layer has a refractive index of at least 1.45. In another example, substrate can include a plurality of detectors. Each of the detectors can define a detection region on a major surface of the substrate. Exemplary detectors include light-sensitive detectors or ion detectors.

[0031] In use, a nucleic acid strand is positioned within the reaction volume defined by the surface layer. In addition, an enzyme, such as a polymerase, can be applied to incorporate nucleic acids complementary to the target nucleic acid strand. Nucleotides are fed to the system either in a reagent solution including a mixture of types of nucleotides or are fed sequentially in different reagent solutions, each including a different type of nucleotide. In an example, the nucleotides are modified with fluorescent labels, which respond to an evanescent field generated by reflected light in a light confining layer of the substrate. When the nucleotide is incorporated or hybridize to the nucleic acid strand, fluorescent emissions from the fluorescent label indicate which type of nucleotide has been incorporated. In another example, the nucleotides are fed sequentially to the device. When the nucleotide is

incorporated, an ionic byproduct is released or formed. Such an ionic byproduct can be detected by the detectors. The byproduct can be hydrogen ions or phosphates released from the incorporation reaction.

[0032] In an embodiment illustrated in FIG. 1, a sequencing device 100 includes a substrate 102 and a surface layer 104 disposed over a major surface 1 14 of the substrate 102. The surface layer 104 defines a plurality of reaction volumes 106. In an example, the reaction volumes 106 are wells surrounded within a plane by the surface layer 104. In another example, the reaction volumes 106 are channels having a width and extending longitudinally perpendicular to the width. For both examples, the refractive index of the surface layer 104 can be approximately that of a background solution 118.

[0033] As illustrated in FIG. 1, a nucleic acid strand 108 is disposed within the reaction volume 106. As depicted, a single nucleic acid strand 108 is disposed within the reaction volume 106. Alternatively, a particle incorporating multiple identical copies of the nucleic acid strand can be disposed within the reaction volume 106. In addition, an enzyme 1 10 is disposed within the reaction volume 106. Nucleotides 112 in a flow region 1 18 above the reaction volumes 106 can migrate or diffuse into the reaction volumes 106. When the enzyme 1 10 encounters a nucleotide complementary to the next nucleotide in a sequence defined by the nucleic acid strand 108, the nucleotide 1 12 can be incorporated or hybridize to the nucleic acid strand 108. In an example, such nucleotide incorporation results in a fluorescent signal. Optionally, a donor group, such as a donor moiety or a qdot, can absorb energy from the evanescent wave and transfer it to a fluorescent acceptor on the nucleotide. In another example, such nucleotide incorporation results in a change in ionic concentration, such as the concentration of phosphate species or the concentration of hydronium or hydrogen ions.

[0034] In an example, the substrate 102 is formed of a material that can propagate or confine light. For example, the substrate 102 can be formed of a transparent material or includes a layer formed of the transparent material adjacent to the major surface 114. A transparent material or layer permits substantial propagation of

electromagnetic radiation having a wavelength of an excitation light provided to the transparent material or layer. Optionally, the layer or substrate 102 is transparent to frequencies of light associated with fluorescent emissions of fluorescent-labeled nucleotides. When fluorescent-labeled nucleotides are utilized for determining a sequence, light reflected within the light-confining layer or substrate 102 can form an evanescent wave region at the interface of the major surface 114 to excite the fluorescent labels on the nucleotide incorporated or hybridized to the nucleic acid strand 108. In such an example, the substrate 102 can further include detectors sensitive to electromagnetic radiation to detect fluorescent emissions. Alternatively, light associated with the fluorescent emissions can pass through a major surface 116 of the substrate 102 and can be collected and focused on a separate detector. In an example, the electromagnetic radiation detectors can include CMOS detectors or charge coupled devices (CCD).

[0035] In another example, the substrate 102 can include a set of ion detectors. When nucleotides are hybridized to the nucleic acid strand 108, a change in ion

concentration can be detected by the ion detector. In such an example, a plurality of identical nucleic acid strands can be disposed within the reaction volume 106, increasing the change in ion concentration and strengthening a signal resulting from detecting the ion concentration.

[0036] In the case of a transparent substrate 102 or transparent layer within the substrate 102, the transparent component can have a refractive index of at least 1.45, such as at least 1.5, or even at least 1.55. In addition, the substrate 102 can be transparent, for example, having a transmission in the visible light spectrum of at least 80%. In an example, the transparent substrate 102 or layer within the substrate 102 is formed of an oxide of a metal or semi-metal, such as silicon, indium, tin, titanium, aluminum, gallium, germanium, arsenic, or any combination thereof. For example, the substrate 102 or layer within the substrate can be formed of silicon dioxide, sapphire, quartz, indium tin oxide, or any combination thereof. In another example, the layer or substrate 102 can be formed of gold, silver, copper, aluminum, titanium, gallium arsenide, or germanium, or any combination thereof. In particular, the transparent layer or substrate 102 can be formed of an oxide of silicon.

[0037] The surface layer 104 is disposed over the substrate 102. In a particular example, the surface layer 104 is in direct contact with the substrate 102 without intervening layers. The surface layer 104 has a refractive index in the range of 1.30 to 1.40. For example, the refractive index of the surface layer 104 can be in a range of 1.32 to 1.40, such as a range of 1.32 to 1.38, a range of 1.32 to 1.37, a range of 1.32 to 1.36, a range of 1.33 to 1.36, or even a range of 1.33 to 135.

[0038] In an example, the surface layer 104 has a refractive index of similar value to that of a solution to fill reaction volumes within the surface layer 104. For example, the surface layer 104 can have an Refractive Matching Index, defined as 100 times the sum of negative one and a ratio of the refractive index of the surface layer 104 to the refractive index of the solution 118 (i.e., 100((RI_surface/RIsoiution) - 1), in a range of -2 to 4, such as a range of -2 to 3.5, a range of -1.5 to 3, a range of -1.5 to 2.5, a range of -1 to 2, or even a range of -1 to 1.5.

[0039] Further, the surface layer 104 can exhibit desirable transmission, such as for wavelengths of 150 nm to 900 nm or in the visible spectrum, of at least 80%. For example, the transmission can be at least 85%, such as at least 90%, at least 92%, at least 94%, or even at least 95%. In addition, the surface layer 104 exhibits a desirable Abbe number of at least 60. For example, the Abbe number of the surface layer 104 can be at least 75, such as at least 85, or even at least 90.

[0040] The surface layer can be formed of a polymeric material. For example, the polymeric material can include acrylic, fluoropolymer, ethylene vinyl acetate (EVA), or any combinations thereof. In an example, the polymeric material is a

fluoropolymer. An exemplary fluoropolymer includes polyvinylidene fluoride (PVDF), polyvinyl fluoride (PVF), fluorinated ethylene propylene (FEP) copolymer, ethylene chlorotrifluoroethylene (ECTFE) copolymer, a copolymer of

tetrafluoroethylene, hexafluoropropylene, and vinylidene fluoride (THV), a copolymer of tetrafluoroethylene and perfluoro methylvinylether (PFA or MFA), a fluoropolymer having a fluorinated oxolane in its backbone, perfluoroether, or any combination thereof. In particular, the fluoropolymer can be a fluoropolymer having fluorinated oxolane in its backbone, for example, Cytop. Further, the polymer coating can be amorphous, exhibiting little or no crystallinity.

[0041] In an example, the surface layer 104 forms reaction volumes 106 in the form of wells surrounded within the plane of the surface layer 104 by the surface layer 104. As illustrated in FIG. 2, a sequencing device 200 can include a plurality of wells, such as at least 10 wells, at least 100 wells, at least 1000 wells, or even at least 10,000 wells. In a particular example, the sequencing device 200 includes at least 100,000 wells or at least 1,000,000 wells. As illustrated in the pattern 202, the wells can be arranged in a row and column configuration. Alternatively, as illustrated in the pattern 204, the wells can be arranged in columns offset, permitting closer positioning of wells.

[0042] In an example, the wells have a characteristic diameter, defined as the square root of four times the cross-sectional area (A) divided by Pi (i.e., sqrt(4*A/7t)), of not greater than 2.5 μιη. For example, the well can have a characteristic diameter of not greater than 2 μιη , such as not greater than 1.5 μιη, not greater than 1.0 μιη, not greater than 0.8 μιη, not greater than 0.6 μιη, not greater than 0.4 μιη, not greater than 0.25 μιη, not greater than 0.2 μιη, or even not greater than 0. lum.

[0043] Further, the surface layer 104 can have a thickness of not greater than 10 μιη. For example, the thickness can be not greater than 5 μιη, not great than 3 μιη, not great than 1 μιη, not greater than 750 nm, not greater than 500 nm, or even not greater than 350 nm. In particular, the surface layer 104 has a thickness of at least 50 nm, such as at least 100 nm.

[0044] Depending on the detection method, the substrate can include a transparent layer. For example, as illustrated in FIG. 3, a sequencing device 300 includes a substrate 302 having a transparent layer 304 adjacent to a major surface 316 of the substrate. A surface layer 306 is disposed over the major surface 316 of the substrate 102 adjacent the transparent layer 304. The surface layer 306 can define a plurality of reaction volumes 310, such as wells. [0045] A light source 312 can provide excitation light 318 to the layer 304. In particular, the light source 312 can provide excitation light 318 at an angle to facilitate internal reflection, such as total internal reflection within the transparent layer 304. As a result, an evanescent wave zone 314 is formed. The surface layer 306 is thicker than the evanescent wave zone 314. When a fluorescent-labeled nucleotide enters the evanescent zone, the fluorescent label can be provided with energy to facilitate fluorescent emission. In particular, when a fluorescent-labeled nucleotide is incorporated or hybridized to the target nucleic acid strand, the fluorescent label of the labeled nucleotide spends sufficient time within the evanescent wave zone or in close proximity to a donor within the evanescent wave zone to provide fluorescent emissions.

[0046] In an example, the excitation light 318 can be coupled into the layer 304 using edge coupling method. As illustrated in FIG. 15, the excitation light 318 is projected toward the edge 1502 of the substrate at an incident angle. The light couples to the substrate and propagates inside the substrate layer 304 or the substrate.

[0047] In another example, the excitation light 318 can be coupled into the layer 304 through prisms. As illustrated in FIG. 16, a prism 1602 is attached at the substrate or the substrate layer 304. The light 318 is refracted through the prism 1602 and into the substrate or the substrate layer 304.

[0048] In a further example, the excitation light 318 can be coupled into the layer 304 through a set of gratings. As illustrated in FIG. 17, a set of gratings 1702 is fabricated on the substrate or the substrate layer 304. The excitation light 318 is incident on the grating 1702 at the incident angle and couples into the substrate or the substrate layer 304.

[0049] In a particular example, the substrate further includes detectors 308, such as optical or light sensing detectors. Such detectors 308 define regions where photons entering such regions excite the detector 308. In a particular example, each reaction volume is uniquely associated with a detection region of a detector 308. For example, each reaction volume 310 can be associated in a one-to-one relationship with a detector 308. [0050] While the detectors 308 are illustrated as being formed as part of the substrate 302, alternatively, the detectors 308 can be separate and the substrate 302 transparent. In such an example, fluorescent emissions emitted from within the reaction volumes 310 can be collected, filtered, and detected using a separate collection and detection apparatus.

[0051 ] In another example, the detection method relies on changes in ion

concentration. In such an example illustrated FIG. 4, a surface layer 404 is disposed over a substrate 402. Particles 410 including a plurality of identical nucleic acid strands 412 can be disposed within the reaction volumes 406 defined by the surface layer 404. In particular, each particle can include a core and plurality of identical nucleic acid strands 412 coupled to a core. The substrate 402 can include ion detectors 408. In a particular example, each reaction volume 406 is uniquely associated with an ion detector 408, such as in a one-to-one relationship. A reagent solution including a type of nucleotide is applied across the surface of the surface layer 404. Nucleotides can diffuse into the reaction volumes 406. When the correct type of nucleotide diffuses into the reaction volume 406, the nucleotides are hybridized to the nucleic acid strands 412, resulting in a change in ion concentration. For example, the change in ion concentration can be a change in phosphate concentration. In another example, the change in ion concentration can be a change in the concentration of hydrogen or hydronium ions, or a change in pH.

[0052] Such sequencing devices can be incorporated into a system for sequencing. An exemplary system 500 is illustrated in FIG. 5. For example, a chamber 502 can incorporate the sequencing device 504 and define a volume in contact with a surface layer of the sequencing device 504. Reagent vessels 508 can selectively provide reagent solutions to the volume disposed in contact with the surface layer of the sequencing device 504. Excess reagent solutions can flow to a waste container 510.

[0053] Depending upon the detection method, the system 500 can include a light source 512 to provide excitation light to the detection device 504. In particular, the light source 512 can provide excitation light at an angle to facilitate internal reflection within a transparent layer of the sequencing device 504. The light source 512 can be disposed at such an angle relative to the sequencing device 504 or a set of mirrors, lenses, gratings, or prisms can adjust the angle. [0054] Furthermore, the system 500 can include controllers or circuitry 514 to control reagent flow, excitation light, temperature, pressure, or any combination thereof. Computational circuitry can also be included to collect data from the sequencing device 504. While not illustrated, the system 500 can further include additional detectors or sensors, flow control devices, pressure control devices, temperature control devices, user interfaces, data input and output devices and ports, power supplies, or any combination thereof.

[0055] The sequencing devices can be formed by coating a substrate with a surface layer and forming reaction volumes within the surface layer. As illustrated in FIG. 6, the method 600 optionally includes forming detectors in a substrate, as illustrated at 602. The detectors can include multilayer semiconductor structures. For example, the detectors can include a set of transistors, resistors, capacitors, leads, or any combination thereof, formed in the substrate. In a particular example, the detectors form detection regions on a major surface of the substrate, such as regions sensitive to ions or light emissions. Such detection regions can be arranged in patterns, for example, corresponding to the patterns illustrated in FIG. 2.

[0056] The method 600 further includes coating the substrate with a surface layer, as illustrated at 604. Coating can include spray coating, dip coating, or spin coating, among other film depositing techniques. In particular, the surface layer includes a polymeric material, such as a fluoropolymer material having a refractive index in a range of 1.3 to 1.4.

[0057] As illustrated at 606, the coating can be patterned to form reaction volumes. For example, the coating can be masked and etched using a plasma etch technique or can be etched using chemical etch techniques. Alternatively, a laser can be used to form the reaction volumes.

[0058] Optionally, the etched surface coating can be treated, as illustrated 608. For example, the surface coating can be treated with a passivation agent. For example, the surface coating can be treated with a BSA/Tween 20/Wax solution, an amino PEG solution, a fluorosurfactant, PEG grafting, or any combination thereof.

[0059] In a particular example illustrated in FIG. 18A-E, a refractive index matching surface layer 1804, for example, a fluoropolymer having a fluorinated oxolane in its backbone, can be deposited to be in contact with the substrate 1802 using spray coating, dip coating, spin coating or another thin film deposition technique, as illustrated in FIG. 18A. A resist layer 1806 can be coated on the surface layer, as illustrated at FIG. 18B. The resist layer 1806 can be exposed and developed to form patterns, like wells or channels, as illustrated at FIG. 18C. For example, the patterns can be formed using photolithography or electrical beam lithography. As illustrated at FIG. 18D, reactive ion etching transfers the patterns into the surface layer 1804. The resist 1806 can be stripped using chemical solvent, as illustrated at FIG. 18E. Additional processing can follow such as surface layer passivation.

[0060] In a further example illustrated at FIG. 19A-F, a surface layer 1904 can be deposited on a substrate 1902, for example, using spray coating, dip coating, or spin coating or another thin film deposition techniques, as illustrated in FIG. 19A. As illustrated at FIG. 19B, a metal layer 1906 is deposited using e-beam evaporation or sputtering, for example, a lOnm -20nm aluminum layer, to function as an etching mask layer. A resist layer 1908 can be coated on the metal layer 1906, as illustrated at FIG. 19C, and the resist 1908 can be exposed and developed to form patterns, like wells or channels, as illustrated at FIG. 19D. The patterns can be formed using photolithography or electrical beam lithography. Reactive ion etching transfers the patterns into the surface layer 1904, as illustrated at FIG. 19E. Both of the resist 1908 and the metal layer 1906 can be stripped using chemical solution, as illustrated at FIG. 19F.

[0061] In another example illustrated in FIG. 20A-F, a surface layer 2004 is deposited on a substrate 2002, using spray coating, dip coating, spin coating or another thin film deposition technique, as illustrated at FIG. 20A. A resist layer 2006 can be coated on the surface layer 2004, as illustrated at FIG. 20B, and the resist 2006 can be exposed and developed to form negative patterns, like posts or pillars, as illustrated at FIG. 20C. The patterns can be formed by photolithography or electrical beam lithography. A metal layer 2008 can be deposited using e-beam evaporation or sputtering, for example, a lOnm -20nm aluminum layer, as illustrated at FIG. 20D. After lift off, the metal wells or channels are formed at the metal layer 2008. Reactive ion etching transfers the patterns into the surface layer 2004, as illustrated at FIG. 20E. The metal layer 2008 can be stripped using chemical solution, as illustrated at FIG. 20F. [0062] Using such techniques, physical structures, for example, wells or channels, can be formed in the surface layer. FIG. 21 and FIG. 22 include scanning electron microscopy images of well structures inside the surface layer.

[0063] The method of operation depends on the detection technique. Using a fluorescent detection technique, the labeled nucleotides can be included in a reagent solution that flows across the surface of the sequencing device. Fluorescent emissions resulting from the incorporation of nucleotides to a nucleic acid strand can be detected. As illustrated in the method 700 of FIG. 7, nucleic acid strands can be applied to the surface of the sequencing device, as illustrated at 702. In particular, the nucleic acid strands are disposed within the reaction volumes defined by the surface layer of the sequencing device. In an example, the nucleic acid strands can be immobilized to a surface within the reaction volume. For example, the nucleic acid strand can be immobilized using an antibiotin-biotin mechanism. The surface can include a biotin-active compound, such as an avidin or derivative thereof, such as streptavidin that binds with biotin-terminated nucleic acid strands. In another example, the surface can include a coating of a nucleic acid binding polymer, such as propylene glycol, dextran, chitosan, or combinations thereof. In a further example, the surface can have a surface incorporating bromoacetyl groups or thiol groups. Such groups can bind with thiol-derivatized oligonucleotides or bromoacetyl- derivatized oligonucleotides, respectively. In an additional example, the surface can bind to a nucleic acid strand using click chemistry, such as an azide interacting with complementary alkylene functionality. In another example, the nucleic acid strands can be immobilized by attaching the strands to bound oligonucleotides that include a section complementary to a terminal end of the target nucleic acid strand. Optionally, a bottom of the reaction volume can be coated with a thin metal layer, such as zinc, to enhance bonding.

[0064] Further, an enzyme can be applied to the surface of the sequencing device, as illustrated 704. The enzyme can be free floating and free to diffuse in and out of the reaction volume. In an alternative example, the enzyme can be tethered or immobilized to the surface of the substrate within the reaction volume. In particular, the enzyme can be immobilized using a technique similar to those described above. Optionally, the nucleic acid strand can be free-floating when the enzyme is immobilized. In a further example, an energy donor can be attached to the enzyme or immobilized on the substrate to provide energy to fluorescent nucleotides.

[0065] As illustrated 706, excitation light is directed at a substrate layer within the sequencing device. In particular, the excitation light is directed from a light source at an angle that provides for internal reflection within the substrate layer, such as total internal reflection within the substrate layer. In particular, the internal reflection results in an evanescent wave zone within a portion of the reaction volume. The thickness of the surface layer is greater than the thickness of the evanescent wave zone.

[0066] A reagent solution including fluorescent-labeled nucleotides can be applied in a flow volume over the reaction volumes, as illustrated at 708. For example, the reagent solution can include a plurality of types of nucleotides. Each type of labeled nucleotide can have a fluorescent label that emits at a uniquely recognizable signature or wavelength. The reagent solution can further include enzymes, cations, and buffering agents to assist with nucleotide hybridization. When a fluorescent-labeled nucleotide, such as a dye-labeled nucleotide, is incorporated or hybridize to the nucleic acid strand within the evanescent zone and the reaction volume, the fluorescent-label of the nucleotide can fluoresce, providing an emission measurable by a detector.

[0067] In an example, the fluorescent emission is detected with a detector, as illustrated at 710. In a particular example, the detectors within the substrate are positioned in a one-to-one relationship with the reaction volume. In an alternative example, the detection system is separate from the substrate. The substrate is transparent within the emission spectrum and a separate collection/detection device detects the fluorescent emissions.

[0068] In an alternative method 800 illustrated in FIG. 8, an ion detector is utilized to detect nucleotide incorporation. As illustrated at 802, particles including a plurality of identical nucleic acid strands are applied to the surface layer of the sequencing device. In particular, the particles are disposed within the reaction volumes defined by a surface layer of the sequencing device. Each reaction volume is associated with a detector, such as in a one-to-one relationship. Optionally, enzymes can be incorporated into the particles or supplied separately, such as in a reagent solution.

[0069] As illustrated at 804, a reagent solution including a single type of nucleotide is applied to the surface of the sequencing device. When the single type of nucleotide is incorporated or hybridized to a nucleic acid strand within the reaction volume, the concentration of ions changes.

[0070] As illustrated at 806, a detector can detect the change in ion concentration, resulting in the detection of the nucleotide incorporation. The reagent solution can be washed from the surface, as illustrated at 808. Repeating 804-808, reagent solutions, each incorporating a different type of nucleotide, can be sequentially applied to the surface of the detection device, followed by washing. As the nucleotides are incorporated into or hybridize to the nucleic acid strands attached to particles in the reaction volumes, the sequence can be determined by measuring the ionic concentration response to the specific nucleotides included in the reagent solution.

[0071] In alternative embodiments, the reaction volumes can include channels. As illustrated in FIG. 9, a sequencing device 900 can include a substrate 902 and a surface coating layer 904 defined over the substrate 902. In particular, the surface coating layer 904 defines channels 906. Nucleic acid strands 908 can be immobilized within the channels 906. One or multiple polymerases 910 can be deposited on the strand of nucleic acid. Reagent solutions flow through the channels 906. In particular, reagent solutions can be applied to flow parallel to the channels 906. Alternatively, the reagent solutions can be applied to flow perpendicular to the channels 906.

[0072] In an alternative example 1000 illustrated in FIG. 10, a substrate 1002 is coated with a surface layer 1004 defining channels 1008. A further layer 1006 can be applied over the surface layer 1004, capping the channels 1008. Nucleic acid strands 1010 or polymerases 1012 can be immobilized within the channels 1008. Reagent solutions can flow through the enclosed channels 1008.

[0073] In particular, the sequencing device including a surface coating as described above exhibits a desirable reduction in background noise. In particular, a Noise Index, defined as the ratio of the noise of the device incorporating the surface coating relative to the noise of a device free of the surface coating, is not greater than 0.8. For example, Noise Index can be not greater than 0.6, such as not greater than 0.4, or not greater than 0.3.

[0074] Further, such devices exhibit a desirable signal-to-background noise ratio of at least 2.0, such as at least 2.5, at least 3.0, at least 5.0, at least 7.5 or even at least 10.0. Such signal-to-background noise ratios indicate great improvement over other systems. In particular, a SBR Index, defined as the ratio of the signal-to-background noise ratio of a device using the above-described surface coating relative to the signal- to-background noise ratio of a device free of the above surface coating, is at least 1.4, such as at least 1.8, at least 2.2, at least 2.6, or even at least 3.

[0075] Particularly when used with fluorescent emission detection techniques, it is been found that the above-described surface coating despite its transparency provides improved signal-to-background noise ratios and reduced noise overall. Contrary to what is expected, a transparent coating having the specified refractive index provides for an enhanced evanescent zone, leading to a signal-to-noise ratio that is significant improved. Similar improvements are observed when using other detection techniques.

[0076] It is believed that when the substrate has a greater refractive index than the refractive indices of the background solution and the surface layer, the propagating light is reflected by back and forth at the interface of the substrate and the surface layer when the incident angle is beyond the critical angle. An electrical field at the interface is diffused and forms an evanescence wave. The evanescence wave exponentially decays along an axis perpendicular to the surface layer and forms a confined excitation volume for sequencing reactions. When the refractive index of the surface layer approximates that of the background solution, the presence of the physical structures made inside the surface layer does not disturb the evanescence wave at the interface, and such physical structures, for example, wells or channels, further reduce the excitation volume of sequencing reactions. Accordingly, the ratio of signal and noise of sequencing reactions is improved.

[0077] EXAMPLE 1

[0078] A simulation is performed for a coated substrate including a 250 nm transparent layer coated with fluoropolymer surface layer having a refractive index of 1.34. The surface layer is 300 nm thick and includes wells having a diameter of 200 nm. Excitation light is projected to the transparent layer at an angle sufficient to cause total internal reflection. The strength of a resulting evanescent electrical field is determined.

[0079] When using water and a surface layer above the substrate, the evanescent wave is not disturbed even in the presence of the confined structures. As illustrated in FIG. 11, the strength of the electrical field is strongest within less than 100 nm from the surface of the transparent layer and weakens to near zero at thickness greater than 250 nm, even in the presence of the surface layer, depicted as broken lines.

[0080] As illustrated in FIG. 12, a similar simulation illustrates the strength of the electric field is greatest within 100 nm of the surface for structures formed of Cytop. When an additional reflective metal layer is disposed on the surface of the Cytop layer, as illustrated in FIG. 13, the field is disturbed and extends beyond the thickness of the Cytop layer. Such a disturbed field would likely produce unwanted background fluorescence.

[0081] EXAMPLE 2

[0082] Single molecule real time sequencing is performed on fused silica substrates with 300nm diameter and 300nm deep Cytop wells. A Microsurface Bio-01 fused silica slide is used as a control. The sequencing polymerase conjugate is SL333 (H370R-T424V). The sequencing polymerase is conjugated with a Qdot that is functioned as energy donor to excite dye molecules during incorporation through fluorescence resonance energy transfer (FRET). In this sequencing experiment, the DNA template is 603. The laser excitation wavelength is 445nm and the power density is 40 - 50W/cm². The sequencing protocol includes:

1. Conjugate 50nM : Primed Template 50nM (primer/template = 1.1) in oxygen scavenging system (OSS) (IX GoKat @ pH = 6.8) for 10 mins at 30°C, with 250 nM single strain bounding protein (SSB).

2. Incubate with TBST+BSA /wax for more than 20 mins incubation.

3. Incubate streptavidin at 5nM for 20 mins incubation. 4. Inject conjugate complex at 4 nM just long enough to get enough density

5. Wash with OSS (2.5X GoKat @ pH = 7.4)

6. Inject reaction mix and image: standard dye set 5 @ 1 uM for each dye

nucleotide Dye Set 5 a (codA6P-AM634, 260 nM; codG6P-AM660, 360 nM; codT6P-AM705, 500 nM; codC6P-AM750, 320 nM)

[0083] The measured donor intensity inside Cytop nanowells is similar to the donor intensity on the control microsurface Bio-01, which indicates the Cytop structures do not disturb the evanescent wave at the interface, as illustrated in FIG. 23 and FIG. 24. The noise background is reduced for different wavelength detection dye signal. The 96 base long template 603 is finished, as illustrated in the trace of FIG. 25. The 300nm Cytop nanowell can improve the signal noise ratio by ~2 times. Smaller Cytop nanowells are expected to improve the signal noise ratio by at the least 3 times.

[0084] The amplitude of noise is measured for each of four labeled nucleotides on the samples. As illustrated in Table 1, the noise is reduced relative to a blank substrate for each of the nucleotides.

[0085] TABLE 1. Noise Reduction for Various Labeled Nucleotides

[0086] EXAMPLE 3

[0087] Samples are prepared having wells of different diameter. Background noise is measured and compared to a blank substrate free of the coating. As illustrated in FIG. 14, the coating provides a Noise Index, defined as the ratio of the background noise on substrates with nanowells relative to that of blank substrates, of less than 0.8 for wells of diameter less than 600 nm. In particular, significant improvement is illustrated for wells of diameter less than 400 nm.

[0088] In a first aspect, a device includes a substrate having a refractive index of at least 1.45 and a surface layer disposed over a major surface of the substrate and defining a plurality of reaction volumes. The surface layer has a refractive index in a range of 1.32 to 1.38.

[0089] In an example of the first aspect, the substrate has transmission of at least 80% in the visible light spectrum. In another example of the first aspect and the above example, the refractive index of the surface layer is in a range of 1.32 to 1.37. For example, the refractive index of the surface layer can be in a range of 1.32 to 1.36. Further, the refractive index of the surface layer can be in a range of 1.33 to 1.36.

[0090] In an additional example of the first aspect and the above examples, the surface layer has a Refractive Matching Index in a range of -2 to 4. For example, the Refractive Matching Index can be in a range of -2 to 3.5. In another example, the Refractive Matching Index is in a range of -1 to 1.5.

[0091] In another example of the first aspect and the above examples, the surface layer has a transmission in the visible light spectrum of at least 80%. For example, the transmission can be at least 85%, such as at least 90%, at least 94%.

[0092] In a further example of the first aspect and the above examples, the surface layer exhibits an Abbe Number of at least 60. For example, the Abbe Number can be at least 75, such as at least 89.

[0093] In an additional example of the first aspect and the above examples, the surface layer includes a polymeric material selected from the group consisting of acrylic, fluoropolymer, ethylene vinyl acetate, or a combination thereof. For example, the surface layer can include fluoropolymer. In an example, the fluoropolymer is selected from the group consisting of polyvinylidene fluoride, polyvinyl fluoride, fluorinated ethylene propylene (FEP) copolymer, ethylene chlorotrifluoroethylene (ECTFE) copolymer, a copolymer of tetrafluoroethylene, hexafluoropropylene, and vinylidene fluoride, a copolymer of tetrafluoroethylene and perfluoro methylvinylether, a fluoropolymer having fluorinated oxolane in its backbone, perfluoroether polymer, or a combination thereof. In a particular example, the fluoropolymer is a fluoropolymer having fluorinated oxolane in its backbone. For example, the fluoropolymer having fluorinated oxolane in its backbone can include a carboxylic terminal group. In another example, the fluoropolymer having fluorinated oxolane in its backbone can include a siloxane terminal group. In a further example of the first aspect and the above examples, the surface layer is amorphous.

[0094] In another example of the first aspect and the above examples, the reaction volume forms a well having a characteristic diameter of not greater than 2 micrometers. For example, the characteristic diameter is not greater than 1.0 micrometers, such as not greater than 0.7 micrometers, not greater than 0.4 micrometers, or not greater than 0.25 micrometers.

[0095] In a further example of the first aspect and the above examples, the surface layer has a thickness of not greater than 5.0 micrometers. For example, the thickness can be not greater than 750 nm, such as not greater than 500 nm, or not greater than 350 nm.

[0096] In an additional example of the first aspect and the above examples, the device exhibits a Noise Index of not greater than 0.8. For example, the Noise Index can be not greater than 0.6, such as not greater than 0.3.

[0097] In another example of the first aspect and the above examples, the device exhibits a signal-to-background noise ratio of at least 2. In an additional example of the first aspect and the above examples, the device exhibits an SBR Index of at least 1.4. For example, the SBR Index can be at least 1.8, such as at least 2.2.

[0098] In a second aspect, a system includes a device comprising a substrate having a refractive index of at least 1.45 and a surface layer disposed over a major surface of the substrate and defining a plurality of reaction volumes. The surface layer has a refractive index in a range of 1.32 to 1.38. The system further includes a chamber defining a volume over the surface layer of the device and a light source to direct excitation light to the transparent substrate. [0099] In an example of the second aspect, the light source is to direct the excitation light to the transparent substrate at an angle to facilitate internal reflection within the transparent substrate.

[00100] In another example of the second aspect and the above example, the substrate has transmission of at least 80% in the visible light spectrum.

[00101] In a further example of the second aspect and the above examples, the refractive index of the surface layer is in a range of 1.32 to 1.36. In an additional example of the second aspect and the above examples, the surface layer has a

Refractive Matching Index in a range of -2 to 4. In a particular example of the second aspect and the above examples, the surface layer has a transmission in the visible light spectrum of at least 80%. In another example of the second aspect and the above examples, the surface layer exhibits an Abbe Number of at least 60.

[00102] In an additional example of the second aspect and the above examples, the surface layer can include a polymeric material selected from the group consisting of acrylic, fluoropolymer, ethylene vinyl acetate, or a combination thereof. For example, the surface layer can include fluoropolymer. In an example, the fluoropolymer is selected from the group consisting of polyvinylidene fluoride, polyvinyl fluoride, fluorinated ethylene propylene (FEP) copolymer, ethylene chlorotrifluoroethylene (ECTFE) copolymer, a copolymer of tetrafluoroethylene, hexafluoropropylene, and vinylidene fluoride, a copolymer of tetrafluoroethylene and perfluoro

methylvinylether, a fluoropolymer having fluorinated oxolane in its backbone, perfluoroether polymer, or a combination thereof. In a particular example, the fluoropolymer is a fluoropolymer having fluorinated oxolane in its backbone. In a further example, the surface layer is amorphous.

[00103] In another example of the second aspect and the above examples, the reaction volume forms a well having a characteristic diameter of not greater than 2

micrometers. In an additional example of the second aspect and the above examples, the surface layer has a thickness of not greater than 1.0 micrometers.

[00104] In a further example of the second aspect and the above examples, the device exhibits a Noise Index of not greater than 0.8. In an addition example of the second aspect and the above examples, the device exhibits a signal-to-background noise ratio of at least 2. In another example of the second aspect and the above examples, the device exhibits an SBR Index of at least 1.4.

[00105] In a third aspect, a method of sequencing a nucleic acid strand includes applying the nucleic acid strand into a reaction volume of a device. The device includes a substrate having a refractive index of at least 1.45 and a surface layer disposed over a major surface of the substrate and defining a plurality of reaction volumes. The surface layer has a refractive index in a range of 1.32 to 1.38. The method further includes flowing a reagent solution over the surface layer of the device. The reagent solution includes nucleotides of a set of nucleotide types. Each type of nucleotide is labeled to provide a unique fluorescence signal. The method also includes directing excitation light to the transparent substrate and detecting fluorescence signals associated with nucleotide incorporation onto the nucleic acid strand.

[00106] In an example of the third aspect, directing the excitation light includes directing the excitation light at an angle to facilitate internal reflection within the transparent substrate.

[00107] In a fourth aspect, a device includes a substrate having a major surface and comprising a plurality of detectors and a surface layer disposed over the major surface. The surface layer defines a plurality of reaction volumes. Each reaction volume of the plurality of reaction volumes is uniquely associated with a detector of the plurality of detectors. The surface layer has an index of refraction in a range of 1.32 to 1.38.

[00108] In an example of the fourth aspect, the plurality of detectors are to detect electromagnetic radiation. In another example of the fourth aspect and the above example, the substrate further includes a transparent layer having a transmission of at least 80% in the visible light spectrum.

[00109] In an additional example of the fourth aspect and the above examples, the plurality of detectors are to detect ions. For example, the ions can be hydronium or hydrogen ions. [00110] In a further example of the fourth aspect and the above examples, the refractive index of the surface layer is in a range of 1.32 to 1.36. In an additional example of the fourth aspect and the above examples, the surface layer has a

Refractive Matching Index in a range of -2 to 4. In another example of the fourth aspect and the above examples, the surface layer has a transmission in the visible light spectrum of at least 80%. In a particular example of the fourth aspect and the above examples, the surface layer exhibits an Abbe Number of at least 60.

[00111] In another example of the fourth aspect and the above examples, the surface layer comprises a polymeric material selected from the group consisting of acrylic, fluoropolymer, ethylene vinyl acetate, or a combination thereof. For example, the surface layer can be fluoropolymer. In a particular example, the fluoropolymer is selected from the group consisting of polyvinylidene fluoride, polyvinyl fluoride, fluorinated ethylene propylene (FEP) copolymer, ethylene chlorotrifluoroethylene (ECTFE) copolymer, a copolymer of tetrafluoroethylene, hexafluoropropylene, and vinylidene fluoride, a copolymer of tetrafluoroethylene and perfluoro

methylvinylether, a fluoropolymer having fluorinated oxolane in its backbone, perfluoroether polymer, or a combination thereof. For example, the fluoropolymer is a fluoropolymer having fluorinated oxolane in its backbone. In a further example, the surface layer is amorphous.

[00112] In an additional example of the fourth aspect and the above examples, the reaction volume forms a well having a characteristic diameter of not greater than 2 micrometers. In another example of the fourth aspect and the above examples, the surface layer has a thickness of not greater than 1.0 micrometers.

[00113] In a further example, the device exhibits a Noise Index of not greater than 0.8. In another example of the fourth aspect and the above examples, the device exhibits a signal-to-background noise ratio of at least 2. In an additional example of the fourth aspect and the above examples, the device exhibits an SBR Index of at least 1.4.

[00114] In a fifth aspect, a system includes a device including a substrate having a major surface and comprising a plurality of detectors and a surface layer disposed over the major surface. The surface layer defines a plurality of reaction volumes. Each reaction volume of the plurality of reaction volumes is uniquely associated with a detector of the plurality of detectors. The surface layer has an index of refraction in a range of 1.32 to 1.38. The system also includes a chamber to receive the device and define a volume over the surface layer.

[00115] In an example of the fifth aspect, the plurality of detectors are to detect electromagnetic radiation.

[00116] In another example of the fifth aspect or the above example, the substrate further includes a transparent layer having a transmission of at least 80% in the visible light spectrum. In an additional example of the fifth aspect or the above examples, the system further includes a light source to direct excitation light at the transparent layer.

[00117] In a further example of the fifth aspect or the above examples, the plurality of detectors are to detect ions. For example, the ions are hydronium or hydrogen ions.

[00118] In another example of the fifth aspect or the above examples, the refractive index of the surface layer is in a range of 1.32 to 1.36. In a further example of the fifth aspect or the above examples, the surface layer has a Refractive Matching Index in a range of -2 to 4. In an additional example of the fifth aspect or the above examples, the surface layer has a transmission in the visible light spectrum of at least 80%. In a particular example of the fifth aspect or the above examples, the surface layer exhibits an Abbe Number of at least 60.

[00119] In addition example of the fifth aspect or the above examples, the surface layer comprises a polymeric material selected from the group consisting of acrylic, fluoropolymer, ethylene vinyl acetate, or a combination thereof. For example, the surface layer comprises fluoropolymer. In a further example, the fluoropolymer is selected from the group consisting of polyvinylidene fluoride, polyvinyl fluoride, fluorinated ethylene propylene (FEP) copolymer, ethylene chlorotrifluoroethylene (ECTFE) copolymer, a copolymer of tetrafluoroethylene, hexafluoropropylene, and vinylidene fluoride, a copolymer of tetrafluoroethylene and perfluoro

methylvinylether, a fluoropolymer having fluorinated oxolane in its backbone, perfluoroether polymer, or a combination thereof. For example, the fluoropolymer is a fluoropolymer having fluorinated oxolane in its backbone. In a particular example, the surface layer is amorphous. [00120] In another example of the fifth aspect or the above examples, the reaction volume forms a well having a characteristic diameter of not greater than 2 micrometers. In an additional example of the fifth aspect or the above examples, the surface layer has a thickness of not greater than 1.0 micrometers.

[00121] In a further example of the fifth aspect or the above examples, the device exhibits a Noise Index of not greater than 0.8. In another example of the fifth aspect or the above examples, the device exhibits a signal-to-background noise ratio of at least 2. In an additional example of the fifth aspect or the above examples, the device exhibits an SBR Index of at least 1.4.

[00122] In a sixth aspect, a method of sequencing a nucleic acid strand includes applying the nucleic acid strand into a reaction volume of a device. The device includes a substrate having a major surface and comprising a plurality of detectors and a surface layer disposed over the major surface. The surface layer defines a plurality of reaction volumes. Each reaction volume of the plurality of reaction volumes is uniquely associated with a detector of the plurality of detectors. The surface layer has an index of refraction in a range of 1.32 to 1.38. The method further includes flowing a reagent solution over the surface layer of the device. The reagent solution includes nucleotides of a set of nucleotide types. Each type of nucleotide is labeled to provide a unique fluorescence signal. The method also includes detecting fluorescent signals associated with nucleotide incorporation onto the nucleic acid strand.

[00123] In an example of the sixth aspect, the method further includes applying an enzyme to the reaction volume of the device.

[00124] In a seventh aspect, a method of sequencing a nucleic acid strand includes applying the nucleic acid strand into a reaction volume of a device. The device includes a substrate having a major surface and comprising a plurality of detectors and a surface layer disposed over the major surface. The surface layer defines a plurality of reaction volumes. Each reaction volume of the plurality of reaction volumes is uniquely associated with a detector of the plurality of detectors. The surface layer has an index of refraction in a range of 1.32 to 1.38. The method further includes sequentially flowing reagent solutions over the surface layer of the device. Each of the reagent solutions includes a type of nucleotide of a set of nucleotide types. The method further includes detecting signals associated with nucleotide

incorporation onto the nucleic acid strand.

[00125] In an example of the seventh aspect, the method further includes applying an enzyme to the reaction volume of the device.

[00126] In an eighth aspect, a method of forming a device includes coating a substrate with a polymeric coating having a refractive index in a range of 1.32 to 1.38. The substrate is transparent and has a refractive index of at least 1.45. The method further includes etching the polymeric coating to define a plurality of reaction volumes. The plurality of reaction volumes includes at least 100 reaction volumes.

[00127] In an example of the eighth aspect, the method further includes treating the etched polymer coating with a passivation agent. In an additional example of the eighth aspect and the above example, the method further includes forming a substrate with a plurality of detectors.

[00128] In a ninth aspect, a method of forming a device includes coating a major surface of a substrate with a polymeric coating having a refractive index in a range of 1.30 to 1.40. The substrate includes a plurality of detectors. The method further includes etching the polymeric coating to define a plurality of reaction volumes. Each reaction volume of the plurality of reaction volumes is uniquely associated with a detector of the plurality of detectors.

[00129] In an example of the ninth, the method further includes treating the etched polymer coating.

[00130] Note that not all of the activities described above in the general description or the examples are required, that a portion of a specific activity may not be required, and that one or more further activities may be performed in addition to those described. Still further, the order in which activities are listed are not necessarily the order in which they are performed.

[00131] In the foregoing specification, the concepts have been described with reference to specific embodiments. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the invention as set forth in the claims below. Accordingly, the specification and figures are to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of invention.

[00132] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of features is not necessarily limited only to those features but may include other features not expressly listed or inherent to such process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive-or and not to an exclusive-or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[00133] Also, the use of "a" or "an" are employed to describe elements and components described herein. This is done merely for convenience and to give a general sense of the scope of the invention. This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise.

[00134] Benefits, other advantages, and solutions to problems have been described above with regard to specific embodiments. However, the benefits, advantages, solutions to problems, and any feature(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature of any or all the claims.

[00135] After reading the specification, skilled artisans will appreciate that certain features are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination. Further, references to values stated in ranges include each and every value within that range.

Claims

WHAT IS CLAIMED IS:

1. A device comprising:

a substrate having a refractive index of at least 1.45; and

a surface layer disposed over a major surface of the substrate and defining a plurality of reaction volumes, the surface layer having a refractive index in a range of 1.32 to 1.38.

2. The device of claim 1, wherein the substrate has transmission of at least 80% in the visible light spectrum.

3. The device of claim 1, wherein the refractive index of the surface layer is in a range of 1.32 to 1.37.

4. The device of claim 3, wherein the refractive index of the surface layer is in a range of 1.32 to 1.36.

5. The device of claim 4, wherein the refractive index of the surface layer is in a range of 1.33 to 1.36.

6. The device of any one of claims 1-3, wherein the surface layer has a Refractive Matching Index in a range of -2 to 4.

7. The device of claim 6, wherein the Refractive Matching Index is in a range of -2 to 3.5.

8. The device of claim 7, wherein the Refractive Matching Index is in a range of -1 to 1.5.

9. The device of any one of claims 1-3, wherein the surface layer has a transmission in the visible light spectrum of at least 80%.

10. The device of claim 9, wherein the transmission is at least 85%.

11. The device of claim 10, wherein the transmission is at least 90%.

12. The device of claim 1 1, wherein the transmission is at least 94%.

13. The device of any one of claims 1-3, wherein the surface layer exhibits an Abbe Number of at least 60.

14. The device of claim 13, wherein the Abbe Number is at least 75.

15. The device of claim 14, wherein the Abbe Number is at least 89.

16. The device of any one of claims 1-3, wherein the surface layer comprises a polymeric material selected from the group consisting of acrylic, fluoropolymer, ethylene vinyl acetate, or a combination thereof.

17. The device of claim 16, wherein the surface layer comprises

fluoropolymer.

18. The device of claim 17, wherein the fluoropolymer is selected from the group consisting of polyvinylidene fluoride, polyvinyl fluoride, fluorinated ethylene propylene (FEP) copolymer, ethylene chlorotrifluoroethylene (ECTFE) copolymer, a copolymer of tetrafluoroethylene, hexafluoropropylene, and vinylidene fluoride, a copolymer of tetrafluoroethylene and perfluoro methylvinylether, a fluoropolymer having fluorinated oxolane in its backbone, perfluoroether polymer, or a combination thereof.

19. The device of claim 18, wherein the fluoropolymer is a fluoropolymer having fluorinated oxolane in its backbone.

20. The device of claim 19, wherein the fluoropolymer having fluorinated oxolane in its backbone includes a carboxylic terminal group.

21. The device of claim 19, wherein the fluoropolymer having fluorinated oxolane in its backbone includes a siloxane terminal group.

22. The device of any one of claims 1-3, wherein the surface layer is amorphous.

23. The device of any one of claims 1-3, wherein the reaction volume forms a well having a characteristic diameter of not greater than 2 micrometers.

24. The device of claim 23, wherein the characteristic diameter is not greater than 1.0 micrometers.

25. The device of claim 24, wherein the characteristic diameter is not greater than 0.7 micrometers.

26. The device of claim 25, wherein the characteristic diameter is not greater than 0.4 micrometers.

27. The device of claim 26, wherein the characteristic diameter is not greater than 0.25 micrometers.

28. The device of any one of claims 1-3, wherein the surface layer has a thickness of not greater than 5.0 micrometers.

29. The device of claim 28, wherein the thickness is not greater than 750 nm.

30. The device of claim 29, wherein the thickness is not greater than 500 nm.

31. The device of claim 30, wherein the thickness is not greater than 350 nm.

32. The device of any one of claims 1-3, wherein the device exhibits a Noise Index of not greater than 0.8.

33. The device of any one of claims 1-3, wherein the Noise Index is not greater than 0.6.

34. The device of claim 33, wherein the Noise Index is not greater than 0.3.

35. The device of any one of claims 1-3, wherein the device exhibits a signal- to-background noise ratio of at least 2.

36. The device of any one of claims 1-3, wherein the device exhibits an SBR Index of at least 1.4.

37. The device of claim 36, wherein the SBR Index is at least 1.8.

38. The device of claim 37, wherein the SBR Index is at least 2.2.

39. A system comprising:

a device comprising:

a substrate having a refractive index of at least 1.45; and

a surface layer disposed over a major surface of the substrate and defining a plurality of reaction volumes, the surface layer having a refractive index in a range of 1.32 to 1.38;

a chamber defining a volume over the surface layer of the device; and a light source to direct excitation light to the substrate.

40. The system of claim 39, wherein the light source is to direct the excitation light to the substrate at an angle to facilitate internal reflection within the substrate.

41. The system of claim 39, wherein the substrate has transmission of at least 80% in the visible light spectrum.

42. The system of claim 39, wherein the refractive index of the surface layer is in a range of 1.32 to 1.36.

43. The system of any one of claims 39-42, wherein the surface layer has a Refractive Matching Index in a range of -2 to 4.

44. The system of any one of claims 39-42, wherein the surface layer has a transmission in the visible light spectrum of at least 80%.

45. The system of any one of claims 39-42, wherein the surface layer exhibits an Abbe Number of at least 60.

46. The system of any one of claims 39-42, wherein the surface layer comprises a polymeric material selected from the group consisting of acrylic, fluoropolymer, ethylene vinyl acetate, or a combination thereof.

47. The system of claim 46, wherein the surface layer comprises fluoropolymer.

48. The system of claim 47, wherein the fluoropolymer is selected from the group consisting of polyvinylidene fluoride, polyvinyl fluoride, fluorinated ethylene propylene (FEP) copolymer, ethylene chlorotrifluoroethylene (ECTFE) copolymer, a copolymer of tetrafluoroethylene, hexafluoropropylene, and vinylidene fluoride, a copolymer of tetrafluoroethylene and perfluoro methylvinylether, a fluoropolymer having fluorinated oxolane in its backbone, perfluoroether polymer, or a combination thereof.

49. The system of claim 48, wherein the fluoropolymer is a fluoropolymer having fluorinated oxolane in its backbone.

50. The system of any one of claims 39-42, wherein the surface layer is amorphous.

51. The system of any one of claims 39-42, wherein the reaction volume forms a well having a characteristic diameter of not greater than 2 micrometers.

52. The system of any one of claims 39-42, wherein the surface layer has a thickness of not greater than 1.0 micrometers.

53. The system of any one of claims 39-42, wherein the device exhibits a Noise Index of not greater than 0.8.

54. The system of any one of claims 39-42, wherein the device exhibits a signal-to-background noise ratio of at least 2.

55. The system of any one of claims 39-42, wherein the device exhibits an SBR Index of at least 1.4.

56. A method of sequencing a nucleic acid strand, the method comprising: applying the nucleic acid strand into a reaction volume of a device, the device comprising:

a substrate having a refractive index of at least 1.45; and

flowing a reagent solution over the surface layer of the device, the reagent solution including nucleotides of a set of nucleotide types, each type of nucleotide labeled to provide a unique fluorescence signal;

directing excitation light to the substrate; and

detecting fluorescent signals associated with nucleotide incorporation onto the nucleic acid strand.

57. The method of claim 56, wherein directing the excitation light includes directing the excitation light at an angle to facilitate internal reflection within the substrate.

58. A device comprising:

a substrate having a major surface and comprising a plurality of detectors; and a surface layer disposed over the major surface, the surface layer defining a plurality of reaction volumes, each reaction volume of the plurality of reaction volumes uniquely associated with a detector of the plurality of detectors, the surface layer having an index of refraction in a range of 1.32 to 1.38.

59. The device of claim 58, wherein the plurality of detectors are to detect electromagnetic radiation.

60. The device of claim 59, wherein the substrate further includes a transparent layer having a transmission of at least 80% in the visible light spectrum.

61. The device of claim 58, wherein the plurality of detectors are to detect ions.

62. The device of claim 61, wherein the ions are hydronium or hydrogen ions.

63. The device of any one of claims 58-62, wherein the refractive index of the surface layer is in a range of 1.32 to 1.36.

64. The system of any one of claims 58-62, wherein the surface layer has a Refractive Matching Index in a range of -2 to 4.

65. The device of any one of claims 58-62, wherein the surface layer has a transmission in the visible light spectrum of at least 80%.

66. The device of any one of claims 58-62, wherein the surface layer exhibits an Abbe Number of at least 60.

67. The device of any one of claims 58-62, wherein the surface layer comprises a polymeric material selected from the group consisting of acrylic, fluoropolymer, ethylene vinyl acetate, or a combination thereof.

68. The device of claim 67, wherein the surface layer comprises

fluoropolymer.

69. The device of claim 68, wherein the fluoropolymer is selected from the group consisting of polyvinylidene fluoride, polyvinyl fluoride, fluorinated ethylene propylene (FEP) copolymer, ethylene chlorotrifluoroethylene (ECTFE) copolymer, a copolymer of tetrafluoroethylene, hexafluoropropylene, and vinylidene fluoride, a copolymer of tetrafluoroethylene and perfluoro methylvinylether, a fluoropolymer having a fluorinated oxolane in its backbone, perfluoroether polymer, or a combination thereof.

70. The device of claim 69, wherein the fluoropolymer is a fluoropolymer having fluorinated oxolane in its backbone.

71. The device of any one of claims 58-62, wherein the surface layer is amorphous.

72. The device of any one of claims 58-62, wherein the reaction volume forms a well having a characteristic diameter of not greater than 2 micrometers.

73. The device of any one of claims 58-62, wherein the surface layer has a thickness of not greater than 1.0 micrometers.

74. The device of any one of claims 58-62, wherein the device exhibits a Noise Index of not greater than 0.8.

75. The device of any one of claims 58-62, wherein the device exhibits a signal-to-background noise ratio of at least 2.

76. The device of any one of claims 58-62, wherein the device exhibits an SBR Index of at least 1.4.

77. A system comprising:

a device comprising:

a substrate having a major surface and comprising a plurality of detectors; and

a surface layer disposed over the major surface, the surface layer defining a plurality of reaction volumes, each reaction volume of the plurality of reaction volumes uniquely associated with a detector of the plurality of detectors, the surface layer having an index of refraction in a range of 1.32 to 1.38; and

a chamber to receive the device and define a volume over the surface layer.

78. The system of claim 77, wherein the plurality of detectors are to detect electromagnetic radiation.

79. The system of claim 78, wherein the substrate further includes a transparent layer having a transmission of at least 80% in the visible light spectrum.

80. The system of claim 79, further comprising a light source to direct excitation light at the transparent layer.

81. The system of claim 77, wherein the plurality of detectors are to detect ions.

82. The system of claim 81, wherein the ions are hydronium or hydrogen ions.

83. The system of any one of claims 77-82, wherein the refractive index of the surface layer is in a range of 1.32 to 1.36.

84. The system of any one of claims 77-82, wherein the surface layer has a Refractive Matching Index in a range of -2 to 4.

85. The system of any one of claims 77-82, wherein the surface layer has a transmission in the visible light spectrum of at least 80%.

86. The system of any one of claims 77-82, wherein the surface layer exhibits an Abbe Number of at least 60.

87. The system of any one of claims 77-82, wherein the surface layer comprises a polymeric material selected from the group consisting of acrylic, fluoropolymer, ethylene vinyl acetate, or a combination thereof.

88. The system of claim 87, wherein the surface layer comprises

fluoropolymer.

89. The system of claim 88, wherein the fluoropolymer is selected from the group consisting of polyvinylidene fluoride, polyvinyl fluoride, fluorinated ethylene propylene (FEP) copolymer, ethylene chlorotrifluoroethylene (ECTFE) copolymer, a copolymer of tetrafluoroethylene, hexafluoropropylene, and vinylidene fluoride, a copolymer of tetrafluoroethylene and perfluoro methylvinylether, a fluoropolymer having fluorinated oxolane in its backbone, perfluoroether polymer, or a combination thereof.

90. The system of claim 89, wherein the fluoropolymer is a fluoropolymer having fluorinated oxolane in its backbone.

91. The system of any one of claims 77-82, wherein the surface layer is amorphous.

92. The system of any one of claims 77-82, wherein the reaction volume forms a well having a characteristic diameter of not greater than 2 micrometers.

93. The system of any one of claims 77-82, wherein the surface layer has a thickness of not greater than 1.0 micrometers.

94. The system of any one of claims 77-82, wherein the device exhibits a Noise Index of not greater than 0.8.

95. The system of any one of claims 77-82, wherein the device exhibits a signal-to-background noise ratio of at least 2.

96. The system of any one of claims 77-82, wherein the device exhibits an SBR Index of at least 1.4.

97. A method of sequencing a nucleic acid strand, the method comprising: applying the nucleic acid strand into a reaction volume of a device, the device comprising:

a substrate having a major surface and comprising a plurality of detectors; and

a surface layer disposed over the major surface, the surface layer defining a plurality of reaction volumes, each reaction volume of the plurality of reaction volumes uniquely associated with a detector of the plurality of detectors, the surface layer having an index of refraction in a range of 1.32 to 1.38;

flowing a reagent solution over the surface layer of the device, the reagent solution including nucleotides of a set of nucleotide types, each type of nucleotide labeled to provide a unique fluorescence signal; and detecting fluorescence signals associated with nucleotide incorporation onto the nucleic acid strand.

98. The method of claim 97, further comprising applying an enzyme to the reaction volume of the device.

99. A method of sequencing a nucleic acid strand, the method comprising: applying the nucleic acid strand into a reaction volume of a device, the device comprising:

a substrate having a major surface and comprising a plurality of detectors; and

sequentially flowing reagent solutions over the surface layer of the device, each the reagent solution including a type of nucleotide of a set of nucleotide types; and

detecting signals associated with nucleotide incorporation onto the nucleic acid strand.

100. The method of claim 99, further comprising applying an enzyme to the reaction volume of the device.

101. A method of forming a device, the method comprising:

coating a substrate with a polymeric coating having a refractive index in a range of 1.32 to 1.38, the substrate being transparent and having a refractive index of at least 1.45; and

etching the polymeric coating to define a plurality of reaction volumes, the plurality of reaction volumes including at least 100 reaction volumes.

102. The method of claim 101, further comprising treating the etched polymer coating with a passivation agent.

103. The method of claim 101, further comprising forming a substrate with a plurality of detectors.

104. A method of forming a device, the method comprising:

coating a major surface of a substrate with a polymeric coating having a

refractive index in a range of 1.30 to 1.40, the substrate including a plurality of detectors;

etching the polymeric coating to define a plurality of reaction volumes, each reaction volume of the plurality of reaction volumes uniquely associated with a detector of the plurality of detectors.

105. The method of claim 104, further comprising treating the etched polymer coating.