Low binding supports for improved solid-phase DNA hybridization and amplification

Abstract

This invention describes a nonspecific low-binding substrate and formulation for solid-phase nucleic acid hybridization and amplification, which provides improved performance for nucleic acid detection, amplification, and sequencing applications. These substrates exhibit a high contrast-to-noise ratio (CNR), contributing to more accurate data collection and more accurate sequence reads.

Description

Low-Binding Substrates for Improved Solid-Phase DNA Hybridization and Amplification Over the past two decades, a variety of DNA sequencing methods have been developed and commercialized (see, e.g., E. Mardis (2008), 「Next-Generation DNA Sequencing Methods」, Annu. Rev. Genomics Hum. Genet. 9:387-402; and J. Heather and B. Chain, (2016), 「The Sequence of Sequencers: The History of Sequencing DNA」, Genomics 107:1-8 for recent reviews). Many 「second-generation」 and 「third-generation」 sequencing technologies utilize a parallel cyclic array approach of massively parallel sequencing by synthesis (SBS), where accurate decoding of the sequence of single-stranded template oligonucleotides tethered to a solid substrate relies on successfully sorting signals generated by stepwise addition of A, G, C, and T nucleotides to a complementary oligonucleotide strand by a polymerase. Such methods typically require the oligonucleotide templates to be modified with a known adapter sequence of a fixed length and adhered to a solid substrate in a random or patterned array by hybridization to surface-tethered probes with known sequences complementary to the surface-tethered probes of the adapter sequence, and then detected using, for example, a single-molecule (unamplified) synchronous synthesis sequencing (smSBS) approach (e.g., Helicos technology) or a single-molecule asynchronous synthesis sequencing (smASBS) approach (e.g., Pacific Biosciences technology). In the smSBS approach, terminator nucleotides encoded with fluorescent tags are used such that the replicase can incorporate only a single base per cycle. For example, the Helicos technology uses a single fluorescent tag and performs sequential introduction of A, G, C, T - one base per cycle. During each cycle, an imaging step is performed to sort the correct 「base」 of each single-molecule template on the array. After the imaging step, the reversibly linked tags are removed such that the replicase (polymerase) can incorporate the next template base. These cycles are repeated multiple times, ultimately decoding the template oligonucleotide strands on the random array and determining their individual sequences. Despite their success, cyclic array approaches generally suffer from two fundamental drawbacks: (i) long cycle times for adding each successive nucleotide to the complementary strand, and (ii) weak signals generated by the stepwise addition of single nucleotides (usually detected by using fluorescent labeling and fluorescence imaging techniques), and as will be discussed in more detail below, exhibit a low contrast-to-noise ratio (CNR), and thus require expensive instrumentation incorporating high-precision optics and long imaging times to achieve accurate base-calling. Attempts have been made to solve the cycle time problem of cyclic array sequencing approaches, e.g., by the emergence of single molecule asynchronous synthesis sequencing (smASBS) approaches such as Pacific Biosciences technology, in which four spectrally distinct fluorescent tags are attached to each A, G, C, and T nucleotide, and the addition of such nucleotides can then be sorted "in real time". In this approach, all four labeled nucleotides are introduced simultaneously and images are obtained during the entire strand replication process. Based on the spectrum of the detected light, each position in the sequence is sorted as 'A', 'G', 'C', and 'T'. Here, the cycle time can theoretically be as fast as the polymerase-catalyzed replication rate, but the trade-off is a reduced CNR, thereby introducing sorting errors that ultimately lead to reduced accuracy and a greater reliance on high-precision optical devices and expensive instrumentation. Attempts have been made to address signal limitations in some cyclic array sequencing approaches (i.e., non-single molecule approaches) by incorporating an amplification step into the method. Solid-phase amplification of template DNA molecules tethered to a solid substrate in a random or patterned array increases the number of copies of the target to be sequenced such that when detectable bases are added stepwise to their respective complementary strands, the signals generated by the "community" of replicated template molecules can be sorted as 'A', 'G', 'C', or 'T'. During each detection event, which is typically limiting, the probability of successful sorting (and thus the accuracy of base identification) depends on the respective CNR. Accordingly, there is a need for improved solid substrates and solid-phase amplification methods for nucleic acid sequencing that will increase the magnitude of the base addition signal, reduce non-specific background signal, and thus improve the CNR, thereby improving the accuracy of base identification, potentially shortening the cycle time, and reducing the reliance of the sequencing process on high-precision optical devices and expensive instrumentation. Some embodiments relate to a method of performing nucleic acid sequencing, the method comprising: a) providing a surface; wherein the surface comprises: i) a substrate; ii) at least one hydrophilic polymer coating; iii) a plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating; and iv) at least one discrete region of the surface that comprises a plurality of clonally amplified sample nucleic acid molecules adhered to the plurality of attached oligonucleotide molecules, wherein the plurality of adhered clonally amplified sample nucleic acid molecules are present at a surface density of at least 5000 molecules / mm 2 and b) performing a nucleic acid amplification reaction on the sample nucleic acid molecules before or after adhering the sample nucleic acid molecules to the plurality of oligonucleotide molecules; and c) performing a cyclic series of single nucleotide binding or incorporation reactions, wherein the nucleotides are labeled with a detectable tag. Disclosed herein is a surface comprising a substrate, at least one hydrophilic low non-specific binding (i.e., low background) coating, and a plurality of oligonucleotide molecules attached to at least one hydrophilic low binding, low background coating. The disclosed low non-specific binding solid substrates can be used in a variety of bioassays, including but not limited to DNA sequencing and genotyping. These substrates comprise temperature and chemically stable functionalized substrates that are resistant to exposure to multiple solvent exchanges and temperature changes, and that confer low non-specific binding properties throughout the duration of the assay. The disclosed substrates can have some or all of the following properties: 1. Surface functionalization using any combination of polar protic, polar aprotic, and / or non-polar solvents, which results in an increase in the efficacy of bioassay performance > 5-fold compared to traditional approaches (e.g., improvements in reaction rate and / or desired product formation, respectively). 2. Minimal contact angle measurements after functionalization (e.g., < 35 degrees), which are maintained by successive solvent and temperature changes. 3. Low non-specific binding of biomolecules relative to specific binding molecules (e.g., > 1 specific binding molecule compared to < 0.25 non-specific binding molecules / region of interest). This can be directly translated into improved contrast-to-noise (CNR) when using any of a variety of detection methods. Disclosed herein is a surface comprising: a) a substrate; b) at least one hydrophilic polymer coating; c) a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating; and d) at least one discrete region of the surface that comprises a plurality of cloned and amplified sample nucleic acid molecules that have been adhered to the plurality of attached oligonucleotide molecules, wherein the surface exhibits a contrast-to-noise ratio (CNR) of at least 20 in a fluorescence image of the surface. In some embodiments, when the sample nucleic acid molecules or their complementary sequences are labeled with a cyanine dye-3 (Cy3) fluorophore, and the surface is immersed in a buffer (e.g., 25 mM ACES, pH 7.4 buffer), the fluorescence image of the surface exhibits a contrast-to-noise ratio (CNR) of at least 20 when obtained using an Olympus IX83 inverted fluorescence microscope equipped with a total internal reflection fluorescence (TIRF) 20×, 0.75 NA objective, a 100W Hg lamp, a bandpass and dichroic mirror filter set optimized for 532 nm excitation and Cy3 fluorescence emission, and a CCD camera (e.g., an Olympus EM-CCD camera) under non-signal saturation conditions. In some embodiments, the contrast noise ratio (CNR) of the fluorescence image of the surface is at least 40. In some embodiments, the contrast noise ratio (CNR) of the fluorescence image of the surface is at least 60. In some embodiments, the substrate comprises glass. In some embodiments, the substrate comprises plastic. In some embodiments, at least one hydrophilic polymer coating comprises PEG. In some embodiments, the surface further comprises a second hydrophilic polymer coating. In some embodiments, at least one hydrophilic polymer layer comprises a branched-chain hydrophilic polymer, such as PEG, having at least 4 branches. In some embodiments, at least one hydrophilic polymer layer comprises a branched-chain hydrophilic polymer, such as PEG, having at least 8 branches. In some embodiments, at least one hydrophilic polymer layer comprises a branched-chain hydrophilic polymer, such as PEG, having at least 16 branches. In some embodiments, at least one hydrophilic polymer layer comprises a branched-chain hydrophilic polymer, such as PEG, and having at least 32 branches. In some embodiments, the plurality of oligonucleotide molecules are present at a surface density of at least 50,000 molecules / μm 2 In some embodiments, the plurality of oligonucleotide molecules are present at a surface density of at least 100,000 molecules / μm 2 In some embodiments, the plurality of oligonucleotide molecules are present at a surface density of at least 500,000 molecules / μm 2 In some embodiments, prior to adhesion and clonal expansion, the sample nucleic acid molecules are administered at a concentration not exceeding 500 nM. In some embodiments, prior to adhesion and clonal expansion, the sample nucleic acid molecules are administered at a concentration not exceeding 20 pM. In some embodiments, the sample nucleic acid molecules comprise single-stranded polynucleic acid molecules comprising repetitive regularly occurring monomer units. In some embodiments, the length of the single-stranded polynucleic acid molecules is at least 10 kb. In some embodiments, the surface further comprises double-stranded monomer replicas of regularly occurring monomer units. In some embodiments, the surface is located on the interior of a flow channel. In some embodiments, the plurality of oligonucleotide molecules are present at a uniform surface density across the surface. In some embodiments, the plurality of oligonucleotide molecules are present at a surface density of at least 100,000 molecules / μm at a first location on the surface 2A local surface density exists and exists at a second position on the surface with a second local surface density. In some embodiments, before the cloning and amplification, the background fluorescence intensity measured in the region where the surface is laterally displaced from the at least one discrete region does not exceed twice the intensity measured in the at least one discrete region. In some embodiments, the surface comprises a first layer, a second layer and a third layer, the first layer comprising a monolayer of polymer molecules tethered to the surface of the substrate; the second layer comprising polymer molecules tethered to the polymer molecules of the first layer; the third layer comprising polymer molecules tethered to the polymer molecules of the second layer, wherein at least one layer comprises branched-chain polymer molecules. In some embodiments, the third layer further comprises oligonucleotides tethered to the polymer molecules of the third layer. In some embodiments, the oligonucleotides tethered to the polymer molecules of the third layer can be distributed at a plurality of depths in the third layer. In some embodiments, the surface further comprises a fourth layer and a fifth layer, the fourth layer comprising branched-chain polymer molecules tethered to the polymer molecules of the third layer, the fifth layer comprising polymer molecules tethered to the branched-chain polymer molecules of the fourth layer. In some embodiments, the polymer molecules of the fifth layer further comprise oligonucleotides tethered to the polymer molecules of the fifth layer. In some embodiments, the oligonucleotides tethered to the polymer molecules of the fifth layer can be distributed at a plurality of depths in the fifth layer. In some embodiments, at least one hydrophilic polymer coating comprises molecules selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin and polyglucose. In some embodiments, the fluorescence intensity ratio of the specifically amplified Cy3-labeled sample nucleic acid molecules or their complementary sequences to the non-specific Cy3 dye adsorption background (B inter ) presented in the image of the surface is at least 3:1. In some embodiments, the fluorescence intensity ratio of the specifically amplified Cy3-labeled sample nucleic acid molecules or their complementary sequences and the non-specific Cy3 dye adsorption background to the combination of the non-specific amplification background (B inter + B intra ) presented in the image of the surface is at least 3:1. In some embodiments, the fluorescence intensity ratio of the specifically amplified Cy3-labeled sample nucleic acid molecules or their complementary sequences to the non-specific dye adsorption background (B inter ) has a fluorescence intensity ratio of at least 5:1. In some embodiments, the image of the surface presents specifically amplified Cy3-labeled sample nucleic acid molecules or their complementary sequences and non-specific Cy3 dye adsorption background and non-specific amplification background (B inter + B intra ) has a fluorescence intensity ratio of at least 5:1. The present disclosure also discloses a surface comprising: a) a substrate; b) at least one hydrophilic polymer coating; and c) a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating, wherein the surface exhibits a non-specific Cy3 dye adsorption level lower than about 0.25 molecules / μm 2 . In some embodiments, the surface exhibits a non-specific Cy3 dye adsorption level lower than about 0.1 molecules / μm 2 . In some embodiments, the surface exhibits a ratio of specific Cy3 oligonucleotide labeling to non-specific Cy3 dye adsorption greater than about 4:1. In some embodiments, the surface exhibits a ratio of specific Cy3 oligonucleotide labeling to non-specific Cy3 dye adsorption greater than about 10:1. In some embodiments, the plurality of oligonucleotide molecules are attached at a surface density of at least 10,000 molecules / μm 2 . In some embodiments, the plurality of oligonucleotide molecules are attached at a surface density of at least 100,000 molecules / μm 2Surface density connection. In some embodiments, the surface further comprises a plurality of cloned and amplified clusters of template molecules that have been adhered to the plurality of oligonucleotide molecules, and wherein the contrast-to-noise ratio (CNR) presented by the fluorescence image of the surface is at least 20. In some embodiments, the contrast-to-noise ratio (CNR) is at least 50. In some embodiments, the contrast-to-noise ratio (CNR) is at least 100. In some embodiments, at least one of the at least two hydrophilic polymer layers comprises branched-chain polyethylene glycol (PEG) molecules. In some embodiments, the surface comprises the surface of a capillary lumen or at least one inner surface of a flow cell. In some embodiments, the capillary lumen or flow cell is structurally designed for performing nucleic acid hybridization, amplification, or sequencing reactions or any combination thereof. In some embodiments, the surface further comprises a branched-chain polymer blocking layer. In some embodiments, the branched-chain polymer blocking layer is a branched-chain PEG blocking layer. In some embodiments, the branched-chain polymer blocking layer is covalently tethered to the top hydrophilic polymer layer. In some embodiments, the polymer of the first layer comprises a primary amine functional group, and the polymer of the second layer comprises an N-hydroxysuccinimide (NHS) ester functional group, and after the deposition of the second layer, the second layer is tethered to the first layer using a covalent amide bond. In some embodiments, the polymer of the first layer comprises an N-hydroxysuccinimide (NHS) ester functional group, and the polymer of the second layer comprises a primary amine functional group, and after the deposition of the second layer, the second layer is tethered to the first layer using a covalent amide bond. In some embodiments, the oligonucleotide is tethered to the polymer molecules of the second or third layer at a molar ratio of oligonucleotide to polymer of about 1:5. In some embodiments, the oligonucleotide is tethered to the polymer molecules of the second or third layer at a molar ratio of oligonucleotide to polymer of about 2:5. In some embodiments, the oligonucleotide is tethered to the polymer molecules of the second or third layer at a molar ratio of oligonucleotide to polymer of about 3:5. In some embodiments, the oligonucleotide is tethered to the polymer molecules of the second or third layer at a molar ratio of oligonucleotide to polymer of about 4:5. In some embodiments, the oligonucleotide is tethered to the polymer molecules of the second or third layer at a molar ratio of oligonucleotide to polymer of about 1:1. In some embodiments, the oligonucleotide is tethered to the polymer molecules of the second or third layer at a molar ratio of oligonucleotide to polymer of about 4:1. In some embodiments, the oligonucleotide is tethered to the polymer molecules of the second or third layer at a molar ratio of oligonucleotide to polymer of about 8:1. In some embodiments, the oligonucleotide is tethered to the polymer molecules of the second or third layer at a molar ratio of oligonucleotide to polymer of about 16:1. In some embodiments, the oligonucleotide is tethered to the polymer molecules of the second or third layer at a molar ratio of oligonucleotide to polymer of about 32:1. In some embodiments, the oligonucleotide is present at a surface density of at least 10,000 molecules per square micron.In some embodiments, the oligonucleotides are present at a surface density of at least 100,000 molecules per square micron. In some embodiments, the oligonucleotides are uniformly distributed within the third layer. Disclosed herein is a method for depositing oligonucleotides on a substrate surface, the method comprising: a) binding a first hydrophilic polymer to the substrate surface in a first layer; b) binding a second hydrophilic polymer to the first layer to form a second layer, wherein the hydrophilic polymer molecules of the second layer are joined to the first layer by at least two covalent bonds per molecule; and c) binding an outermost hydrophilic polymer to the second layer, wherein the outermost hydrophilic polymer molecules comprise oligonucleotide molecules covalently linked thereto prior to binding to the second layer. In some embodiments, the hydrophilic polymer molecules of the second layer are bonded to the first layer by at least four covalent bonds per molecule. In some embodiments, the hydrophilic polymer molecules of the second layer are bonded to the first layer by at least eight covalent bonds per molecule. In some embodiments, the method further comprises directly bonding the outermost hydrophilic polymer to the second layer. In some embodiments, the method further comprises bonding a third hydrophilic polymer to the second layer to form a third layer, and bonding the outermost hydrophilic polymer to the second layer via the third layer. In some embodiments, the method further comprises bonding a third hydrophilic polymer to the second layer to form a third layer, bonding a fourth hydrophilic polymer to the third layer to form a fourth layer, and bonding the outermost hydrophilic polymer to the second layer via the fourth layer and the third layer. In some embodiments, the first hydrophilic polymer comprises PEG. In some embodiments, the first hydrophilic polymer comprises PGA. In some embodiments, at least one of the hydrophilic polymer layers comprises a branched-chain polymer. In some embodiments, the branched-chain polymer comprises at least 4 branches. In some embodiments, the branched-chain polymer comprises at least 8 branches. In some embodiments, the branched-chain polymer comprises 16 to 32 branches. In some embodiments, the hydrophilic polymer molecules of the fourth layer are bonded to the third layer by at least two covalent bonds per molecule. In some embodiments, the hydrophilic polymer molecules of the fourth layer are bonded to the third layer by at least four covalent bonds per molecule. In some embodiments, the hydrophilic polymer molecules of the fourth layer are bonded to the third layer by at least eight covalent bonds per molecule. In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent comprising ethanol. In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent comprising methanol. In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent comprising dimethyl sulfoxide (DMSO). In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent comprising acetonitrile. In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent comprising buffered phosphate. In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent comprising buffered 3-(N-morpholino)propanesulfonic acid (MOPS). In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent comprising 75% acetonitrile and 25% phosphate buffer. In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent comprising 90% methanol and 10% MOPS buffer. Disclosed herein is a surface comprising oligonucleotides at a density of at least 10,000 molecules per square micron, wherein the oligonucleotides are tethered to the surface via a multi-layer hydrophilic polymer layer, and wherein the oligonucleotides are uniformly distributed in the outermost layer of the multi-layer hydrophilic polymer layer. In some embodiments, the oligonucleotides are distributed at a surface density of at least 50,000 molecules per square micron. In some embodiments, the oligonucleotides are distributed at a surface density of at least 100,000 molecules per square micron. In some embodiments, the oligonucleotides are distributed at a surface density of at least 500,000 molecules per square micron. In some embodiments, at least 10% of the tethered oligonucleotides are adhered to the target (or sample) oligonucleotides. In some embodiments, the multi-layer hydrophilic polymer layer is saturated with a hydrophilic solvent. In some embodiments, the surface comprises the surface of a glass, fused silica, silicon or polymer (e.g., plastic) substrate. In some embodiments, the multi-layer hydrophilic polymer layer comprises three or more polymer layers. In some embodiments, the multi-layer hydrophilic polymer layer comprises five or more polymer layers. In some embodiments, one or more of the hydrophilic polymer layers comprises branched-chain PEG, branched-chain PVA, branched-chain poly(vinyl pyridine), branched-chain PVP, branched-chain PAA, branched-chain PNIPAM, branched-chain PMA, branched-chain PHEMA, branched-chain PEGMA, branched-chain PGA, branched-chain polylysine, branched-chain glucoside or dextran. In some embodiments, one or more of the hydrophilic polymer layers comprises branched-chain PEG molecules. In some embodiments, the branched-chain PEG molecules comprise at least 4 branches. In some embodiments, the branched-chain PEG molecules comprise at least 8 branches. In some embodiments, the branched-chain PEG molecules comprise 16 to 32 branches. In some embodiments, the at least first and second layers of the hydrophilic polymer layer are tethered to each other using covalent amide bonds. In some embodiments, the at least first and second layers of the hydrophilic polymer layer are tethered to each other by at least two covalent bonds per polymer molecule. In some embodiments, the at least first and second layers of the hydrophilic polymer layer are tethered to each other by at least four covalent bonds per polymer molecule. In some embodiments, the at least first and second layers of the hydrophilic polymer layer are tethered to each other by at least eight covalent bonds per polymer molecule. In some embodiments, the surface density of the tethered oligonucleotides is at least 50,000 molecules per square micron. In some embodiments, the surface density of the tethered oligonucleotides is at least 100,000 molecules per square micron. In some embodiments, the surface exhibits a non-specific binding of less than 0.25 molecules / μm of the CY3 dye 2. In some embodiments, the surface further comprises a cluster of cloned amplified copies of the ligated target oligonucleotides, wherein substantially all of the cloned amplified copies of the ligated target oligonucleotides comprise Cy3-labeled nucleotides ligated at a first position, and wherein the fluorescence image of the surface exhibits a contrast-to-noise (CNR) ratio of at least 20. In some embodiments, the contrast-to-noise ratio (CNR) is at least 50. In some embodiments, the contrast-to-noise ratio (CNR) is at least 100. In some embodiments, the contrast-to-noise ratio (CNR) is at least 150. In some embodiments, the contrast-to-noise ratio (CNR) is at least 200. In some embodiments, the cloned amplified copies of the ligated target oligonucleotides are prepared using a bridge amplification protocol. In some embodiments, the cloned amplified copies of the ligated target oligonucleotides are prepared using an isothermal bridge amplification protocol. In some embodiments, the cloned amplified copies of the ligated target oligonucleotides are prepared using a rolling circle amplification (RCA) protocol. In some embodiments, the cloned amplified copies of the ligated target oligonucleotides are prepared using a helicase-dependent amplification protocol. In some embodiments, the cloned amplified copies of the ligated target oligonucleotides are prepared using a recombinase-dependent amplification protocol. In some embodiments, the cloned amplified copies of the ligated target oligonucleotides are prepared using a single-strand binding (SSB) protein-dependent amplification protocol. In some embodiments, the surface comprises the surface of a capillary lumen or at least one inner surface of a flow cell. In some embodiments, the capillary lumen or flow cell is configured for performing nucleic acid hybridization, amplification, or sequencing reactions or any combination thereof. Methods for performing solid-phase nucleic acid hybridization are disclosed herein, the methods comprising: a) providing any one of the surfaces disclosed herein; and b) performing a solid-phase nucleic acid hybridization reaction, wherein a template nucleic acid molecule is ligated to an anchored oligonucleotide. Methods for performing solid-phase nucleic acid amplification are also disclosed herein, the methods comprising: a) providing any one of the surfaces disclosed herein; and b) performing a solid-phase nucleic acid amplification reaction using a template nucleic acid molecule hybridized to an anchored oligonucleotide. In some embodiments, solid-phase nucleic acid amplification includes thermal cycling. In some embodiments, solid-phase nucleic acid amplification includes isothermal amplification. In some embodiments, solid-phase nucleic acid amplification includes rolling circle amplification. In some embodiments, solid-phase nucleic acid amplification includes bridge amplification. In some embodiments, solid-phase nucleic acid amplification includes isothermal bridge amplification. In some embodiments, solid-phase nucleic acid amplification includes multiple displacement amplification. In some embodiments, solid-phase nucleic acid amplification includes helicase treatment. In some embodiments, solid-phase nucleic acid amplification includes recombinase treatment. In some embodiments, the surface density of the tethered oligonucleotides does not change within at least 30 cycles of the solid-phase nucleic acid amplification reaction. In some embodiments, the surface density of the tethered oligonucleotides does not change within at least 40 cycles of the solid-phase nucleic acid amplification reaction. In some embodiments, the surface density of the tethered oligonucleotides does not change within at least 50 cycles of the solid-phase nucleic acid amplification reaction. Disclosed herein are methods for performing nucleic acid sequencing, the methods comprising: a) providing any one of the surfaces disclosed herein; b) performing a solid-phase nucleic acid amplification reaction using a template nucleic acid molecule that hybridizes to a tethered oligonucleotide; and c) performing a cyclic series of single nucleotide binding or incorporation reactions, wherein the nucleotides are labeled with a detectable label. In some embodiments, the detectable label is a fluorophore. In some embodiments, the fluorophore is Cy3, and wherein a fluorescence image of the surface is obtained under non-signal saturation conditions as described elsewhere herein after the contrast-to-noise ratio (CNR) of the binding or incorporation of the first Cy3-labeled nucleotide is at least 20. In some embodiments, the contrast-to-noise ratio (CNR) is at least 50. In some embodiments, the contrast-to-noise ratio (CNR) is at least 100. In some embodiments, the contrast-to-noise ratio (CNR) is at least 150. In some embodiments, the contrast-to-noise ratio (CNR) is at least 200. In some embodiments, the solid-phase nucleic acid amplification reaction includes a bridge amplification reaction. In some embodiments, the solid-phase nucleic acid amplification reaction includes an isothermal bridge amplification reaction. In some embodiments, the solid-phase nucleic acid amplification reaction includes a rolling circle amplification (RCA) reaction. In some embodiments, the solid-phase nucleic acid amplification reaction includes a helicase-dependent amplification reaction. In some embodiments, the solid-phase nucleic acid amplification reaction includes a recombinase-dependent amplification reaction. Disclosed herein is an apparatus for performing nucleic acid amplification, the apparatus comprising: a) any one of the surfaces disclosed herein; wherein the surface includes the surface of a capillary lumen or at least one inner surface of a flow cell. In some embodiments, the device further comprises at least one fluid inlet for entering a capillary lumen. In some embodiments, the device further comprises at least one fluid outlet. In some embodiments, the device further comprises at least one pump. In some embodiments, the device further comprises at least one fluid mixing manifold. In some embodiments, the device further comprises at least one temperature control element. In some embodiments, the device further comprises at least one optical window. Disclosed herein is a system for performing nucleic acid sequencing, the system comprising: a) at least one of the devices disclosed herein; b) a fluid control module; and c) an imaging module. Incorporated by reference All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference in their entirety, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. If there is a conflict between the terms herein and the terms of the incorporated references, the terms herein shall control. Cross-reference This application claims the benefit of U.S. Provisional Application No. 62 / 767,343, filed Nov. 14, 2018, U.S. Provisional Application No. 62 / 776,898, filed Dec. 7, 2018, and U.S. Application 16 / 363,842, filed Mar. 25, 2019, each of which is hereby incorporated by reference in its entirety. Disclosed herein are novel solid substrates for solid-phase nucleic acid amplification and sequencing or other bioanalytical applications. The solid substrates disclosed herein exhibit low non-specific binding of proteins and other amplification reaction components, and improved stability upon repeated exposure to different solvents, temperature variations, chemical affronts such as low pH, or long-term storage. Alone or in combination with improved nucleic acid hybridization and amplification protocols, some of the substrates disclosed herein cause one or more of the following: (i) a reduction in the amount of starting material required, (ii) a reduction in the temperature required for isothermal or slow temperature ramp amplification protocols, (iii) an increase in amplification rate, (iv) an increase in amplification specificity (i.e., more selective amplification of single-stranded template molecules of the amplified population while reducing non-specific amplification of surface primers and primer-dimers), and (v) allowing a greater degree of discrimination between sequence-specific signals and background signals (such as signals generated by interstitial and in-gap background), thereby providing an improved contrast-to-noise ratio (CNR) and base calling accuracy compared to conventional nucleic acid amplification and sequencing methods. The starting point for achieving the foregoing improvements or any combination thereof is the disclosed low non-specific binding substrate, which comprises one or more polymer coatings, such as a PEG polymer film, which minimizes the non-specific binding of proteins and labeled nucleotides to the solid substrate. Subsequent confirmation of improved nucleic acid hybridization and amplification rates and specificities can be achieved by one or more of the following additional aspects of the present invention: (i) primer design (sequence and / or modification), (ii) control of the tethered primer density on the solid substrate, (iii) the surface composition of the solid substrate, (iv) the surface polymer density of the solid substrate, (v) the use of improved hybridization conditions before and during amplification, and / or (vi) the use of improved amplification formulations that reduce non-specific primer amplification or increase template amplification efficiency. The advantages of the disclosed low non-specific binding substrates and related hybridization and amplification methods confer one or more of the following additional advantages to any sequencing system: (i) reduced fluid wash times (due to reduced non-specific binding and thus faster sequencing cycle times), (ii) reduced imaging times (and thus faster turnaround times for analysis reads and sequencing cycles), (iii) reduced total workflow time required (due to reduced cycle times), (iv) reduced detector instrument costs (due to improved CNR), (v) improved read (base calling) accuracy (due to improved CNR), (vi) improved reagent stability and reduced reagent usage requirements (and thus reduced reagent costs), and (vii) fewer run time failures due to nucleic acid amplification failures. A low binding hydrophilic surface (multi-layer and / or single-layer) for surface bioanalysis (such as genotyping and sequencing analysis) is formed by using any combination of the following. Depositing and / or coupling polar protons, polar aprotic, and / or non-polar solvents of linear or multi-branched chain hydrophilic polymer subunits on the substrate surface. Some multi-branched chain hydrophilic polymer subunits may contain functional end groups that promote covalent coupling or non-covalent binding interactions with other polymer subunits. Examples of suitable functional end groups include biotin, methoxy ether, carboxylate, amine, ester compounds, azide, alkyne, maleimide, thiol, and silyl. Any combination of linear, branched, or multi-branched chain polymer subunits is coupled via a modified coupling chemistry / solvent / buffer system that may include individual subunits by subsequent layer-by-layer addition to an orthogonal end coupling chemistry or any one of the individual combinations, such that the resulting surface is hydrophilic and exhibits low non-specific binding of proteins and other molecular analysis components. In some cases, the hydrophilic functionalized substrate surface of the present invention exhibits a contact angle measurement of no more than 35 degrees. A compatibility buffer system other than the aforementioned solvents, with a required pH range of 5 - 10. Examples include, but are not limited to, phosphate buffered saline, phosphate buffer, TAPS, MES, MOPS, or any combination thereof. Subsequent biomolecule ligation (e.g., of proteins, peptides, nucleic acids, oligonucleotides, or cells) on a low-binding / hydrophilic substrate via various individual conjugation chemistries described below or any combination thereof. Layer deposition and / or binding reactions can be carried out using a solvent mixture that may contain any ratio of the following components: ethanol, methanol, acetonitrile, acetone, DMSO, DMF, H 2 O and its analogs. Additionally, a compatibility buffer system in the required pH range of 5 - 10 can be used to control the rate and efficiency of deposition and coupling, whereby a coupling rate exceeding 5 times the coupling rate of methods based on conventional aqueous buffers can be achieved. Definitions : Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless the context clearly indicates otherwise, the singular forms "a / an" and "the" as used in this specification and the appended claims include plural references. Any reference to "or" herein is intended to cover "and / or" unless otherwise stated. As used herein, the term 'about' a number means that number plus or minus 10% of that number. The term 'about' when used in the context of a range means that range minus 10% of its lowest value and plus 10% of its highest value. As used herein, in a series of contexts, the phrase 'at least one' encompasses a list including a single member of the series, two members of the series up to and including all members of the series, alone or in some cases in combination with components not listed. As used herein, if fluorescence is generated by a fluorophore, the fluorescence is '特异的' (should be'specific' in English), and such fluorophores adhere or are otherwise tethered to a surface, such as by nucleic acid adhesion with a reverse complementary region or otherwise tethered to a corresponding segment of an oligomer on the surface, and adhere to the corresponding segment. This fluorescence contrasts with the fluorescence generated by fluorophores that are not tethered to the surface by such adhesion methods, or in some cases, the background fluorescence of fluorophores tethered to the surface. Nucleic acid As used herein, "nucleic acid" (also referred to as "polynucleotide", "oligonucleotide", ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is a linear polymer of two or more nucleotides joined by covalent internucleoside bonds, or variants or functional fragments thereof. In naturally occurring examples of nucleic acids, the internucleoside bond is typically a phosphodiester bond. However, other examples may include other internucleoside bonds, such as phosphorothioate bonds, and may or may not include phosphate groups. Nucleic acids include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA, DNA / RNA hybrids, peptide nucleic acids (PNAs), hybrids between PNAs and DNA or RNA, and may also include other types of nucleic acid modifications. As used herein, "nucleotide" refers to a nucleotide, nucleoside or analog thereof. In some cases, a nucleotide is an N-glycoside or C-glycoside of a purine or pyrimidine base (e.g., a deoxynucleoside containing 2-deoxy-D-ribose or a nucleoside containing D-ribose). Examples of other nucleotide analogs include, but are not limited to, phosphorothioates, aminophosphates, methylphosphonates, enantiomeric methylphosphonates, 2-O-methyl ribonucleotides and their analogs. Nucleic acids may optionally be linked to one or more non-nucleotide moieties, such as labels and other small molecules, macromolecules (such as proteins, lipids, sugars, etc.), and solid or semi-solid substrates, for example by covalent or non-covalent bonds to the 5' or 3' end of the nucleic acid. Labels include any moiety that can be detected using any of a variety of detection methods known to those skilled in the art, and thus render the linked oligonucleotide or nucleic acid similarly detectable. Some labels emit electromagnetic radiation that is optically detectable or visible. Alternatively or in combination, some labels include mass tags that render the labeled oligonucleotide or nucleic acid visible in mass spectrometry data, or redox tags that render the labeled oligonucleotide or nucleic acid detectable by amperometry or voltammetry. Some labels include magnetic tags that facilitate the separation and / or purification of the labeled oligonucleotide or nucleic acid. Nucleotides or polynucleotides are generally not linked to labels, and the presence of the oligonucleotide or nucleic acid is detected directly. The disclosed low non-specific binding substrates and related nucleic acid hybridization and amplification methods can be used to analyze nucleic acid molecules derived from any of a variety of different cell, tissue, or sample types known to those skilled in the art. For example, nucleic acids can be extracted from cells or tissue samples derived from eukaryotes (such as animals, plants, fungi, protists), archaebacteria, or eubacteria, or from tissue samples containing one or more types of cells. In some cases, nucleic acids can be extracted from prokaryotic or eukaryotic cells, such as adherent or non-adherent eukaryotic cells. Nucleic acids can be extracted differently from, for example, primary or immortalized rodent, porcine, feline, canine, bovine, equine, primate, or human cell lines. Nucleic acids can be extracted from any of a variety of different cell, organ, or tissue types (e.g., white blood cells, red blood cells, platelets, epithelial cells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts, skeletal muscle cells, smooth muscle cells, gametes, or cells from the heart, lung, brain, liver, kidney, spleen, pancreas, thymus, bladder, stomach, colon, or small intestine). Nucleic acids can be extracted from normal or healthy cells. Alternatively or in combination, acids can be extracted from diseased cells, such as cancer cells, or from pathogenic cells that infect a host. Some nucleic acids can be extracted from different subpopulations of cell types, such as immune cells (such as T cells, cytotoxic (killer) T cells, helper T cells, αβ T cells, γδ T cells, T cell progenitors, B cells, B cell progenitors, lymphoid stem cells, bone marrow progenitors, lymphocytes, granulocytes, natural killer cells, plasma cells, memory cells, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells, and / or macrophages or any combination thereof), undifferentiated human stem cells, induced differentiated human stem cells, rare cells (e.g., circulating tumor cells (CTCs), circulating epithelial cells, circulating endothelial cells, circulating endometrial cells, bone marrow cells, progenitors, foam cells, mesenchymal cells, or trophoblasts). Other cells are encompassed within and consistent with the disclosure herein. Nucleic acid extraction from cells or other biological samples can be performed using any of a variety of techniques known to those skilled in the art. For example, a typical DNA extraction procedure includes (i) collecting a cell sample or tissue sample from which DNA is to be extracted, (ii) disrupting the cell membrane (i.e., cell lysis) to release DNA and other cytoplasmic components, (iii) treating the lysed sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, followed by centrifugation to separate the precipitated proteins, lipids, and RNA, and (iv) purifying the DNA from the supernatant to remove detergents, proteins, salts, or other reagents used during the cell membrane lysis step. A variety of suitable commercial nucleic acid extraction and purification kits are consistent with the disclosure herein. Examples include, but are not limited to, the QIAamp kits from Qiagen (Germantown, MD) (for isolating genomic DNA from human samples) and the DNAeasy kits (for isolating genomic DNA from animal or plant samples), or the Maxwell® and ReliaPrep™ series of kits from Promega (Madison, WI). Solid-phase nucleic acid hybridization and amplification substrates with low non-specific binding : Disclosed herein are solid substrates comprising a low non-specific binding surface composition that allows for improved nucleic acid hybridization and amplification performance. Generally, the disclosed substrates can comprise a substrate (or substrate structure), one or more covalently or non-covalently linked low-binding chemical modification layers (such as silane layers, polymer films), and one or more covalently or non-covalently linked primer sequences that can be used to tether single-stranded template oligonucleotides to the substrate surface ( Figure 1). In some cases, the formulation of the surface, such as the chemical composition of one or more layers, the coupling chemicals used to crosslink one or more layers to the substrate surface and / or to each other, and the total number of layers can vary such that non-specific binding of proteins, nucleic acid molecules, and other hybridization and amplification reaction components to the substrate surface is minimized or reduced relative to a comparable single layer. Generally, the formulation of the surface can vary such that non-specific hybridization on the substrate surface is minimized or reduced relative to a comparable single layer. The formulation of the surface can vary such that non-specific amplification on the substrate surface is minimized or reduced relative to a comparable single layer. The formulation of the surface can vary such that the rate and / or yield of specific amplification on the substrate surface is maximized. In some cases disclosed herein, an amplification level suitable for detection is achieved in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30 amplification cycles. Examples of materials from which the substrate or substrate structure can be made include, but are not limited to, glass, fused silica, silicon, polymers (such as, polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. A variety of compositions encompassing both glass and plastic substrates are contemplated. The substrate or base material structure can be presented in any of various geometric structures and dimensions known to those skilled in the art, and can include any of various materials known to those skilled in the art. For example, in some cases, the substrate or base material structure can be partially planar (e.g., including the surface of a microscope slide or a microscope slide). Generally, the substrate or base material structure can be cylindrical (e.g., including the inner surface of a capillary or a capillary), spherical (e.g., including the outer surface of a non-porous bead), or irregular (e.g., including the outer surface of an irregularly shaped, non-porous bead or particle). In some cases, the surface of the substrate or base material structure for nucleic acid hybridization and amplification can be a solid non-porous surface. In some cases, the surface of the substrate or base material structure for nucleic acid hybridization and amplification can be porous, such that the coatings described herein penetrate the porous surface, and nucleic acid hybridization and amplification reactions carried out thereon can occur within the pores. A substrate or base material structure comprising one or more chemically modified layers (such as a low non-specific binding polymer layer) can be self-standing or integrated into another structure or assembly. For example, in some cases, the substrate or base material structure can include the surface within one or more integrated or assembled microfluidic flow channels. The substrate or base material structure can include the surface within one or more microtiter plate formats, such as the lower surface of the wells in a microtiter plate. As noted above, in some preferred embodiments, the substrate or base material structure includes the inner surface of a capillary (such as the lumen surface). In alternative preferred embodiments, the substrate or base material structure includes the inner surface of a capillary (such as the lumen surface) etched into a planar wafer. The chemically modified layer can be uniformly coated on the surface of the substrate or base material structure. Alternatively, the surface of the substrate or base material structure can be non-uniformly distributed or patterned such that the chemically modified layer is restricted to one or more discrete regions of the substrate. For example, the substrate surface can be patterned using photolithography techniques to form an ordered array or a random pattern of chemically modified regions on the surface. Alternatively or in combination, the substrate surface can be patterned using techniques such as contact printing and / or inkjet printing. In some cases, the ordered array or random pattern of discrete chemically modified regions can include at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000 or more discrete regions, or any intermediate number across the ranges herein. To achieve a surface with low non-specific binding (also referred to herein as a "low-binding" or "passivated" surface), hydrophilic polymers can be non-specifically adsorbed or covalently grafted to the surface of a substrate or a base material. Generally, passivation is carried out using poly(ethylene glycol) (PEG, also known as poly(ethylene oxide) (PEO) or poly(ethylene oxide)), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucosides, streptavidin, polyglucose or other hydrophilic polymers with different molecular weights and end groups connected to the surface using, for example, silane chemistry. The end groups away from the surface can include, but are not limited to, biotin, methoxy ether, carboxylate, amine, NHS ester, maleimide and disilane. In some cases, two or more layers of hydrophilic polymers, such as linear polymers, branched-chain polymers or multi-branched-chain polymers, can be deposited on the surface. In some cases, two or more layers can be covalently coupled or internally cross-linked to each other to improve the stability of the resulting surface. In some cases, oligonucleotide primers (or other biomolecules, such as enzymes or antibodies) with different base sequences and base modifications can be tethered to the resulting surface layer at various surface densities. In some cases, for example, both the surface functional group density and the oligonucleotide concentration can be varied to target a certain primer density range. Additionally, the primer density can be controlled by diluting the oligonucleotide with other molecules carrying the same functional group. For example, amine-labeled oligonucleotides can be diluted with amine-labeled polyethylene glycol in the reaction with a surface coated with NHS ester to reduce the final primer density. Primers with linkers of different lengths between the hybridization region and the surface attachment functional group can also be applied to control the surface density. Examples of suitable linkers include poly-T and poly-A strands (e.g., 0 to 20 bases) at the 5' end of the primer, PEG linkers (e.g., 3 to 20 monomer units) and carbon chains (e.g., C6, C12, C18, etc.). To measure the primer density, fluorescently labeled primers can be tethered to the surface, and then the fluorescence readings are compared with the fluorescence readings of a dye solution with a known concentration. In some embodiments, the hydrophilic polymer can be a crosslinked polymer. In some embodiments, the crosslinked polymer can include one type of polymer crosslinked with another type of polymer. Examples of crosslinked polymers can include poly(ethylene glycol) crosslinked with another polymer selected from: poly(ethylene oxide) (PEO) or poly(ethylene oxide), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, dextran, or other hydrophilic polymers. In some embodiments, the crosslinked polymer can be poly(ethylene glycol) crosslinked with polyacrylamide. Due to the surface passivation techniques disclosed herein, proteins, nucleic acids, and other biomolecules do not "stick" to these substrates, i.e., they exhibit low non-specific binding (NSB). Examples are shown below using standard monolayer surface agents under different glass preparation conditions. Hydrophilic surfaces that have been passivated to achieve ultra-low NSB for proteins and nucleic acids require novel reaction conditions to improve primer deposition reaction efficiency, hybridization performance, and induce efficient amplification. All of these methods require oligonucleotide ligation and subsequent protein binding and delivery to low-binding surfaces. As described below, the combination of a novel primer surface binding formulation (Cy3 oligonucleotide graft titration) and the resulting ultra-low non-specific background (NSB functionality testing using red and green fluorescent dyes) gives results that demonstrate the feasibility of the disclosed approach. Some of the surfaces disclosed herein exhibit specificity such as a Cy3 fluorophore (e.g., hybridizing with tethered primers or probes) inter ) in a ratio of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate value across the ranges herein. Some of the surfaces disclosed herein exhibit specificity of a fluorophore such as Cy3 and non-specific fluorescence signals (e.g., oligonucleotides labeled for specific hybridization and non-specific binding, or for specific amplification and non-specific binding (B inter ) or non-specific amplification (B intra ) labeled oligonucleotides or combinations thereof (B inter + B intra )) The ratio is at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1 or greater than 100:1, or any intermediate value across the ranges herein. To scale the primer surface density and add additional dimensions to hydrophilic or amphiphilic surfaces, substrates comprising multiple layers of PEG and other hydrophilic polymer coatings have been developed. By using hydrophilic and amphiphilic surface layering approaches, which include but are not limited to the polymer / copolymer materials described below, it is possible to significantly increase the primer loading density on the surface. Conventional PEG coating approaches use single-layer primer deposition, which has generally been reported for single-molecule applications but does not yield high copy numbers for nucleic acid amplification applications. "Layering" as described herein can be achieved using conventional crosslinking approaches utilizing any compatible polymer or monomer subunits such that surfaces comprising two or more highly crosslinked layers can be sequentially constructed. Examples of suitable polymers include but are not limited to streptavidin, polyacrylamide, polyester, dextran, polylysine and copolymers of polylysine and PEG. In some cases, the different layers can be connected to each other by any of a variety of binding reactions, including but not limited to biotin-streptavidin binding, azide-alkyne click reaction, amine-NHS ester reaction, thiol-maleimide reaction and ionic interactions between positively charged and negatively charged polymers. In some cases, high primer density materials can be constructed in solution and subsequently layered onto the surface in multiple steps. Figure 2 provides a schematic illustration of one non-limiting example of grafting a first hydrophilic polymer layer to a substrate, such as a glass substrate. After cleaning the glass surface using any of a variety of methods known to those skilled in the art (e.g., treatment with Piranha solution, plasma cleaning, etc.), the substrate is treated with a silane solution (e.g., silane PEG 5K solution), rinsed, dried and cured at high temperature to form a covalent bond with the surface. The ends of the polymer away from the surface can comprise any of a variety of chemically reactive or protected functional groups. In Figure 2 illustrates amine-reactive NHS groups. Figure 3 provides for making a composition comprising, such as Figure A schematic diagram of a non - limiting example in which a derivatized substrate of a first polymer layer of the NHS functional group described in 2 is coupled with a primary amine - functionalized branched - chain polymer (e.g., a 16 - branched or 32 - branched PEG polymer, also referred to as a 16 - arm or 32 - arm PEG respectively) to form a second hydrophilic polymer layer containing an excess of unreacted functional groups. Figure Figure 4 provides a schematic diagram of a non - limiting example of reacting a branched - chain polymer containing reactive functional groups (e.g., 4 - branched NHS - PEG) with one or more oligonucleotide linkers or primer sequences in solution (e.g., an oligonucleotide containing a primary amine as illustrated by the dotted and dashed lines), and then depositing on the substrate surface to form a hydrophilic layer containing covalently - linked oligonucleotide molecules. By varying the molar ratio of the oligonucleotide molecules (or other biomolecules to be tethered, such as peptides, proteins, enzymes, antibodies, etc.) to the branched - chain polymer, one can vary the resulting surface density of the linked oligonucleotide sequences in a controlled manner. In some cases, after the layer has been deposited on the surface, one or more oligonucleotide molecules (or other biomolecules) can be covalently tethered to the existing polymer layer. Figure Figure 5 provides a non - limiting example of coupling a branched - chain polymer containing covalently - linked oligonucleotide primers with a Figure A schematic diagram of a non - limiting example of coupling to a stratified hydrophilic surface as described in Figure 3. In this example, a branched - chain polymer containing two different oligonucleotide primers (represented by dotted and dashed lines) and amine - reactive NHS groups is coupled with the primary amines of the previous layer to form a multi - layer three - dimensional hydrophilic surface that contains a controlled surface density of the tethered oligonucleotide primers. The connecting chemical used to graft the first chemically modified layer to the substrate surface will generally depend on both the material from which the substrate is made and the chemical nature of the layer. In some cases, the first layer can be covalently attached to the substrate surface. In some cases, the first layer can be non - covalently attached, such as by electrostatic interactions, hydrogen bonding, or van der Waals interactions between the surface and the molecular components of the first layer, e.g., adsorbed to the surface. In either case, the substrate surface can be treated before attaching or depositing the first layer. Any of a variety of surface preparation techniques known to those skilled in the art can be used to clean or treat the substrate surface. For example, glass or silicon surfaces can be treated with a strong piranha solution (sulfuric acid (H 2 SO 4 ) and hydrogen peroxide (H 2 O 2 ) mixture) is subjected to pickling and / or cleaned using an oxygen plasma treatment method. Silane chemistry constitutes a non-limiting route for covalently modifying silanol groups on glass or silicon surfaces to attach more reactive functional groups (e.g., amines or carboxyl groups), which can subsequently be used to couple linker molecules (e.g., linear hydrocarbon molecules of various lengths such as C6, C12, C18 hydrocarbons or linear polyethylene glycol (PEG) molecules) or layer molecules (e.g., branched-chain PEG molecules or other polymers) to the surface. Examples of suitable silanes that can be used to form any of the disclosed low-binding substrate surfaces include, but are not limited to, (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), various PEG-silanes (e.g., having molecular weights including 1K, 2K, 5K, 10K, 20K, etc.), amino-PEG silanes (i.e., containing free amino functional groups), maleimide-PEG silanes, biotin-PEG silanes, and any of their analogs. Any of a variety of molecules known to those skilled in the art (including, but not limited to, amino acids, peptides, nucleotides, oligonucleotides, other monomers or polymers, or combinations thereof) can be used to form one or more chemically modified layers on the substrate surface, where the choice of components used can vary to alter one or more properties of the substrate surface, such as the functional groups and / or the surface density of tethered oligonucleotide primers, the hydrophilicity / hydrophobicity of the substrate surface, or the three-dimensional properties (i.e., "thickness") of the substrate surface. Examples of preferred polymers that can be used to form one or more layers of non-specific low-binding material in any of the disclosed substrate surfaces include, but are not limited to, polyethylene glycol (PEG) of various molecular weights and branched-chain structures, streptavidin, polyacrylamide, polyester, dextran, polylysine, and polylysine copolymers, or any combination thereof. Examples of conjugation chemistries that can be used to graft one or more layers of material (e.g., polymer layers) to the substrate surface and / or crosslink such layers to each other include, but are not limited to, biotin-streptavidin interactions (or variations thereof), his-tag-Ni / NTA conjugation chemistry, methoxy ether conjugation chemistry, carboxylate conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxies, azides, hydrazides, alkynes, isocyanates, and silanes. One or more of the multi-layers may comprise branched-chain polymers or may be linear. Examples of suitable branched-chain polymers include, but are not limited to, branched-chain PEG, branched-chain poly(vinyl alcohol) (branched-chain PVA), branched-chain poly(vinyl pyridine), branched-chain poly(vinyl pyrrolidone) (branched-chain PVP), branched poly(acrylic acid) (branched-chain PAA), branched-chain polyacrylamide, branched-chain poly(N-isopropylacrylamide) (branched-chain PNIPAM), branched-chain poly(methyl methacrylate) (branched-chain PMA), branched-chain poly(2-hydroxyethyl methacrylate) (branched-chain PHEMA), branched-chain poly(oligo(ethylene glycol) methyl ether methacrylate) (branched-chain POEGMA), branched-chain polyglutamic acid (branched-chain PGA), branched-chain polylysine, branched-chain polyglucoside, and dextran. In some cases, the branched-chain polymers used to form one or more of the multi-layers disclosed herein may comprise at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, at least 32, at least 34, at least 36, at least 38, or at least 40 branches. Molecules typically exhibit a '2 to the power' number of branches, such as 2, 4, 8, 16, 32, 64, or 128 branches. Exemplary PEG multi-layers include PEG (8K,16K,8K) on PEG-amine-APTES that was exposed to two layers of 7 uM oligonucleotide primer pre-loaded and presented a total of 2,000,000 to 10,000,000 molecules on the surface. Similar concentrations were observed using star PEG-amine to replace dumbbell-shaped 16-mer and 64-mer polymers on 3 layers of multi-arm PEG (8K,16K,8K) and (8K,16K,8K) and 3 layers of multi-arm PEG (8,8,8) on PEG-amine-APTES exposed to 8 uM primer. Also encompassed are PEG multi-layers having equivalent first, second, and third PEG layers. The molecular weight of the linear, branched, or multi-branched chain polymers used to form one or more layers of any of the multi-layer surfaces disclosed herein can be at least 500, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 7,500, at least 10,000, at least 12,500, at least 15,000, at least 17,500, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, or at least 50,000 Daltons. In some cases, the molecular weight of the linear, branched, or multi-branched chain polymers used to form one or more layers of any of the multi-layer surfaces disclosed herein can be at most 50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000, at most 20,000, at most 17,500, at most 15,000, at most 12,500, at most 10,000, at most 7,500, at most 5,000, at most 4,500, at most 4,000, at most 3,500, at most 3,000, at most 2,500, at most 2,000, at most 1,500, at most 1,000, or at most 500 Daltons. Any of the lower and higher values described in this paragraph can be combined to form a range included within the present invention. For example, in some cases, the molecular weight of the linear, branched, or multi-branched chain polymers used to form one or more layers of any of the multi-layer surfaces disclosed herein can be in the range of about 1,500 to about 20,000 Daltons. Those skilled in the art will recognize that any value of the molecular weight of the linear, branched, or multi-branched chain polymers used to form one or more layers of any of the multi-layer surfaces disclosed herein can be within this range, such as about 1,260 Daltons. In some cases, for example, where at least one layer of the multilayer surface comprises a branched-chain polymer, the number of covalent bonds between the branched-chain polymer molecules of the deposited layer and the molecules of the previous layer can range from about one covalent bond per molecule to about 32 covalent bonds per molecule. In some cases, the number of covalent bonds between the branched-chain polymer molecules of the novel layer and the molecules of the previous layer can be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, or at least 32 or more than 32 covalent bonds per molecule. In some cases, the number of covalent bonds between the branched-chain polymer molecules of the novel layer and the molecules of the previous layer can be at most 32, at most 30, at most 28, at most 26, at most 24, at most 22, at most 20, at most 18, at most 16, at most 14, at most 12, at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 covalent bond per molecule. Any of the lower and higher values described in this paragraph can be combined to form a range included within the present invention. For example, in some cases, the number of covalent bonds between the branched-chain polymer molecules of the novel layer and the molecules of the previous layer can range from about 4 to about 16. Those skilled in the art will recognize that any value of the number of covalent bonds between the branched-chain polymer molecules of the novel layer and the molecules of the previous layer can be within this range, such as about 11 in some cases, or an average number of about 4.6 in other cases. Any reactive functional groups remaining after coupling the material layer to the substrate surface can optionally be blocked by coupling small inert molecules using high-yield coupling chemistry. For example, in the case where amine coupling chemistry is used to attach a novel material layer to the previous layer, any remaining amino groups can subsequently be acetylated or inactivated by coupling with a small amino acid such as glycine. The number of layers of a low non-specific binding material, such as a hydrophilic polymer material, deposited on the surface of the disclosed low-binding substrate can range from 1 to about 10. In some cases, the number of layers is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. In some cases, the number of layers can be at most 10, at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1. Any of the lower and higher values described in this paragraph can be combined to form a range included within the present invention. For example, in some cases, the number of layers can range from about 2 to about 4. In some cases, all layers can comprise the same material. In some cases, each layer can comprise a different material. In some cases, the plurality of layers can comprise a plurality of materials. In some cases, at least one layer can comprise a branched-chain polymer. In some cases, all layers can comprise a branched-chain polymer. One or more layers of a low non-specific binding material can be deposited on and / or coupled to the surface of a substrate using, in some cases, a polar protic solvent, a polar aprotic solvent, a non-polar solvent, or any combination thereof. In some cases, the solvent used for layer deposition and / or coupling can comprise an alcohol (e.g., methanol, ethanol, propanol, etc.), another organic solvent (e.g., acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), etc.), water, a buffered aqueous solution (e.g., phosphate buffer, phosphate buffered saline, 3-(N-morpholino)propanesulfonic acid (MOPS), etc.), or any combination thereof. In some cases, the organic component of the solvent mixture used can comprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, or any percentage spanning or near the ranges herein, with the remainder consisting of water or a buffered aqueous solution. In some cases, the aqueous component of the solvent mixture used can comprise at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, or any percentage spanning or near the ranges herein, with the remainder consisting of an organic solvent. The pH of the solvent mixture used can be less than 5, 5, 5, 5, 6, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or greater than 10, or any value spanning or near the ranges described herein. In some cases, one or more low non-specific binding material layers can be deposited on and / or bound to a substrate surface using a mixture of organic solvents, where at least one component has a dielectric constant of less than 40 and constitutes at least 50% by volume of the total mixture. In some cases, the dielectric constant of at least one component can be less than 10, less than 20, less than 30, less than 40. In some cases, at least one component constitutes at least 20%, at least 30%, at least 40%, at least 50%, at least 50%, at least 60%, at least 70% or at least 80% by volume of the total mixture. As noted, the low non-specific binding substrates of the present invention exhibit reduced non-specific binding of proteins, nucleic acids, and other components of hybridization and / or amplification formulations for solid-phase nucleic acid amplification. The degree of non-specific binding presented by a given substrate surface can be evaluated qualitatively or quantitatively. For example, in some cases, exposing the surface to fluorescent dyes (e.g., Cy3, Cy5, etc.), fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g., polymerase) under a set of standardized conditions, followed by a specified wash protocol and fluorescence imaging, can be used as a qualitative tool for comparing non-specific binding on substrates containing different surface formulations. In some cases, exposing the surface to fluorescent dyes, fluorescently labeled nucleotides, fluorescently labeled oligonucleotides, and / or fluorescently labeled proteins (e.g., polymerase) under a set of standardized conditions, followed by a specified wash protocol and fluorescence imaging, can be used as a quantitative tool for comparing non-specific binding on substrates containing different surface formulations - provided that conditions are noted to ensure that the fluorescence signal is linearly related (or related in a predictable manner) to the number of fluorophores on the substrate surface (e.g., under conditions where signal saturation and / or self-quenching of the fluorophores are not issues), and suitable calibration standards are used. In some cases, other techniques known to those skilled in the art, such as radioisotope labeling and counting methods, can be used to quantitatively evaluate the degree of non-specific binding presented by different substrate surface formulations of the present invention. The ratio of specific to non-specific binding of some surfaces disclosed herein presenting fluorophores such as Cy3 is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value across the ranges herein. The ratio of specific to non-specific fluorescence of some surfaces disclosed herein presenting fluorophores such as Cy3 is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value across the ranges herein. As noted, in some cases, for contacting a surface with a labeled protein (e.g., bovine serum albumin (BSA), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-stranded binding protein (SSB), etc. or any combination thereof), a labeled nucleotide, a labeled oligonucleotide, etc. under a set of standardized culturing and washing conditions, followed by detecting the amount of label remaining on the surface and comparing the signal obtained therefrom with an appropriate calibration standard, the degree of non-specific binding presented by the disclosed low-binding substrate can be evaluated using a standardized protocol. In some cases, the label may comprise a fluorescent label. In some cases, the label may comprise a radioisotope. In some cases, the label may comprise any other detectable label known to those skilled in the art. In some cases, the degree of non-specific binding presented by a given substrate surface formulation can thus be evaluated in terms of the number of non-specifically bound protein molecules (or other molecules) per unit area. In some cases, the low-binding substrate of the present invention may exhibit non-specific protein binding (or non-specific binding of other designated molecules, such as Cy3 dye) of less than 0.001 molecules / µm 2 , less than 0.01 molecules / µm 2 , less than 0.1 molecules / µm 2 , less than 0.25 molecules / µm 2 , less than 0.5 molecules / µm 2 , less than 1 molecule / µm 2 , less than 10 molecules / µm 2 , less than 100 molecules / µm 2 or less than 1,000 molecules / µm 2 . Those skilled in the art will recognize that a given substrate surface of the present invention may exhibit non-specific binding anywhere within this range, e.g., less than 86 molecules / µm 2 . For example, after contacting with a 1 uM solution of Cy3-labeled streptavidin (GE Amersham) in phosphate-buffered saline (PBS) buffer for 15 minutes, followed by washing 3 times with deionized water, some of the modified surfaces disclosed herein exhibit non-specific protein binding of less than 0.5 molecules / um 2. Some of the modified surfaces disclosed herein exhibit non-specific binding of Cy3 dye molecules of less than 0.25 molecules / μm 2。In the non-dependent non-specific binding analysis, a 384-well plate was incubated at 37 °C on a low-binding substrate with 1 μM labeled Cy3 SA (ThermoFisher), 1 μM Cy5 SA dye (ThermoFisher), 10 μM aminoallyl-dUTP-ATTO-647N (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rho11 (Jena Biosciences), 10 μM aminoallyl-dUTP-ATTO-Rho11 (Jena Biosciences), 10 μM 7-propynylamino-7-deaza-dGTP-Cy5 (Jena Biosciences), and 10 μM 7-propynylamino-7-deaza-dGTP-Cy3 (Jena Biosciences) for 15 minutes. Each well was washed 2-3 times with 50 μl of deionized RNase / DNase-free water and 2-3 times with 25 mM ACES buffer pH 7.4. The 384-well plate was imaged on a GE Typhoon (GE Healthcare Lifesciences, Pittsburgh, PA) instrument using a Cy3, AF555, or Cy5 filter set as specified by the manufacturer (depending on the dye test performed), with the PMT gain set to 800 and a resolution of 50-100 μm. For higher-resolution imaging, images were collected on an Olympus IX83 microscope (Olympus Corp., Center Valley, PA) with a total internal reflection fluorescence (TIRF) objective (20×, 0.75 NA or 100×, 1.5 NA, Olympus), a CCD camera (e.g., Olympus EM-CCD monochrome camera, Olympus XM-10 monochrome camera, or Olympus DP80 color and monochrome camera), an illumination source (e.g., Olympus 100W Hg lamp, Olympus 75W Xe lamp, or Olympus U-HGLGPS fluorescence light source), and an excitation wavelength of 532 nm or 635 nm. Dichroic mirrors were purchased from (IDEX Health & Science, LLC, Rochester, New York), e.g., 405, 488, 532, or 633 nm dichroic reflectors / beam splitters, and bandpass filters were selected as 532 LP or 645 LP consistent with the appropriate excitation wavelength. Some of the modified surfaces disclosed herein exhibit non-specific binding of dye molecules lower than 0.25 molecules / μm 2 。 In some cases, the ratio of specific to non-specific binding of some fluorophores such as Cy3 presented herein is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value across the ranges herein. In some cases, the ratio of specific to non-specific fluorescence signals of some fluorophores such as Cy3 presented herein is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value across the ranges herein. A low-background surface consistent with the disclosure herein can exhibit a ratio of specific dye attachment (e.g., Cy3 attachment) to non-specific dye adsorption (e.g., Cy3 dye adsorption) of at least 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50 specific dye molecules attached per non-specifically adsorbed molecule. Similarly, when subjected to excitation energy, a low-background surface consistent with the disclosure herein to which a fluorophore such as Cy3 has been attached can exhibit a ratio of specific fluorescence signal (e.g., generated by a Cy3-labeled oligonucleotide attached to the surface) to non-specifically adsorbed dye fluorescence signal of at least 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50:1. In some cases, the hydrophilicity (or "wettability" with an aqueous solution) of the disclosed substrate surface can be evaluated, for example, by measuring the water contact angle, where a small water droplet is placed on the surface and its contact angle with the surface is measured using, for example, an optical tensiometer. In some cases, the electrostatic contact angle can be measured. In some cases, the advancing or receding contact angle can be measured. In some cases, the water contact angle of the hydrophilic low-binding substrate surface disclosed herein can be in the range of about 0 degrees to about 50 degrees. In some cases, the water contact angle of the hydrophilic low-binding substrate surface disclosed herein can be no more than 50 degrees, 45 degrees, 40 degrees, 35 degrees, 30 degrees, 25 degrees, 20 degrees, 18 degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6 degrees, 4 degrees, 2 degrees, or 1 degree. In many cases, the contact angle does not exceed any value within this range, for example, does not exceed 40 degrees. Those skilled in the art will recognize that a given hydrophilic low-binding substrate surface of the present invention can exhibit a water contact angle value anywhere within this range, for example, about 27 degrees. In some cases, the hydrophilic surfaces disclosed herein help reduce the wash time for bioanalysis, typically due to reduced non-specific binding of biomolecules to low-binding surfaces. In some cases, sufficient wash steps can be performed in less than 60, 50, 40, 30, 20, 15, 10, or less than 10 seconds. For example, in some cases, sufficient wash steps can be performed in less than 30 seconds. Some of the low-binding surfaces of the present invention exhibit a significantly improved stability or durability against repeated cycles of long-term exposure to solvents and high temperatures or solvent exposure or temperature changes. For example, in some cases, the stability of the disclosed surfaces can be tested by fluorescently labeling functional groups on the surface or biomolecules tethered to the surface (e.g., oligonucleotide primers) and monitoring the fluorescent signal before, during, and after repeated cycles of long-term exposure to solvents and high temperatures or solvent exposure or temperature changes. In some cases, the degree of fluorescent change used to assess surface quality can be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% (or any combination of such percentages measured within such time periods) within a time period of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 100 hours of exposure to solvents and / or high temperatures. In some cases, the degree of fluorescent change used to assess surface quality can be less than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% (or any combination of such percentages measured within such cycle ranges) within 5 cycles, 10 cycles, 20 cycles, 30 cycles, 40 cycles, 50 cycles, 60 cycles, 70 cycles, 80 cycles, 90 cycles, 100 cycles, 200 cycles, 300 cycles, 400 cycles, 500 cycles, 600 cycles, 700 cycles, 800 cycles, 900 cycles, or 1,000 cycles of repeated exposure to solvent changes and / or temperature changes. In some cases, the surfaces disclosed herein can exhibit a relatively high ratio of specific signal to non-specific signal or other background. For example, when used for nucleic acid amplification, some surfaces can exhibit an amplified signal that is at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold greater than the signal in adjacent non-dense regions of the surface. Similarly, some surfaces exhibit an amplified signal that is at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold greater than the signal in adjacent amplified nucleic acid population regions of the surface. The fluorescence excitation energy varies for specific fluorophores and protocols and can range from wavelengths below 400 nm to above 800 nm, which is consistent with the fluorophore selection or other parameters for the use of the surfaces disclosed herein. Thus, the low-background surfaces disclosed herein exhibit a low background fluorescence signal or a high contrast-to-noise (CNR) ratio relative to surfaces known in the art. For example, in some cases, the background fluorescence of the surface at spatially distinct locations or at locations removed from labeled features (e.g., labeled spots, clusters, discrete regions, sub-parts, or sub-groups of the surface) overlying hybridized clusters of nucleic acid molecules or clonally amplified clusters of nucleic acid molecules generated by, for example, 20 cycles of nucleic acid amplification via thermal cycling can be no more than 20-fold, 10-fold, 5-fold, 2-fold, 1-fold, 0.5-fold, 0.1-fold, or less than 0.1-fold greater than the background fluorescence measured at the same location during those 20 cycles of hybridization or nucleic acid amplification. In some cases, when used in nucleic acid hybridization or amplification applications to form hybridized or clonally amplified clusters of nucleic acid molecules (e.g., which have been directly or indirectly labeled with a fluorophore), the fluorescence images of the disclosed low-background surfaces exhibit a contrast-to-noise ratio (CNR) of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250, or greater than 250. Oligonucleotide primers and adapter sequences Generally, one or more layers of low non-specific binding material can comprise functional groups for covalently or non-covalently linking oligonucleotide molecules, such as adapter or primer sequences, or at least one layer can already comprise covalently or non-covalently linked oligonucleotide adapters or primer sequences when deposited on the substrate surface. In some cases, oligonucleotides of polymer molecules tethered to at least a third layer can be distributed at multiple depths within the layer. In some cases, oligonucleotide adaptors or primer molecules are covalently coupled to a polymer in solution, i.e., the polymer is then coupled or deposited on a surface. In some cases, the oligonucleotide adaptor or primer molecule is covalently coupled to the polymer after it has been coupled to or deposited on the surface. In some cases, at least one hydrophilic polymer layer comprises a plurality of covalently linked oligonucleotide adaptors or primer molecules. In some cases, at least two, at least three, at least four, or at least five layers of the hydrophilic polymer comprise a plurality of covalently linked adaptors or primer molecules. In some cases, any of a variety of suitable conjugation chemistries known to those of skill in the art can be used to couple the oligonucleotide adaptor or primer molecule to one or more layers of the hydrophilic polymer. For example, the oligonucleotide adaptor or primer sequence can comprise moieties that can react with amino, carboxyl, thiol, and similar groups. Examples of suitable amine-reactive conjugation chemistries include, but are not limited to, reactants involving isothiocyanate groups, isocyanate groups, acyl azide groups, NHS ester groups, sulfonyl chloride groups, aldehyde groups, glyoxal groups, epoxide groups, oxirane groups, carbonate groups, aryl halide groups, imidoester groups, carbodiimide groups, anhydride groups, and fluorophenyl ester groups. Examples of suitable carboxyl-reactive conjugation chemistries include, but are not limited to, reactions involving carbodiimide compounds, such as water-soluble EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide·HCl). Examples of suitable thiol-reactive conjugation chemistries include maleimide, haloacetyl, and pyridyl disulfide. One or more types of oligonucleotide molecules can be linked or tethered to a substrate surface. In some cases, one or more types of oligonucleotide adaptors or primers can comprise spacer sequences, adaptor sequences for hybridizing to a template library nucleic acid sequence conjugated to the adaptor, forward amplification primers, reverse amplification primers, sequencing primers, and / or molecular barcode sequences, or any combination thereof. In some cases, 1 primer or adaptor sequence can be tethered to at least one layer of the surface. In some cases, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 different primer or adaptor sequences can be tethered to at least one layer of the surface. In some cases, the length of the tethered oligonucleotide adaptor and / or primer sequence can range from about 10 nucleotides to about 100 nucleotides. In some cases, the length of the tethered oligonucleotide adaptor and / or primer sequence can be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides. In some cases, the length of the tethered oligonucleotide adaptor and / or primer sequence can be at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, or at most 10 nucleotides. Any of the lower and higher values described in this paragraph can be combined to form a range included within the present invention. For example, in some cases, the length of the tethered oligonucleotide adaptor and / or primer sequence can range from about 20 nucleotides to about 80 nucleotides. Those skilled in the art will recognize that any value of the length of the tethered oligonucleotide adaptor and / or primer sequence can be within this range, such as about 24 nucleotides. In some cases, the tethered adaptor or primer sequence can contain modifications designed to facilitate the specificity and efficiency of nucleic acid amplification, such as on a low-binding substrate. For example, in some cases, the primer can contain a polymerase stop point such that the segment of the primer sequence between the surface-binding site and the modification site remains single-stranded and serves as a loading site for a 5' to 3' helicase in some helicase-dependent isothermal amplification methods. Other examples of primer modifications that can be used to form a polymerase stop point include, but are not limited to, inserting a PEG chain into the backbone of the primer towards the 5' end between two nucleotides, inserting an abasic nucleotide (i.e., a nucleotide that has neither a purine nor a pyrimidine base), or a lesion site that can be bypassed by a helicase. As will be further discussed in the examples below, it may be necessary to vary the surface density of oligonucleotide adaptors or primers tethered to the substrate surface and / or the distance of the tethered adaptors or primers from the substrate surface (e.g., by varying the length of the linking molecule used to tether the adaptor or primer to the surface) to "tune" the substrate for optimal performance when using a given amplification method. As noted below, adjusting the surface density of the tethered oligonucleotide adaptors or primers can affect the degree of specific and / or non-specific amplification observed on the substrate in a manner that varies according to the selected amplification method. In some cases, the surface density of the tethered oligonucleotide adaptors or primers can be varied by adjusting the ratio of the molecular components used to form the substrate surface. For example, in the case where an oligonucleotide primer-PEG conjugate is used to form the final layer of a low-binding substrate, the ratio of the oligonucleotide primer-PEG conjugate to the unbound PEG molecules can be varied. The resulting surface density of the tethered primer molecules can then be estimated or measured using any of a variety of techniques known to those skilled in the art. Examples include, but are not limited to, using radioisotope labeling and counting methods, covalently coupling a cleavable molecule containing an optically detectable label (e.g., a fluorescent label) that can be cleaved from the substrate surface in a defined area, collected in a fixed volume of an appropriate solvent, and then quantified by comparing the fluorescence signal to the fluorescence signal of a calibration solution of known optical label concentration, or using fluorescence imaging techniques, with the proviso that care is taken with the labeling reaction conditions and the image acquisition settings to ensure that the fluorescence signal is linearly related to the number of fluorophores on the surface (e.g., there is no significant self-quenching of the fluorophores on the surface). In some cases, the resulting surface density of oligonucleotide adaptors or primers on the low-binding substrate surface of the present invention can be from about 100 primer molecules / μm 2 to about 1,000,000 primer molecules / μm 2Within a range. In some cases, the surface density of the oligonucleotide linker or primer can be at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500, at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least 9,000, at least 9,500, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000 or at least 1,000,000 molecules / µm 2。In some cases, the surface density of oligonucleotide adaptors or primers can be at most 1,000,000, at most 950,000, at most 900,000, at most 850,000, at most 800,000, at most 750,000, at most 700,000, at most 650,000, at most 600,000, at most 550,000, at most 500,000, at most 450,000, at most 400,000, at most 350,000, at most 300,000, at most 250,000, at most 200,000, at most 150,000, at most 100,000, at most 95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most 9,500, at most 9,000, at most 8,500, at most 8,000, at most 7,500, at most 7,000, at most 6,500, at most 6,000, at most 5,500, at most 5,000, at most 4,500, at most 4,000, at most 3,500, at most 3,000, at most 2,500, at most 2,000, at most 1,500, at most 1,000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 400, at most 300, at most 200 or at most 100 molecules / µm 2 。Any of the lower and higher values described in this paragraph can be combined to form a range included within the present invention. For example, in some cases, the surface density of the adaptor or primer can be between about 10,000 molecules / µm 2 and about 100,000 molecules / µm 2 The person skilled in the art will recognize that any value of the surface density of the adaptor or primer molecule can be within this range. For example, in some cases, about 3,800 molecules / µm 2 or, in other cases, about 455,000 molecules / µm 2。In some cases, as will be further discussed below, the surface density of template library nucleic acid sequences (e.g., sample DNA molecules) that are first hybridized to adaptor or primer sequences on a substrate surface can be less than or equal to the surface density specified for the surface density of the tethered oligonucleotide primers. In some cases, as will also be further discussed below, the surface density of the cloned and amplified template library nucleic acid sequences hybridized to adaptor or primer sequences on a substrate surface can span the same range or a different range as the surface density specified for the surface density of the tethered oligonucleotide adaptors or primers. The local surface density of the adaptor or primer molecules as listed above does not exclude density changes on the surface such that the surface can contain regions with an oligomer density of, for example, 500,000 / μm 2 while also containing at least a second region with a substantially different local density. Hybridizing nucleic acid molecules to a low-binding substrate : In some aspects of the present invention, hybridization buffer formulations are described that, in combination with the disclosed low-binding substrates, provide improved hybridization rates, hybridization specificity (or stringency), and hybridization efficiency (or yield). As used herein, hybridization specificity is a measure of the ability of the tethered adaptor sequence, primer sequence, or oligonucleotide sequence to typically hybridize only correctly to a fully complementary sequence, and hybridization efficiency is a measure of the percentage of the total available tethered adaptor sequence, primer sequence, or oligonucleotide sequence that typically hybridizes to a complementary sequence. Improved hybridization specificity and / or efficiency can be achieved by optimizing the hybridization buffer formulation used with the disclosed low-binding surface and will be discussed in more detail in the following examples. Examples of hybridization buffer components that can be adjusted to achieve improved performance include, but are not limited to, buffer type, organic solvent mixtures, buffer pH, buffer viscosity, detergents and zwitterionic components, ionic strength (including adjusting monovalent and divalent ion concentrations), antioxidants and reducing agents, carbohydrates, BSA, polyethylene glycol, dextran sulfate, betaine, other additives, and the like. By way of non-limiting example, suitable buffers for formulating hybridization buffers can include but are not limited to phosphate buffered saline (PBS), succinate, citrate, histidine, acetate, Tris, TAPS, MOPS, PIPES, HEPES, MES, and the like. The choice of appropriate buffer will generally depend on the target pH of the hybridization buffer solution. Generally speaking, the desired pH of the buffer solution will be in the range of about pH 4 to about pH 8.4. In some embodiments, the buffer pH can be at least 4.0, at least 4.5, at least 5.0, at least 5.5, at least 6.0, at least 6.2, at least 6.4, at least 6.6, at least 6.8, at least 7.0, at least 7.2, at least 7.4, at least 7.6, at least 7.8, at least 8.0, at least 8.2, or at least 8.4. In some embodiments, the buffer pH can be at most 8.4, at most 8.2, at most 8.0, at most 7.8, at most 7.6, at most 7.4, at most 7.2, at most 7.0, at most 6.8, at most 6.6, at most 6.4, at most 6.2, at most 6.0, at most 5.5, at most 5.0, at most 4.5, or at most 4.0. Any of the lower and higher values described in this paragraph can be combined to form a range included within the present invention. For example, in some cases, the desired pH can be in the range of about 6.4 to about 7.2. Those skilled in the art will recognize that any value of buffer pH can be within this range, such as about 7.25. Detergents suitable for hybridization buffer formulations include but are not limited to zwitterionic detergents (e.g., 1-dodecanoyl-sn-glycero-3-phosphocholine, 3-(4-tert-butyl-1-pyridinio)propanesulfonate, 3-(N,N-dimethyltetradecylammonio)propanesulfonate, 3-(N,N-dimethyltetradecylammonio)propanesulfonate, ASB-C80, C7BzO, CHAPS, CHAPS hydrate, CHAPSO, DDMAB, dimethylethylammonium propane sulfonate, N,N-dimethyldodecylamine N-oxide, N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate or N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) and anionic, cationic, and nonionic detergents. Examples of nonionic detergents include poly(ethylene oxide) ethers and related polymers (e.g., Brij®, TWEEN®, TRITON®, TRITON X-100, and IGEPAL® CA-630), bile salts, and glycoside detergents. The low-binding substrate disclosed, used alone or in combination with an optimized buffer formulation, can produce a relative hybridization rate that is in the range of about 2 to about 20 times faster than the hybridization rate of conventional hybridization protocols. In some cases, the relative hybridization rate can be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 14 times, at least 16 times, at least 18 times, at least 20 times, at least 25 times, at least 30 times, or at least 40 times the hybridization rate of a conventional hybridization protocol. In some cases, the low-binding substrate disclosed, used alone or in combination with an optimized buffer formulation, can produce a total hybridization reaction time (i.e., the time required to reach 90%, 95%, 98%, or 99% completion of the hybridization reaction) of less than 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes for any of these completion metrics. In some cases, the low-binding substrate disclosed, used alone or in combination with an optimized buffer formulation, can produce improved hybridization specificity compared to conventional hybridization protocols. In some cases, a hybridization specificity can be achieved that is better than 1 base mismatch in 10 hybridization events, 1 base mismatch in 20 hybridization events, 1 base mismatch in 30 hybridization events, 1 base mismatch in 40 hybridization events, 1 base mismatch in 50 hybridization events, 1 base mismatch in 75 hybridization events, 1 base mismatch in 100 hybridization events, 1 base mismatch in 200 hybridization events, 1 base mismatch in 300 hybridization events, 1 base mismatch in 400 hybridization events, 1 base mismatch in 500 hybridization events, 1 base mismatch in 600 hybridization events, 1 base mismatch in 700 hybridization events, 1 base mismatch in 800 hybridization events, 1 base mismatch in 900 hybridization events, 1 base mismatch in 1,000 hybridization events, 1 base mismatch in 2,000 hybridization events, 1 base mismatch in 3,000 hybridization events, 1 base mismatch in 4,000 hybridization events, 1 base mismatch in 5,000 hybridization events, 1 base mismatch in 6,000 hybridization events, 1 base mismatch in 7,000 hybridization events, 1 base mismatch in 8,000 hybridization events, 1 base mismatch in 9,000 hybridization events, or 1 base mismatch in 10,000 hybridization events. In some cases, the low-binding substrates disclosed, used alone or in combination with an optimized buffer formulation, can produce improved hybridization efficiency (e.g., the oligonucleotide primer moieties obtainable on the substrate surface that have successfully hybridized with the target oligonucleotide sequence) compared to conventional hybridization protocols. In some cases, for any of the input target oligonucleotide concentrations specified below and for any of the hybridization reaction times specified above, a hybridization efficiency of greater than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% can be achieved. In some cases, such as where the hybridization efficiency is less than 100%, the resulting surface density of the target nucleic acid sequence hybridized to the substrate surface can be less than the surface density of the oligonucleotide linker or primer sequences on the surface. In some cases, the use of a low-binding substrate as disclosed in conventional hybridization (or amplification) protocols or optimized hybridization (or amplification) protocols for nucleic acid hybridization (or amplification) applications can result in a reduced requirement for the input concentration of target (or sample) nucleic acid molecules in contact with the substrate surface. For example, in some cases, the target (or sample) nucleic acid molecules can be contacted with the substrate surface at a concentration in the range of about 10 pM to about 1 μM (i.e., prior to adhesion or amplification). In some cases, the target (or sample) nucleic acid molecules can be administered at a concentration of at least 10 pM, at least 20 pM, at least 30 pM, at least 40 pM, at least 50 pM, at least 100 pM, at least 200 pM, at least 300 pM, at least 400 pM, at least 500 pM, at least 600 pM, at least 700 pM, at least 800 pM, at least 900 pM, at least 1 nM, at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM or at least 1 μM. In some cases, the target (or sample) nucleic acid molecules can be administered at a concentration of at most 1 μM, at most 900 nM, at most 800 nM, at most 700 nM, at most 600 nM, at most 500 nM, at most 400 nM, at most 300 nM, at most 200 nM, at most 100 nM, at most 90 nM, at most 80 nM, at most 70 nM, at most 60 nM, at most 50 nM, at most 40 nM, at most 30 nM, at most 20 nM, at most 10 nM, at most 1 nM, at most 900 pM, at most 800 pM, at most 700 pM, at most 600 pM, at most 500 pM, at most 400 pM, at most 300 pM, at most 200 pM, at most 100 pM, at most 90 pM, at most 80 pM, at most 70 pM, at most 60 pM, at most 50 pM, at most 40 pM, at most 30 pM, at most 20 pM or at most 10 pM. Any of the lower and higher values described in this paragraph can be combined to form a range included within the present invention. For example, in some cases, the target (or sample) nucleic acid molecules can be administered at a concentration in the range of about 90 pM to about 200 nM. Those skilled in the art will recognize that the target (or sample) nucleic acid molecules can be at any value within this range, such as at a concentration of about 855 nM. In some cases, the low-binding substrate disclosed, alone or in combination with an optimized hybridization buffer formulation, can result in a surface density of hybridization target (or sample) oligonucleotide molecules (i.e., prior to any subsequent solid-phase or cloning amplification reactions) in the range of about 0.0001 target oligonucleotide molecules / μm 2 to about 1,000,000 target oligonucleotide molecules / μm 2 In some cases, the surface density of the hybridization target oligonucleotide molecules can be at least 0.0001, at least 0.0005, at least 0.001, at least 0.005, at least 0.01, at least 0.05, at least 0.1, at least 0.5, at least 1, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500, at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least 9,000, at least 9,500, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000 or at least 1,000,000 molecules / μm 2. In some cases, the surface density of the hybridized target oligonucleotide molecules can be at most 1,000,000, at most 950,000, at most 900,000, at most 850,000, at most 800,000, at most 750,000, at most 700,000, at most 650,000, at most 600,000, at most 550,000, at most 500,000, at most 450,000, at most 400,000, at most 350,000, at most 300,000, at most 250,000, at most 200,000, at most 150,000, at most 100,000, at most 95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most 9,500, at most 9,000, at most 8,500, at most 8,000, at most 7,500, at most 7,000, at most 6,500, at most 6,000, at most 5,500, at most 5,000, at most 4,500, at most 4,000, at most 3,500, at most 3,000, at most 2,500, at most 2,000, at most 1,500, at most 1,000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 400, at most 300, at most 200, at most 100, at most 90, at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, at most 20, at most 10, at most 5, at most 1, at most 0.5, at most 0.1, at most 0.05, at most 0.01, at most 0.005, at most 0.001, at most 0.0005 or at most 0.0001 molecules / µm 2 . Any one of the lower and higher values described in this paragraph can be combined to form a range included in the present invention. For example, in some cases, the surface density of the hybridized target oligonucleotide molecules can be about 3,000 molecules / µm 2 to about 20,000 molecules / µm 2 . Those skilled in the art will recognize that any value of the surface density of the hybridized target oligonucleotide molecules can be within this range, such as about 2,700 molecules / µm 2 . In other words, in some cases, the low-binding substrate disclosed, used alone or in combination with an optimized hybridization buffer formulation, can result in a surface density of hybridization target (or sample) oligonucleotide molecules (i.e., prior to any subsequent solid-phase or cloning amplification reactions) in the range of about 100 hybridization target oligonucleotide molecules / mm 2 to about 1 × 10 12 hybridization target oligonucleotide molecules / mm 2 In some cases, the surface density of hybridization target oligonucleotide molecules can be at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1 × 10 7 at least 5 × 10 7 at least 1 × 10 8 at least 5 × 10 8 at least 1 × 10 9 at least 5 × 10 9 at least 1 × 10 10 at least 5 × 10 10 at least 1 × 10 11 at least 5 × 10 11or at least 1 × 10 12 molecules / mm 2 . In some cases, the surface density of the hybridized target oligonucleotide molecules can be at most 1 × 10 12 , at most 5 × 10 11 , at most 1 × 10 11 , at most 5 × 10 10 , at most 1 × 10 10 , at most 5 × 10 9 , at most 1 × 10 9 , at most 5 × 10 8 , at most 1 × 10 8 , at most 5 × 10 7 , at most 1 × 10 7 , at most 5,000,000, at most 1,000,000, at most 950,000, at most 900,000, at most 850,000, at most 800,000, at most 750,000, at most 700,000, at most 650,000, at most 600,000, at most 550,000, at most 500,000, at most 450,000, at most 400,000, at most 350,000, at most 300,000, at most 250,000, at most 200,000, at most 150,000, at most 100,000, at most 95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most 5,000, at most 1,000, at most 500 or at most 100 molecules / mm 2。Any one of the lower and higher values described in this paragraph can be combined to form a range included in the present invention. For example, in some cases, the surface density of the hybridized target oligonucleotide molecules can be from about 5,000 molecules / mm 2 to about 50,000 molecules / mm 2 . Those skilled in the art will recognize that any value of the surface density of the hybridized target oligonucleotide molecules can be within this range, such as about 50,700 molecules / µm 2 . In some cases, the length of the target (or sample) oligonucleotide molecule (or nucleic acid molecule) hybridized to the oligonucleotide linker or primer molecule attached to the low-binding substrate surface can be in the range of about 0.1 kilobases (kb) to about 20 kb. In some cases, the length of the target oligonucleotide molecule can be at least 0.1 kb, at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 0.6 kb, at least 0.7 kb, at least 0.8 kb, at least 0.9 kb, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 15 kb, at least 20 kb, at least 30 kb, or at least 40 kb, or any intermediate value across the range described herein, such as at least 0.85 kb. In some cases, the target (or sample) oligonucleotide molecule (or nucleic acid molecule) can comprise a single-stranded or double-stranded polynucleic acid molecule, which further comprises repeating regularly occurring monomer units. In some cases, the length of the single-stranded or double-stranded polynucleic acid molecule can be at least 0.1 kb, at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 15 kb, or at least 20 kb, at least 30 kb, or at least 40 kb, or any intermediate value across the range described herein, such as about 2.45 kb. In some cases, a target (or sample) oligonucleotide molecule (or nucleic acid molecule) can comprise a single-stranded or double-stranded polynucleic acid molecule that includes a copy of from about 2 to about 100 regularly repeating monomer units. In some cases, the number of copies of the regularly repeating monomer units can be at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, and at least 100. In some cases, the number of copies of the regularly repeating monomer units can be at most 100, at most 95, at most 90, at most 85, at most 80, at most 75, at most 70, at most 65, at most 60, at most 55, at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, at most 5, at most 4, at most 3, or at most 2. Any of the lower and higher values described in this paragraph can be combined to form ranges included within the present invention. For example, in some cases, the number of copies of the regularly repeating monomer units can be in the range of from about 4 to about 60. One of ordinary skill in the art will recognize that any value of the number of copies of the regularly repeating monomer units can be within this range, such as about 17. Thus, in some cases, with respect to the number of copies of the target sequence per unit area of the substrate surface, the surface density of the hybridized target sequences can exceed the surface density of the oligonucleotide primers, even if the hybridization efficiency is less than 100%. Nucleic Acid Surface Amplification ( NASA ) : As used herein, the phrase "Nucleic Acid Surface Amplification" (NASA) can be used interchangeably with the phrase "solid-phase nucleic acid amplification" (or simply "solid-phase amplification"). In some aspects of the present invention, nucleic acid amplification formulations are described that, in combination with the disclosed low-binding substrates, provide improved amplification rates, amplification specificity, and amplification efficiency. As used herein, specific amplification refers to the amplification of a library of template oligonucleotide strands that have been covalently or non-covalently tethered to a solid substrate. As used herein, non-specific amplification refers to the amplification of primer-dimers or other non-template nucleic acids. As used herein, amplification efficiency is a measure of the percentage of oligonucleotides tethered to the surface of the substrate that are successfully amplified during a given amplification cycle or amplification reaction. Nucleic acid amplification performed on the surfaces disclosed herein can achieve an amplification efficiency of at least 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%, such as 98% or 99%. Any one of various thermal cycling or isothermal nucleic acid amplification procedures can be used with the disclosed low-binding substrates. Examples of nucleic acid amplification methods that can be used with the disclosed low-binding substrates include, but are not limited to, polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification, circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, or single-strand binding (SSB) protein-dependent amplification. Generally, the disclosed low-binding substrates can be used alone or in combination with a formulation of amplification reaction components to achieve improvements in amplification rate, amplification specificity, and amplification efficiency. In addition to including nucleotides, one or more polymerases, helicases, single-strand binding proteins, etc. (or any combination thereof), the amplification reaction mixture can be adjusted in a variety of ways to achieve improved performance, including but not limited to selecting buffer type, buffer pH, organic solvent mixture, buffer viscosity, detergent and zwitterionic components, ionic strength (including adjusting monovalent and divalent ion concentrations), antioxidants and reducing agents, carbohydrates, BSA, polyethylene glycol, dextran sulfate, betaine, other additives, and the like. Using the disclosed low-binding substrates alone or in combination with an optimized amplification reaction formulation can produce an increased amplification rate compared to those obtained using conventional substrates and amplification protocols. In some cases, the relative amplification rate that can be achieved can be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 12-fold, at least 14-fold, at least 16-fold, at least 18-fold, or at least 20-fold the amplification rate of a conventional substrate and amplification protocol for any one of the methods of amplification described above. In some cases, for any one of these completion metrics, using the disclosed low-binding substrates alone or in combination with an optimized buffer formulation can produce an amplification reaction total time of less than 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes (i.e., the time required to reach 90%, 95%, 98%, or 99% completion of the amplification reaction). Some of the low-binding substrates disclosed herein exhibit a ratio of specific binding to non-specific binding of a fluorophore such as Cy3 of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate value spanning the ranges herein. Some of the surfaces disclosed herein exhibit a ratio of specific to non-specific fluorescent signals of a fluorophore such as Cy3 of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate value spanning the ranges herein. In some cases, use of the disclosed low-binding substrates alone or in combination with an optimized amplification buffer formulation can permit a total amplification reaction time of no more than 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, or 10 minutes (i.e., the time required to reach 90%, 95%, 98%, or 99% completion of the amplification reaction). Similarly, in some cases, use of the disclosed low-binding substrates alone or in combination with an optimized buffer formulation can permit the amplification reaction to be completed in no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or no more than 30 cycles. In some cases, use of the disclosed low-binding substrates alone or in combination with an optimized amplification reaction formulation can produce enhanced specific amplification and / or reduced non-specific amplification compared to that obtained using conventional substrates and amplification protocols. In some cases, the resulting ratio of specific amplification to non-specific amplification that can be achieved is at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, or 1,000:1. In some cases, use of the low-binding substrates alone or in combination with an optimized amplification reaction formulation can produce an increased amplification rate compared to the amplification efficiency obtained using conventional substrates and amplification protocols. In some cases, the amplification efficiency that can be achieved is greater than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% during any of the amplification reaction times specified above. In some cases, the length of the cloned and amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) hybridized to an oligonucleotide linker or primer molecule attached to a low-binding substrate surface can range from about 0.1 kilobases (kb) to about 20 kb. In some cases, the length of the cloned and amplified target oligonucleotide molecule can be at least 0.1 kb, at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 15 kb or at least 20 kb, or any intermediate value across the ranges described herein, such as at least 0.85. In some cases, the cloned and amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) can comprise a single-stranded or double-stranded polynucleotide molecule, which further comprises repetitive regularly occurring monomeric units. In some cases, the length of the cloned and amplified single-stranded or double-stranded polynucleotide molecule can be at least 0.1 kb, at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 1 kb, at least 2 kb, at least 3 kb, at least 4 kb, at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, at least 10 kb, at least 15 kb or at least 20 kb, or any intermediate value across the ranges described herein, such as about 2.45 kb. In some cases, the cloned and amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) can comprise a single-stranded or double-stranded polynucleotide molecule that comprises about 2 to about 100 copies of a regularly repeating monomer unit. In some cases, the number of copies of the regularly repeating monomer unit can be at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, and at least 100. In some cases, the number of copies of the regularly repeating monomer unit can be at most 100, at most 95, at most 90, at most 85, at most 80, at most 75, at most 70, at most 65, at most 60, at most 55, at most 50, at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, at most 5, at most 4, at most 3, or at most 2. Any of the lower and higher values described in this paragraph can be combined to form a range that is included within the present invention. For example, in some cases, the number of copies of the regularly repeating monomer unit can be in the range of about 4 to about 60. One of ordinary skill in the art will recognize that any value of the number of copies of the regularly repeating monomer unit can be within this range, such as about 12. Thus, in some cases, with respect to the number of copies of the target sequence per unit area of the substrate surface, the surface density of the cloned and amplified target sequence can exceed the surface density of the oligonucleotide primer, even if the hybridization and / or amplification efficiency is less than 100%. In some cases, the low-binding substrates disclosed, used alone or in combination with an optimized amplification reaction formulation, can produce increased numbers of clonal replicates compared to the numbers of clonal replicates obtained using conventional substrates and amplification protocols. In some cases, such as where the target (or sample) oligonucleotide molecules being clonally amplified contain tandem polymeric repeat monomer target sequences, the number of clonal replicates may be substantially smaller compared to the numbers of clonal replicates obtained using conventional substrates and amplification protocols. Thus, in some cases, the number of clonal replicates can range from about 1 molecule to about 100,000 molecules (e.g., target sequence molecules) per amplification community. In some cases, the number of clonal replicates can be at least 1, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, or at least 100,000 molecules per amplification community. In some cases, the number of clonal replicates can be at most 100,000, at most 95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most 9,000, at most 8,000, at most 7,000, at most 6,000, at most 5,000, at most 4,000, at most 3,000, at most 2,000, at most 1,000, at most 500, at most 100, at most 50, at most 10, at most 5, or at most 1 molecule per amplification community. Any of the lower and higher values described in this paragraph can be combined to form ranges that are included within the invention, e.g., in some cases, the number of clonal replicates can range from about 2,000 molecules to about 9,000 molecules. Those skilled in the art will recognize that any value of the number of clonal replicates can be within this range, e.g., about 2,220 molecules in some cases, or about 2 molecules in other cases. As noted above, in some cases, the amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) may contain tandemly repeated monomer target sequences. In some cases, the amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) may contain a plurality of molecules, each containing a single monomer target sequence. Thus, the low-binding substrate disclosed herein, used alone or in combination with an optimized amplification reaction formulation, can achieve a surface density of target sequence replicas in the range of about 100 target sequence replicas / mm 2 to about 1 × 10 12 target sequence replicas / mm 2 . In some cases, the surface density of target sequence replicas can be at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1 × 10 7 or at least 5 × 10 7 or at least 1 × 10 8 or at least 5 × 10 8 or at least 1 × 10 9 or at least 5 × 10 9 or at least 1 × 10 10 or at least 5 × 10 10 or at least 1 × 10 11 , at least 5 × 10 11 or at least 1 × 10 12 target sequence copies / mm 2 . In some cases, the surface density of the target sequence copies can be at most 1 × 10 12 , at most 5 × 10 11 , at most 1 × 10 11 , at most 5 × 10 10 , at most 1 × 10 10 , at most 5 × 10 9 , at most 1 × 10 9 , at most 5 × 10 8 , at most 1 × 10 8 , at most 5 × 10 7 , at most 1 × 10 7 , at most 5,000,000, at most 1,000,000, at most 950,000, at most 900,000, at most 850,000, at most 800,000, at most 750,000, at most 700,000, at most 650,000, at most 600,000, at most 550,000, at most 500,000, at most 450,000, at most 400,000, at most 350,000, at most 300,000, at most 250,000, at most 200,000, at most 150,000, at most 100,000, at most 95,000, at most 90,000, at most 85,000, at most 80,000, at most 75,000, at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most 5,000, at most 1,000, at most 500 or at most 100 target sequence copies / mm 2。Any one of the lower and higher values described in this paragraph can be combined to form a range included in the present invention. For example, in some cases, the surface density of target sequence replicas can be from about 1,000 target sequence replicas / mm 2 to about 65,000 target sequence replicas / mm 2 within the range. Those skilled in the art will recognize that any value of the surface density of target sequence replicas can be within this range, such as about 49,600 target sequence replicas / mm 2 。 In some cases, the disclosed low-binding substrate used alone or in combination with an optimized amplification buffer formulation can result in a surface density of the clonally amplified target (or sample) oligonucleotide colonies (or clusters) in the range of about 100 colonies / mm 2 to about 1 × 10 12 colonies / mm 2 within the range. In some cases, the surface density of the clonally amplified colonies can be at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1 × 10 7 、 at least 5 × 10 7 、 at least 1 × 10 8 、 at least 5 × 10 8, at least 1 × 10 9 , at least 5 × 10 9 , at least 1 × 10 10 , at least 5 × 10 10 , at least 1 × 10 11 , at least 5 × 10 11 or at least 1 × 10 12 colonies / mm 2 . In some cases, the surface density of the colonies after cloning and amplification can be at most 1 × 10 12 , at most 5 × 10 11 , at most 1 × 10 11 , at most 5 × 10 10 , at most 1 × 10 10 , at most 5 × 10 9 , at most 1 × 10 9 , at most 5 × 10 8 , at most 1 × 10 8 , at most 5 × 10 7 , at most 1 × 10 7, up to 5,000,000, up to 1,000,000, up to 950,000, up to 900,000, up to 850,000, up to 800,000, up to 750,000, up to 700,000, up to 650,000, up to 600,000, up to 550,000, up to 500,000, up to 450,000, up to 400,000, up to 350,000, up to 300,000, up to 250,000, up to 200,000, up to 150,000, up to 100 ,000, up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500 or up to 100 colonies / mm 2 Any of the lower and higher values ​​described in this paragraph may be combined to form a range included in the present invention. For example, in some cases, the surface density of the colony expanded by selection may be about 5,000 colonies / mm 2 To about 50,000 colonies / mm 2 Those skilled in the art will recognize that the surface density of the colonies selected for expansion can be anywhere within this range, such as about 48,800 colonies / mm 2 . In some cases, the disclosed low binding substrates, alone or in combination with optimized expansion buffer formulations, can allow for the selection and expansion of target (or sample) oligonucleotide colonies (or clusters) at a surface density of about 100 colonies / cm 2 to about 1 × 10 12 Colonies / cm 2within the range. In some cases, the surface density of the colonized and amplified colonies can be at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1 × 10 7 、 at least 5 × 10 7 、 at least 1 × 10 8 、 at least 5 × 10 8 、 at least 1 × 10 9 、 at least 5 × 10 9 、 at least 1 × 10 10 、 at least 5 × 10 10 、 at least 1 × 10 11 、 at least 5 × 10 11 or at least 1 × 10 12 colonies / cm 2 。 In some cases, the surface density of the colonized and amplified colonies can be at most 1 × 10 12 、 at most 5 × 10 11 、 at most 1 × 10 11, up to 5 × 10 10 , up to 1 × 10 10 , up to 5 × 10 9 , up to 1 × 10 9 , up to 5 × 10 8 , up to 1 × 10 8 , up to 5 × 10 7 , up to 1 × 10 7 , up to 5,000,000, up to 1,000,000, up to 950,000, up to 900,000, up to 850,000, up to 800,000, up to 750,000, up to 700,000, up to 650,000, up to 600,000, up to 550,000, up to 500,000, up to 450,000, up to 400,000, up to 350,000, up to 300,000, up to 250,000, up to 200,000, up to 150,000, up to 100 ,000, up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500 or up to 100 colonies / cm 2 Any of the lower and higher values ​​described in this paragraph may be combined to form a range encompassed by the present invention. For example, in some cases, the surface density of the colony expanded by selection may be about 5,000 colonies / cm 2 Up to about 50,000 colonies / cm 2 Those skilled in the art will recognize that the surface density of the colonies selected for expansion can be anywhere within this range, such as about 48,800 colonies / cm 2 . In some cases, the low-binding substrates disclosed herein, used alone or in combination with optimized amplification reaction formulations, can generate a signal (e.g., a fluorescent signal) from an amplified and labeled nucleic acid population with a coefficient of variation of no more than 50%, such as 50%, 40%, 30%, 20%, 15%, 10%, 5% or less than 5%. In some cases, amplification is achieved on the substrate surfaces and by the methods disclosed herein at elevated extension temperatures, such as at 15°C, 20°C, 25°C, 30°C, 40°C or higher temperatures, or for example at about 21°C or 23°C. In some cases, simplified amplification reactions are achieved using the substrate surfaces and methods disclosed herein. For example, in some cases, the amplification reaction is carried out using no more than 1, 2, 3, 4 or 5 discrete reagents. In some cases, the substrate surfaces and methods disclosed herein allow for a simplified temperature profile to be used during amplification such that the reaction is carried out at temperatures in the range from a low temperature of 15°C, 20°C, 25°C, 30°C or 40°C to a high temperature of 40°C, 45°C, 50°C, 60°C, 65°C, 70°C, 75°C, 80°C or higher than 80°C, for example in the range from 20°C to 65°C. The amplification reaction is also improved such that a lower amount of template (e.g., target or sample molecules) is sufficient to generate a distinguishable signal on the surface, such as a sample of 1 pM, 2 pM, 5 pM, 10 pM, 15 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1,000 pM, 2,000 pM, 3,000 pM, 4,000 pM, 5,000 pM, 6,000 pM, 7,000 pM, 8,000 pM, 9,000 pM, 10,000 pM or greater than 10,000 pM, such as 500 nM. In an exemplary embodiment, an input of about 100 pM is sufficient to generate a signal for reliable signal determination. Fluorescent imaging of the substrate surface : The solid-phase nucleic acid amplification reaction formulations and low-binding substrates disclosed herein can be used in any of a variety of nucleic acid analysis applications, such as nucleic acid base discrimination, nucleic acid base classification, nucleic acid base identification, nucleic acid detection applications, nucleic acid sequencing applications and nucleic acid (gene and genome)-based diagnostic applications. In many of these applications, fluorescent imaging techniques can be used to monitor hybridization, amplification and / or sequencing reactions carried out on the low-binding substrate. Fluorescent imaging can be performed using any of a variety of fluorophores, fluorescent imaging techniques, and fluorescent imaging instruments known to those skilled in the art. Examples of suitable fluorescent dyes that can be used (e.g., by binding to nucleotides, oligonucleotides, or proteins) include, but are not limited to, fluorescein, rhodamine, coumarin, cyanine, and their derivatives, including the cyanine derivatives cyanine dye-3 (Cy3), cyanine dye-5 (Cy5), cyanine dye-7 (Cy7), etc. Examples of fluorescent imaging techniques that can be used include, but are not limited to, fluorescence microscopy imaging, fluorescence confocal imaging, two-photon fluorescence, and similar techniques. Examples of fluorescent imaging instruments that can be used include, but are not limited to, fluorescence microscopes, confocal fluorescence microscopes, two-photon fluorescence microscopes, or custom instruments equipped with an image sensor or camera, which include an appropriate selection of a light source, lenses, mirrors, prisms, dichroic reflectors, apertures, and an image sensor or camera, etc. A non-limiting example of a fluorescence microscope equipped to obtain an image of the selected and amplified colonies (or clusters) of the low-binding substrate surface and the target nucleic acid sequences hybridized thereon is the Olympus IX83 inverted fluorescence microscope, which is equipped with a total internal reflection fluorescence (TIRF) 20× objective lens, a 100W Hg lamp, a bandpass and dichroic mirror filter set optimized for Cy3 fluorescence excitation and emission filters (e.g., using a 532 nm broadband or 532 nm short-pass filter, a Semrock 532 nm dichroic reflector, etc.) and a CCD camera (e.g., an Olympus EM-CCD camera), wherein the excitation light intensity is adjusted to avoid signal saturation. Typically, the substrate surface can be immersed in a buffer (e.g., 25 mM ACES, pH 7.4 buffer) when obtaining the image. In some cases, the efficacy of the nucleic acid hybridization and / or amplification reactions using the disclosed reaction formulations and low-binding substrates can be evaluated using fluorescent imaging techniques, where the contrast-to-noise ratio (CNR) of the image provides a key metric in assessing amplification specificity and non-specific binding on the substrate. CNR is typically defined as: CNR = (signal - background) / noise. The background term is generally considered to be the signal measured for the gap region around a specific feature (diffraction-limited spot, DLS) in a designated region of interest (ROI). Although the signal-to-noise ratio (SNR) is generally considered a benchmark for the overall signal quality, it can be shown that improving the CNR can provide a significant advantage over the SNR as a benchmark for signal quality in applications that require rapid image capture (e.g., sequencing applications where the cycle time must be minimized), as shown in the following examples. As Figure 6A and Figure As described in 6B, at high CNR, even in the case of moderate improvement in CNR, the imaging time required to achieve accurate discrimination (and thus accurate base identification in the case of sequencing applications) can be significantly reduced. Figure 6A and Figure 6B provide simulated data of signal and background intensity (solid line) and integrated mean (dashed line) variations measured with CNR (CNR = 1.25 in Figure 6A and 12.49 in Figure 6B) and integration time (SNR = 2 in both figures), which illustrate that discrimination achievable by using CNR as a signal quality metric can be improved. Figure 6A and at Figure 6B at 12.49) and integration time (SNR = 2 in both figures), which illustrate that discrimination achievable by using CNR as a signal quality metric can be improved. Figure 7 provides an example of the effect of improved CNR in the image data on the imaging integration time required for accurate detection features such as the nucleic acid community that has been clonally amplified on the substrate surface. In most ensemble-based sequencing methods, the background term is typically measured as the signal associated with the "interstitial" region (see Figure 8). In addition to the "interstitial" background (B inter ), an "intrastitial" background (B intra ) exists within the region occupied by the amplified DNA community. The combination of these two background signals indicates the achievable CNR and subsequently directly affects the optical instrument requirements, infrastructure costs, reagent costs, operation time, cost / genome, and ultimately the accuracy and data quality of the cyclic array-based sequencing application. B inter The background signal is generated from various sources; several examples include autofluorescence from disposable flow cells, non-specific adsorption of detection molecules that produce false fluorescence signals that may confound the signal with the ROI, the presence of non-specific DNA amplification products (e.g., products generated by primer dimers). In typical next-generation sequencing (NGS) applications, this background signal in the current field of view (FOV) is averaged and subtracted over time. The signal generated by individual DNA communities (i.e., (S)-B in the FOV inter ) produces distinguishable features that can be classified. In some cases, the intrastitial background (B intra) may contribute to confounding fluorescence signals, which are not specific to the relevant target but are present in the same ROI, thus making it extremely difficult to average and subtract them. As demonstrated in the following examples, the implementation of nucleic acid amplification on the low-binding substrates of the present invention can reduce background signal B by reducing non-specific binding, improve specific nucleic acid amplification, and can lead to a reduction in non-specific amplification that may affect the background signal generated from the gap and intra-gap regions. In some cases, the disclosed low-binding substrate surface, optionally in combination with the disclosed hybridization and / or amplification reaction formulations, can result in a 2-, 5-, 10-, 100-, or 1000-fold improvement in CNR compared to that achieved using conventional substrates and hybridization, amplification, and / or sequencing protocols. Although fluorescence imaging is described herein as a readout or detection mode, the same principles also apply to the use of the disclosed low-binding substrates and nucleic acid hybridization and amplification formulations in other detection modes, including both optical and non-optical detection modes. inter The disclosed low-binding substrates, optionally in combination with the disclosed hybridization and / or amplification schemes, produce solid-phase reactions that exhibit: (i) negligible non-specific binding of proteins and other reaction components (thus minimizing substrate background), (ii) negligible non-specific nucleic acid amplification products, and (iii) provide tunable nucleic acid amplification reactions. Although described herein primarily in nucleic acid hybridization, amplification, and sequencing assays, those skilled in the art will understand that the disclosed low-binding substrates can be used in a variety of other bioanalytical formats, including (but not limited to) any of sandwich immunoassays, enzyme-linked immunosorbent assays (ELISA), etc. Systems: Systems for performing base discrimination or base classification reactions using the surfaces described herein are provided. Systems for performing one or more steps of any of the sequencing methods disclosed herein are also provided. Optionally, the systems include components and reagents necessary for coupling oligonucleotide molecules, hybridizing samples or target nucleic acids to the coupled oligonucleotide molecules, and detecting or imaging signals on the surface. ​Also provided are systems for performing one or more steps of any of the sequencing methods disclosed herein. Optionally, the system includes components and reagents necessary for analyzing the sequence of a nucleic acid in such sequencing techniques based on the detection of fluorescent nucleotides or oligonucleotides. The detection instrument for reading the fluorescent signals on such arrays can be based on evanescent fluorescence or total internal reflection microscopy. One detection instrument using an optical synthesis sequencer has been proposed. The reader can include a laser that induces fluorescence in a sample within a water channel of a flow cell. The fluorescence is emitted and collected by imaging optics including one or more objective lenses and tube lenses. Among other things, the optical imager includes a light source that illuminates the sample in a relevant area, one or more detectors, and an optical component that directs light directly into the detector from the relevant area. The optical imager can also include a focusing mechanism that maintains the focus of the optical component on the relevant area to receive the light received at the detector in focus. Method for base pair characterization: Provided herein are methods for performing nucleic acid base pair discrimination or base pair characterization, the method comprising: a) providing a surface; wherein the surface comprises: i) a substrate; ii) at least one hydrophilic polymer coating; iii) a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating; and iv) at least one discrete region of the surface, which comprises a plurality of clonally amplified sample nucleic acid molecules adhered to the plurality of attached oligonucleotide molecules, wherein the plurality of adhered clonally amplified sample nucleic acid molecules are present at a surface density of at least 10,000 molecules / mm 2 and b) performing a nucleic acid amplification reaction on the sample nucleic acid molecules before or after adhering the sample nucleic acid molecules to the plurality of oligonucleotide molecules; and c) performing a cyclic series of single nucleotide binding or incorporation reactions, wherein the nucleotides are labeled with a detectable label. Also provided herein are methods for nucleic acid sequencing by utilizing the surfaces or systems described herein. In some embodiments, the detectable label is a fluorophore. In some embodiments, the detectable label is Cy3 (cyanine dye - 3), and wherein after the binding or incorporation of the first Cy3 - labeled nucleotide, while the surface is immersed in a buffer, a fluorescence image of the surface is obtained using an Olympus IX83 inverted fluorescence microscope equipped with a total internal reflection fluorescence (TIRF) 20×, 0.75 NA objective lens, a 100W Hg lamp, a band - pass and dichroic filter set optimized for 532 nm excitation and Cy3 fluorescence emission, and a CCD camera under non - signal - saturation conditions with a contrast - to - noise ratio (CNR) of at least 20. In some embodiments, the nucleic acid amplification reaction comprises a bridge amplification reaction. In some embodiments, the nucleic acid amplification reaction comprises an isothermal bridge amplification reaction. In some embodiments, the nucleic acid amplification reaction comprises a rolling circle amplification (RCA) reaction. In some embodiments, the nucleic acid amplification reaction comprises a helicase-dependent amplification reaction. In some embodiments, the nucleic acid amplification reaction comprises a recombinase-dependent amplification reaction. In some embodiments, at least one hydrophilic polymer coating exhibits a water contact angle of less than 50 degrees. Examples These examples are provided for illustrative purposes only and do not limit the scope of the patent applications provided herein. Examples 1 - Hydrophilic substrate Studies were conducted to prepare and evaluate low non-specific binding substrate surfaces using poly(ethylene glycol) (PEG) molecules of different molecular weights and functional end groups. One or more PEG layers were attached to the glass surface via a silane coupled to the functional end group. Examples of functional groups that can be used for coupling include, but are not limited to, biotin, methoxy ether, carboxylate, amine, NHS ester, maleimide, and disilane. Oligonucleotide primers with different base sequences and base modifications were then tethered to the surface layer at various densities. Both the surface functional group density and the oligonucleotide concentration were varied to target certain primer density ranges. Additionally, the primer density can be controlled by diluting the oligonucleotide with other molecules carrying the same functional group. For example, amine-labeled oligonucleotides can be diluted with amine-labeled polyethylene glycol in a reaction with an NHS ester-coated surface to reduce the final primer density. Primers with linkers of different lengths between the hybridization region and the surface-linked functional group can also be applied to control the density. Example linkers include poly-T and poly-A strands (lengths from 0 to 20 bases) at the 5' end of the primer, PEG linkers (lengths from 3 to 20 units), and hydrocarbon chain linkers of various lengths (e.g., C6, C12, C18, etc.). To measure the primer density, fluorescently labeled primers were immobilized on the surface, and the fluorescence readings were compared with the fluorescence readings of a dye solution of known concentration. The surface of the substrate needs to be low in non-specific binding (also referred to herein as "passivation") so that biomolecules such as proteins and nucleic acids do not "stick" to the surface. Examples of low non-specific binding (low NSB) surfaces prepared using standard monolayer surface agents and various glass surface agents are provided below. Successfully performing nucleic acid amplification on a passivated surface poses a unique challenge. Since passivated hydrophilic surfaces exhibit ultra-low NSB for proteins and nucleic acids, novel conditions must be used to achieve high passivation, improve primer deposition reaction efficiency, hybridization conditions, and induce effective nucleic acid amplification. Solid-phase nucleic acid hybridization and amplification methods require nucleic acid templates to be linked to a low-binding or passivated surface, and subsequent protein delivery and binding to the surface. The combination of a novel primer surface binding formulation (identified by Cy3 oligonucleotide graft titration) and the resulting ultra-low non-specific background (evaluated by NSB functionality test results using red and green fluorescent dyes) demonstrates the feasibility of these approaches. To scale primer density and add additional dimensions to hydrophilic or amphiphilic surfaces, multilayer coatings have been applied and tested using PEG and other hydrophilic polymers. By using hydrophilic and amphiphilic surface layering approaches, which include but are not limited to the polymer / copolymer materials described herein, it is possible to significantly increase the primer loading on the substrate surface. Conventional PEG-coated substrates using monolayer primer deposition methods have been reported for single molecule sequencing applications, but they do not produce high copy numbers for nucleic acid amplification. Herein, we disclose that "layering" can be achieved using conventional crosslinking approaches and chemically compatible monomer or polymer subunits such that one or more highly crosslinked layers can be sequentially constructed. Non-limiting examples of polymers suitable for the use include streptavidin, polyacrylamide, polyester, dextran, polylysine, polyethylene glycol, and polylysine copolymers polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(2-hydroxyethyl methacrylate), (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate (POEGMA), polyester, polyglucose, polylysine, polyethylene glycol (PEG), polyacrylic acid (PAA), poly(vinylpyridine), poly(vinylimidazole), and polylysine copolymers. Different layers can be connected to each other using any of a variety of covalent or non-covalent reactions, including but not limited to biotin-streptavidin binding, azide-alkyne click reaction, amine-NHS ester reaction, thiol-maleimide reaction, and ionic interactions between positively charged polymers and negatively charged polymers. It is also contemplated that such high primer density materials can be constructed in solution and subsequently layered onto the surface in multiple steps. By this method, it is possible to produce a low NSB / low background substrate surface ( Figure 9 - 13) Chemical substances for performing solid-phase nucleic acid amplification and sequencing, thereby providing significantly improved nucleic acid amplification to enable a tunable signal-to-background ratio to meet the requirements of specific sequencing applications ( Figure 14 and Figure 15). Figure Figure 9 provides an example of image data from a study for determining the relative content of non-specific binding of a green fluorescent dye to the surface of a glass substrate treated according to different surface modification schemes. Figure Figure 10 provides an example of image data from a study for determining the relative content of non-specific binding of a red fluorescent dye to the surface of a glass substrate treated according to different surface modification schemes. Figure Figure 11 provides an example of oligonucleotide primer grafting data on the surface of a substrate treated according to different surface modification schemes. Using thiol - Preparation of maleimide chemical substances 2 Layers PEG Method for the surface : The glass slide was cleaned with a 2M KOH treatment agent at room temperature for 30 minutes, washed, and then the surface silanol groups were activated using oxygen plasma. Silane-PEG5K-Thiol (Creative PEGWorks, Inc., Durham, NC) was applied at a concentration of 0.1% in ethanol. After a 2-hour coating reaction, the slide was thoroughly washed with ethanol and water, and then reacted with 2.5 mM of N-hydroxysuccinimide ester of maleimide-PEG-succinimide ester (MW = 20K) in dimethylformamide (DMF) for 30 minutes. The resulting surface was washed and reacted immediately with 5'-amine-labeled oligonucleotide primers at room temperature for 2 hours. The excess succinimide ester was inactivated by reacting with 100 mM glycine (pH 9) after primer immobilization. Using NHS Ester - Preparation of multilayer using amine chemical substances PEG Method for the surface : The glass slides were cleaned with 2M KOH treatment agent at room temperature for 30 minutes, washed, and then the surface silanol groups were activated using oxygen plasma. Silane-PEG2K-amine (Nanocs, Inc., New York, NY) was applied at a concentration of 0.5% in ethanol. After a 2-hour coating reaction, the slides were thoroughly washed with ethanol and water. 100 uM of 8-arm PEG NHS (MW = 10K, Creative PEGWorks, Inc., Durham, NC) was introduced in the solvent composition at room temperature for 20 minutes. The solvent composition can include 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90% organic solvent and 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90% low ionic strength buffer. The resulting surface was washed and reacted with 20 µM multi-arm PEG amine (MW = 10K, Creative PEGWorks, Inc., Durham, NC) for 2 hours. The resulting amine-PEG surface was then reacted with a mixture of multi-arm PEG-NHS and amine-labeled oligonucleotide primers at different concentrations. This process can be repeated to generate additional PEG layers on the surface. Solid-phase isothermal amplification : Considering various isothermal amplification methods, it can be shown that each isothermal amplification method has a unique optimal primer density range, which requires a tunable surface coating to maximize the amplification efficiency. In some cases, the higher primer surface density scales proportionally with the larger number of template copies ( Figure 15). In some cases, although a higher surface density of primers can generate a high foreground signal (i.e., sequence-specific signal) for some amplification pathways, it can generate a high background signal and thus prove harmful to other amplification pathways. In Figure 16, the primer deposition concentration is shown at the top of the figure, while the various helicase isothermal amplification formulations tested are designated in the images obtained for the surfaces after the isothermal amplification reaction. If specific amplification occurs, the images of each surface are expected to appear red. As from Figure The image series of formulation "58" in 16 is obvious. As the primer density increases, the color fades from red to green, thus indicating a decrease in specific amplification. In addition, it can be seen that including betaine (a common buffer additive) in the amplification reaction formulation reduces the degree of non-specific amplification in favor of specific amplification, thereby making the signal brighter and improving the CNR. The combination of the low-binding stratified substrate surface, the tunable surface density of the primer, and the improvement of the hybridization and / or amplification reaction formulation (including the adjustment of buffer components and additives (such as the selection of buffers, pH, solvents, ionic strength, detergents, formamide, betaine, crowding agents, and other additives, etc.)) and the resulting improvement in amplification rate and specificity should lead to unprecedented improvement in next-generation sequencing of nucleic acids. The present invention solves the problem of achieving highly monoclonal amplification of library nucleic acid strands on a hydrophilic substrate for various applications requiring signal enhancement, such as nucleic acid detection, sequencing, and diagnostic applications. Conventional isothermal methods for nucleic acid amplification for generating monoclonal clusters of library nucleic acid strands are limited and have defects. Examples of their performance limitations include long clustering times (e.g., 2+ hours), high temperature (e.g., 60 °C or higher) requirements, inability to amplify / cluster effectively on certain surfaces, high cost, polyclonality problems, reagent stability problems, etc. Although a large number of isothermal amplification methods are described in the literature, only one or two methods have been successfully applied to commercial sequencing applications. Herein, we propose an isothermal amplification strategy that successfully generates clusters of monoclonal replicas of library DNA fragments (or other nucleic acids) for applications such as DNA sequencing and eliminates or alleviates the aforementioned problems. Developing overall DNA amplification / DNA sequencing composition variations to reduce background signal (B inter and B intra ) and the design criteria contributing to controlled DNA amplification on a low-binding substrate include: (i) reducing non-specific DNA amplification in the gap region (B inter ) compared to traditional / prior art approaches (e.g., due to primer dimer amplification), (ii) reducing the amount of non-specific DNA amplification products (e.g., primer dimers) within the specific DNA community (B intra ) compared to traditional / prior art approaches, and (iii) increasing the control of specific DNA amplification on a low-binding substrate (e.g., reaction time, cycle time, primer surface density titration, primer surface density, primer sequence, etc.) such that the signal-to-background (S / B) ratio is reduced, even in the absence of improvements in hybridization and amplification formulations. Examples 2 - Helicase-Dependent Amplification on a Low-Binding Surface with Improved Specificity It is well known that helicase-dependent amplification is highly prone to non-specific amplification, such as primer dimer formation. We can reduce this non-specific amplification on the surface by any combination of the following methods: (i) designing oligonucleotide primers that generate fewer primer dimers, (ii) adjusting the primer surface density on the multi-layer substrate surface, (iii) performing the reaction with thermophilic enzymes at higher temperatures, (iv) using amplification buffer additives and non-self-annealing primer sequences such as those mentioned above in combination, and (v) introducing one or more complete nucleic acid denaturation and primer hybridization steps. In the absence of SSB protein, low NSB Specific helicase-dependent amplification on the surface: Helicase-dependent amplification of linear template strands can be achieved using reduced non-specific amplification and highly efficient cloning amplification on a low-binding surface. Forward primers on the surface are extended using an amplification reaction mixture containing polymerase and helicase on single-stranded template library strands. Subsequently, the template strands are optionally denatured and washed away. Alternatively, the double helix can be unwound by helicase activity. Either method results in the forward strand, which is the part of the primer extended from the surface binding, being partially or completely in single-stranded form. Subsequently, surface-tethered reverse primers hybridize to this forward strand and are also extended, forming a double-stranded bridge structure. The helicase present in the reaction mixture unwinds the intermediate double-stranded amplicon strands, which can then be used to re-hybridize to other free surface-tethered primers for subsequent rounds of amplification. For this to occur, the degree of unwinding does not need to be extensive - just sufficient to convert the primer hybridization region into a single-stranded component (end abrasion) to allow hybridization of subsequent surface-bound primers. The helicase used for this reaction should be able to unwind from the ends of the bridge structure and can be a 3' to 5' helicase or a 5' to 3' helicase. In some cases, it can be a helicase of superfamily 1, 2, 3, 4, 5, or 6. In some cases, it can be a highly processive helicase (i.e., able to unwind multiple consecutive base pairs without releasing the single-stranded or double-stranded structure) or a helicase with limited processive synthesis ability. Certain mutants of superfamily 1 helicases with higher processive synthesis ability, such as the UvrD303 mutant of helicase UvrD, can be used in this amplification protocol. To facilitate unwinding by a 5'-to-3' helicase, all or a portion of one or two surface-tethered primers may include modifications to form a specific loading site for the 5'-to-3' helicase. On the template nucleic acid extending from such primers, the modification site will act as a polymerase termination point, such that the segment of the primer sequence between the surface binding site and the modification site will always be in single-stranded form. This segment will act as a loading site for the 5'-to-3' helicase with 5'-to-3' directionality, because many helicases have much better single-stranded nucleic acid binding affinities, guiding and facilitating the 5'-to-3' helicase unwinding activity required for helicase-dependent amplification on the substrate surface. Examples of primer modifications that can be used include, but are not limited to, inserting a PEG chain into the backbone of the primer towards the 5'-end between two nucleotides, inserting a abasic nucleotide (i.e., a nucleotide that has neither a purine nor a pyrimidine base), or a lesion site that can be bypassed by the helicase. A variety of helicases have cofactor proteins and specific conformations that activate or enhance helicase activity. Examples include, but are not limited to, the RepD protein of the PcrA helicase from Bst, the φX gene protein A of the E. coli E . coli) Rep helicase, and the MutL protein of the UvrD helicase. The addition of such accessory proteins can make the required unwinding activity more efficient and thus further facilitate helicase-dependent isothermal amplification. Some of these cofactors have specific binding sequences or moieties that can be added to the primers to direct the unwinding activity. When using a thermophilic helicase and a strand displacement polymerase formulation lacking a single-stranded binding (SSB) protein (e.g., internal reference formulation 58), the helicase amplification formulation exhibits reduced non-specific amplification. Different from most helicase-dependent amplification pathways, it was observed that excluding the single-stranded binding protein from the formulation (which is commonly used to disrupt transient non-specific hybridization) reduces non-specific amplification. Various formulation variations are shown to mitigate the increase in non-specific hybridization on low-binding surfaces. Formulation compositions for improving helicase-dependent amplification are altered : Additives such as betaine are generally known to reduce non-specific amplification in isothermal amplification reactions carried out in solution. The criteria become more restrictive because high primer surface density is required to support tunable amplification and high copy numbers of the template nucleic acid population. At high primer densities, non-specific amplification begins to outweigh the benefits of the high template copy numbers in the resulting population ( Figure 16). Thus, additional additive formulations have been found that promote non-specific amplification within the template nucleic acid population. In Figure In the example shown in Fig. 16, it can be clearly seen that adding betaine to Formulation No. 58 results in more specific amplification (indicated in red) on the surface with a higher primer density. In addition to betaine, it is possible to combine a variety of different reaction formulation components to achieve higher amplification specificity in solution and on low-binding surfaces ( Fig 17 and Fig 18). Organic solvents such as acetonitrile, DMSO, DMF, ethanol, methanol, and similar compounds change the structure of single-stranded and double-stranded nucleic acids by oligonucleotide dehydration methods. Compounds such as 2-pyrrolidone and formamide are known to lower the melting temperature of nucleic acids and reduce secondary structures from oligonucleotides with high GC content. Crowding agents are also known to stabilize nucleic acid structures. In low-pH buffers, hybridization and hydrogen bonding in base pairing become more favorable. When combining the different properties of each of these individual formulations, it is possible to increase the specific ligation of oligonucleotide sequences by more than two orders of magnitude compared to traditional approaches. By combining these compounds and adding them to the amplification formulation described below, non-specific amplification such as that generated by primer dimers is significantly attenuated, and specific amplification products with good yields are still observed in solution ( Fig 17) and on the low-NSB surface ( Fig 18) disclosed. Example 3 -[[-END]] Modified Rolling circle Multiple displacement amplification ( Modified RCA -[[-END]] MDA ) Nucleic acid library fragments are ligated to adapter sequences (which contain forward, reverse, and sequencing primers, and any identification / barcode sequences) and circularized in solution or on a low-binding surface. Circular ssDNA is then captured or hybridized to the forward surface primer in solution or on a surface and extended in an RCA reaction by strand displacement polymerase, which yields a single-stranded concatemeric copy of the library nucleic acid and the adapter sequence. Reverse primers hybridize to the concatemeric forward template at multiple positions and are extended by the RCA reaction mixture, thereby yielding a concatemeric reverse copy. During this process, the reverse strands displace each other. Upstream reverse strand extension will displace downstream extension, thus forming a single-stranded concatemeric reverse strand. Addition of a helicase may generate single-stranded regions of the nucleic acid that persist long enough to re-initiate hybridization and trigger a displacement cascade, which can increase the number of amplified copies in a relatively controlled manner. Alternatively, addition of recombinase and accessory proteins can hybridize primers to homologous regions of double-stranded DNA in a method called strand invasion. This will re-initiate displacement and hybridization cascades and increase the number of community copies. The number of copies of the RCA-MDA community is determined by primer surface density, which indicates how often and successfully the initial concatemer or displaced concatemer hybridizes to the forward and reverse primers. Increased primer density on a low-binding surface has been shown to yield higher numbers of amplified copies in such clusters ( Fig 15). In summary, it is possible to increase the number of copies or specific amplification and reduce non-specific amplification on low-binding surfaces using one or a combination of the following methods: (i) the number of specific copies can be increased by enhancing the efficiency of primer-template hybridization by formulation changes ( Fig 16), (ii) the number of specific copies can be increased by increasing primer density on a low-binding substrate ( Fig 14 and Fig 15), (iii) non-specific amplification of primer dimers or chimeric DNA production can be reduced by using the additives described above, (iv) the amplification incubation temperature can be increased using thermostable enzymes and formulation changes as previously described to reduce non-specific amplification, (v) primer compositions containing non-self-hybridizing primer sequences can be used in combination with additives and / or elevated amplification incubation temperatures to reduce non-specific primer dimer amplification. Example 4 - single-stranded DNA binding ( SSB ) protein-mediated isothermal amplification SSB proteins can resolve secondary structures in nucleic acid strands, stabilize single-stranded DNA after unfolding, prevent or disrupt transient non-extensive hybridization of two nucleic acid strands (precursor to primer dimer amplification), promote specific hybridization of short oligonucleotides to the correct complementary regions in target oligonucleotides, and enable enzymes to interact with the fork junctions of single-stranded and double-stranded nucleic acids and direct the enzymes to the fork junctions of single-stranded and double-stranded nucleic acids. C-terminal truncation mutations in SSB are reported to remove cooperative binding to ssDNA, while its monomers exhibit stronger binding to ssDNA and reduce the melting temperature of dsDNA. For example, compared to the efficiency of wild-type proteins, the C-terminal truncation mutant T4 gp32 of the phage SSB protein produces a protein (gp32ΔC) that reduces the melting temperature of dsDNA by dozens of degrees. In this amplification protocol, we use these proteins in formulations containing both truncated and wild-type SSB proteins (and strand displacement polymerases) to transiently melt the ends of double-stranded nucleic acid bridge intermediates and allow surface primer hybridization and primer extension. Even though SSB slows down nucleic acid hybridization, it is known to generally promote specific hybridization. Thus, for example, using truncated SSB proteins such as T4 gp32ΔC can make the 3'-ends in the bridging nucleic acid structure hybridize more efficiently to other freely surface-tethered primers. Although this will also disrupt newly formed primer-template complexes, the optimized formulation should extend these complexes by strand displacement polymerases. SSB proteins are found in a variety of phages (e.g., T4 gp32), bacteria, archaea, fungi, and eukaryotes. Some are thermostable SSB proteins, where the C-terminal truncated forms can allow more efficient nucleic acid end melting at optimized higher temperatures, thus allowing SSB-dependent thermophilic amplification with lower non-specific amplification at higher temperatures. By using additives such as those mentioned above, primer sequence design, and specific hybridization and / or amplification formulations and optimized temperatures used in conjunction with appropriately temperature- and chemical-tolerant enzymes and proteins, this amplification method can be adjusted to drive highly specific amplification while eliminating non-specific amplification. This protocol can be used to amplify circular DNA as well as linear DNA, where independent initiation of extension by strand displacement polymerases can lead to multiple subsequent multiple displacement amplification events, which produce concatemeric replicas of circular DNA templates (see the section on modified RCA-MDA above). We have found that SSB-mediated amplification of circular library DNA (using, for example, phi29 SSB) is much faster than traditional RCA (e.g., requiring only 30 minutes to 1 hour of amplification time compared to 2 to 3 hours for traditional RCA). The products of SSB-mediated amplification carried out in solution migrate on gels in a concatemer ladder form. Examples 5 - Low-Temperature Thermal Cycling Bridging Amplification on a Low-Binding Surface By using a combination of additives that improve hybridization and / or amplification formulations, thermostable SSB proteins, and / or truncated SSB proteins, thermal cycling can be developed at temperatures lower than those of traditional methods outlined in Mullis' original PCR disclosure. In this protocol, additives that drive nucleic acid hybridization and a dehybridization temperature lower than its traditional value can be used. For example, formamide is commonly used to lower the melting temperature (T m ) of DNA, and subsequent DNA dehybridization can be carried out at a temperature of about 60 degrees. On the other hand, the reannealing temperature typically also requires a high temperature (95°C) to a uniform change in temperature close to room temperature. It is possible to form a formulation such that the reannealing temperature stringency can be significantly reduced. Using such a formulation would constitute a significant improvement over traditional bridging amplification methods such that the temperature uniform change can be carried out between 20 and 60 degrees. The need for temperature uniform change on a low-binding substrate and the reduction in improved hybridization stringency can produce the following advantages over traditional bridging amplification: (i) reduced amplification time; (ii) simplified instrument design, (iii) reduced reagent use by faster and more specific hybridization, resulting in a more efficient amplification rate, and (iv) reduced reagent costs. It is also possible to show that improving the stringency of hybridization will reduce the number of amplification cycles required for amplification on the substrate surface. Figure 19A - B provides an example of data demonstrating the improvement of hybridization efficiency, which can be obtained using a low non-specific binding substrate and the improved hybridization formulation of the present invention compared to those of conventional hybridization formulations ( Figure 19B). Figure 19A). Figure 20 illustrates a non-limiting example of the workflow of nucleic acid sequencing using the disclosed low-binding substrate and the hybridization / amplification reaction formulation of the present invention and the processing time that can thereby be achieved. Examples 6 - Preparation with thiol - Maleimide chemistry 2 Layers PEG Surface The glass slides were chemically treated to remove organics and activate the hydroxyl groups for silane coupling (various methods including plasma treatment, piranha solution etching, alkali washing, alkali bath, high-temperature glass bonding, and any combination thereof). Silane-PEG5K-Thiol (Creative PEGWorks, Inc) was applied at a concentration of 0.1% in an ethanol solution. After a 2-hour coating reaction, the slides were thoroughly washed with ethanol and water, and then reacted with 2.5 mM of maleimide-PEG-succinimidyl ester (MW 20K) containing valeric acid in DMF for 30 minutes. The resulting surface was washed and reacted with 5'-amine-labeled oligonucleotide primers immediately at room temperature for 2 hours. The excess succinimidyl ester was inactivated with 100 mM glycine at pH 9 after primer immobilization. This approach confers negligible low binding to the solid substrate surface by more than two orders of magnitude via efficient primer and polymer iterative coupling over traditional methods. Example 7 - with NHS Preparation of multilayer with ester-amine chemistry PEG surface The glass slides were chemically treated to remove organics and activate the hydroxyl groups for silane coupling (various methods including plasma treatment, piranha solution etching, alkali washing, alkali bath, high-temperature glass bonding, and any combination thereof). Silane-PEG-amine (Nanocs, Inc) was applied at a concentration of 0.1% - 2% in a clean ethanol solution. After a 2-hour coating reaction, the slides were thoroughly washed with ethanol and water. Multifunctional PEG NHS was introduced into the solvent composition at room temperature for 5 - 30 minutes, and the solvent composition could include 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90% organic solvent and 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90% low ionic strength buffer. The resulting surface was washed and reacted with multifunctional PEG amine (MW 10k, Creative PEGWorks, Inc). The resulting amine-PEG surface was then reacted with a mixture of multifunctional PEG NHS and amine-labeled oligonucleotide primers at different concentrations. This process can be repeated to generate additional PEG layers on the surface. This approach confers negligible low binding to the solid substrate surface by more than two orders of magnitude via efficient primer and polymer iterative coupling over traditional methods. Example 8 - on cluster data CNR calculation of Typically, when fluorescence detection is used for solid-phase assays, the signal generated by tethering and / or amplifying molecules is coupled to or followed by coupling a reporter dye molecule. This method generates specific and non-specific signals. The non-specific component is commonly referred to as non-specific noise or background (resulting from interstitial or intrasite contributions), which interferes with the measurement of the specific signal and reduces the contrast-to-noise ratio (CNR). Non-specific background can arise from dye molecules non-specifically adsorbed to the substrate surface or from non-specific amplification such as primer-dimer pairing on the surface. When a fluorescent reporter is used to label a specific molecule of interest, both mechanisms generate a substantial fluorescent background. The disclosed low-binding substrate surfaces and related methods employed demonstrate a significant improvement in minimizing non-specific background. As shown in the examples described below, the estimated non-specific background is far less than 10% of the total signal using the specified amplification method and substrate surface. On the other hand, conventional amplification methods and substrate surfaces typically generate background signals of 30% to 50% of the total signal. It should be noted that, as used herein, non-specific background or noise is only one component of the total system noise, which may also include other contributions from the detection system, such as photon shot noise, autofluorescence background, image sensor noise, illumination noise (e.g., resulting from fluctuations in illumination intensity), etc. In this regard, it may be possible to extend the disclosed approach for CNR improvement with novel substrate surfaces and related hybridization and amplification methods to achieve even greater improvements for NGS and other bioanalytical techniques by, for example, correcting signal impurities that can be generated by traditional synthesis sequencing such as by using a predetermined phase and phasing over multiple assay reaction cycles, and correcting errors that occur due to DNA strand loss and / or DNA damage resulting from stringent wash conditions or deblocking (reversible terminator removal). Generally, assays for measuring the CNR of solid-phase bioassays include the following steps: (1) Prepare the disclosed low-binding substrate functionalized with relevant receptors, targets, and / or capture oligonucleotides. (2) Capture the receptor, target, and / or capture oligonucleotide, which may be directly labeled or may be a precursor for subsequent labeling reactions. If no amplification or additional probe labeling step is required, we proceed to step 5. If a probe labeling step is required to form a reporter, we proceed to step 4. Otherwise, we proceed to step 3. (3) Amplify the receptor, target, and / or capture oligonucleotide via conventional immunoassay signal amplification, oligonucleotide replication amplification (e.g., using bridge amplification, isothermal amplification, RCA amplification strategy, HDA amplification, or RCA-MDA amplification strategy). (4) Detect the amplified target using a reporter label (e.g., by using a fluorescent species or other types of reporters). This step is applicable to bioanalysis based on any surface, including but not limited to genotyping, nucleic acid sequencing, or surface-based target / receptor identification. (5) Perform an appropriate detection method. For fluorescence imaging, the detection method can be structurally designed in various ways. For example, conventional optical microscopy will include all or a subgroup of the following components: an illumination or excitation light source, an objective lens, a sample, other optical components (such as tube lenses, optical filters, dichroic reflectors, etc.), and a detection mode (e.g., using an EMCCD camera, a CCD camera, an sCMOS, a CMOS, a PMT, an APD, or other conventional methods for measuring the light level). For non-light-based detection, various ways can be used to measure electrical signals, including but not limited to field-effect transistor (FET) detection, electrode-based measurement of electrical signals (DC or AC), tunneling current, measurement of magnetic signals, etc. Use imaging and signal processing from a specified field of view (FOV) to calculate the CNR as illustrated in Figure 8, where, as illustrated in the figure, CNR = (signal - background) / (noise), and where background = (B 隙間 + B 隙內 ). For the following example of calculating the CNR of clusters of cloned and amplified nucleic acid sequences on the low-binding substrate of the present invention, an image analysis program is used to find representative foreground bright spots ("clusters"). Generally, a spot is defined as a small connected area of image pixels presenting a light intensity higher than a certain intensity threshold value. Only the connected areas containing the total pixel count within a specified range are considered as spots or clusters. Areas that are too large or too small in terms of the number of pixels are ignored. Once several spots or clusters have been identified, the average spot or foreground intensity and other signal statistics are calculated. For example, the maximum intensity, the average intensity, and / or the interpolated maximum intensity can be calculated. The median or average of all spot intensities is used to represent the spot foreground intensity. A representative estimate of the background region intensity can be determined using one of several different methods. One method is to divide the image into multiple small "tiles" each including, for example, 25 × 25 pixels. Within each tiled area, a certain percentage (e.g., 25%) of the brightest pixels are discarded, and intensity statistics are calculated for the remaining pixels. Another method for determining the background intensity is to select a region of at least 500 pixels or larger that does not contain any foreground "dots" (as defined in the previous step), and then calculate the intensity statistics. For either of these methods, a representative background intensity (median or mean) and standard deviation are then calculated. The standard deviation of the intensity in the selected region is used as the representative background variation. The contrast-to-noise ratio (CNR) is then calculated as (foreground intensity - background intensity) / (background standard deviation). Figure 21 - 23 provides examples of the raw image data and intensity data histograms that are used to calculate the CNR for the differential combinations of nucleic acid amplification methods and the low-binding substrates described herein. In each of these examples, the upper histogram is the background pixel intensity histogram, the lower histogram is the foreground dot intensity histogram, and also includes a portion of the original image. It should be noted that the images are not on the same intensity scale, so the visual brightness perception does not represent the actual intensity. For these examples, the low-binding solid substrates were formed using the methods described previously. Oligonucleotide primers (one or two primer sequences depending on the amplification protocol used) were grafted at different densities. The surface density for each of these experiments was estimated to be approximately 100K primers / μm 2 . The primer surface density was estimated using the following method: (i) a fluorescence titration curve was prepared using a GE Typhoon (GE Healthcare Lifesciences, Pittsburgh, PA) and a capillary flow cell with a known area (40 mm 2 ), height (0.5 mm), and volume (200 μl) containing a known concentration of Cy3-dCTP, (ii) the primers grafted to the low-binding substrate were hybridized to Cy3-labeled complementary oligonucleotides using a conventional hybridization protocol (3 times sodium citrate saline (SSC) at 37°C or at room temperature (RT); the hybridization conditions should characterize the integrity), and the fluorescence intensity of the resulting signal on the surface was measured using the same GE Typhoon instrument used to generate the calibration curve, (iii) and the number of primer molecules tethered per unit area of the surface was calculated based on the comparison of the measured surface signal to the calibration curve. The DNA library sequences are then hybridized to the tethered primers. The hybridization protocol for the library hybridization step can vary depending on the surface characteristics, but requires controlled library input to form resolvable DNA amplification colonies. DNA amplification was performed for this example using the following protocol: (i) bridge amplification for 28 cycles, where the primer density was approximately 100K primers / μm 2 , (ii) bridge amplification for 28 cycles, where the higher primer density > 500K primers / μm 2 , and (iii) rolling circle amplification (RCA) for 90 minutes, where the primer density was approximately 100K primers / μm 2 . After amplification, the amplified DNA was hybridized to complementary "sequencing" primers, and a sequencing reaction mixture containing Cy3-labeled dNTPs ("first base" analysis) was added to determine the first base CNR for each of the individual methods. The sequencing reaction mixture for "first base analysis" can include any combination of labeled nucleotides (to enable differentiation of the 4 bases), enzymes incorporating modified deoxynucleoside triphosphates (dNTPs), and associated incorporation buffers, metal cations, cofactors, etc. After the first base incorporation, the sequencing reaction mixture was buffer-exchanged, imaged using the same GE Typhoon instrument, and the CNR was calculated on the resulting image. Figure 21 provides an example of the fluorescence image and intensity data of the low-binding substrate of the present invention. Solid-phase nucleic acid amplification on the low-binding substrate of the present invention was performed using bridge amplification for 28 cycles with a primer density of approximately 100K primers / μm 2 to form a clonal amplification cluster of the templated oligonucleotide sequence. In this example, the background intensity was 592 counts (standard deviation of 66.5 counts), the foreground intensity was 1047.3 counts, and the calculated CNR = (1047.3 - 592) / 66.5 = 455.3 / 66.5 = 6.8. The estimated non-specific noise = (592 - 100) / (1047 - 100) = 52%. Figure 22 provides a second example of the fluorescence image and intensity data of the low-binding substrate of the present invention. Solid-phase nucleic acid amplification on the low-binding substrate of the present invention was performed using a higher primer density > 500K primers / μm 2Bridge amplification was performed 28 times to form a cloned and amplified cluster of the template oligonucleotide sequence. In this example, the background intensity was 680 counts (with a standard deviation of 118.2 counts), the foreground intensity was 1773 counts, and the calculated CNR = (1773 - 680) / 118.2 = 1093 / 118.2 = 9.2. The estimated non-specific noise = (680 - 100) / (1773 - 100) = 35%. Figure 23 provides an example of the fluorescence image and intensity data of the low-binding substrate of the present invention. Solid-phase nucleic acid amplification on the low-binding substrate of the present invention uses a primer density of approximately 100 K primers / μm 2 Rolling circle amplification (RCA) was performed for 90 minutes to form a cloned and amplified cluster of the template oligonucleotide sequence. In this example, the background intensity was 254 counts (with a standard deviation of 22.7 counts), the foreground intensity was 6161 counts, and the calculated CNR = (6161 - 254) / 22.7 = 5907 / 22.7 = 260. Note that a significant improvement in CNR was achieved through this combination of using a low-binding surface area and an amplification protocol. The estimated non-specific noise = (254 - 100) / (6161 - 100) = 3%. Example 9 - Modification of the polymer substrate surface The modification of the surface for the purposes disclosed herein involves creating surfaces that are reactive towards a variety of chemical groups (-R), including amines. When prepared on a suitable substrate, these reactive surfaces can be stored at room temperature for extended periods, such as at least 3 months or longer. These surfaces can be further grafted with R-PEG and R-primer oligomers for surface amplification of nucleic acids as described elsewhere herein. Plastic surfaces, such as cycloolefin polymers (COP), can be modified using any of the methods known in many of these techniques. For example, it can be treated with Ti:sapphire laser ablation, UV-mediated photografting of ethylene glycol methacrylate, plasma treatment, or mechanical agitation (e.g., sandblasting or polishing, etc.) to form a hydrophilic surface that remains reactive towards a variety of chemical groups, such as amines, for several months. These groups can then allow the binding of passivating polymers (such as PEG) or biomolecules (such as DNA or proteins) without loss of biochemical activity. For example, the ligation of DNA primer oligomers allows for the passivation of DNA amplification on plastic surfaces while minimizing the non-specific adsorption of proteins, fluorophore molecules, or other hydrophobic molecules. In addition, surface modification can be combined with, for example, laser printing or UV masking to form a patterned surface. This allows for the patterned attachment of DNA oligomers, proteins, or other moieties, providing surface-based enzymatic activity, binding, detection, or processing. For example, DNA oligomers can be used to amplify DNA only within the patterned features, or to capture the amplified DNA concatemers in a patterned manner. In some embodiments, enzyme islands can be generated in the patterned regions capable of reacting with solution-based substrates. Since plastic surfaces are particularly susceptible to such processing modalities, in some embodiments, as covered herein, plastic surfaces can be identified as particularly advantageous. In addition, plastics can be injection molded, embossed, or 3D printed to form any shape, including microfluidic devices that are much easier than glass substrates, and can thus be used to form surfaces for binding and analyzing biological samples in multiple structural designs, such as sample-derived microfluidic chips for biosensor detection or DNA sequencing. Specific local DNA amplification on modified plastic surfaces has been achieved, which produces spots with an ultra-high contrast-to-noise ratio and very low background when detected with fluorescently labeled probes. We have grafted representative hydrophilic and amine-reactive cycloolefin polymer surfaces with amine-primers and amine-PEGs and found that they support rolling circle amplification. We then found that when detected with fluorophore-labeled primers, or when labeled dNTPs were added to hybridized primers by polymerase, bright spots of DNA amplicons showed a noise ratio higher than 100, with an extremely low background, indicating highly specific amplification and an ultra-low level of protein and hydrophobic fluorophore binding, which characterizes a high-accuracy detection system such as a fluorescence-based DNA sequencer. Herein, plastic flow cells were tested, which were filled with tethered DNA clusters and probed for the first base of the library sequence. For a population of surfaces with DNA, as previously described in Examples 1 and 7 and elsewhere herein, for PEG-NHS-coated glass surfaces, hydrophilic cycloolefin polymer (COP) plastic surfaces were grafted with 25-mer amine-primers 1, amine-primers 2, and amine-5K PEG. A 5 pM circularized DNA library containing, in addition to the library insert sequence, the primer 2 sequence, the sequencing primer sequence, and a sequence complementary to primer 1 was then hybridized to the surface for 15 minutes. Rolling circle amplification (RCA) was then performed as described in Examples 2-5 and elsewhere herein to form concatemeric sequence DNA rolls up to 0.5-1 Mb in length. The sequencing primer was hybridized, and the first base was incorporated with polymerase using a fluorophore-labeled dNTP set to label as FIG clusters with three different colors as shown in 25A. Parallel experiments were conducted using the same parameters, starting with glass instead of the COP surface (with the surface agent as described in Example 7) to provide a comparison between the passivated glass and the passivated COP surface. As Figure 25A - shown in B, by combining the signals generated from the first fluorescently labeled COP-based surface with those obtained on a similarly treated glass surface, both in terms of the intensity and resolution of the observed points. This indicates that the method disclosed herein provides a general method for preparing surfaces for immobilizing, amplifying, and detecting nucleic acids. Intensity and CNR were measured for glass and plastic. We see at Figure 26A that both glass and plastic exhibit signal intensity under detection conditions substantially above the background. For both glass and plastic, the signal intensity is on the left and the background is depicted on the right. We see at Figure 26B that the CNR of glass and plastic is higher than 50 under the analyzed conditions. Example 10 - Surface generation Surfaces that exhibit negligible non-specific binding to organic dyes and proteins, exhibit stability up to at least 95 °C, chemical stability to high pH (0.1 M NaOH), low pH (> 5.0); organic solvents (methanol, ethanol, acetonitrile, formamide, oxidants, phosphines), long-term storage stability, low input library requirements, and adjustable primer loading are generated as follows. The method includes cleaning and silanizing or passivating the surface. The surface is washed with 2 M KOH and alconox / hellmanex detergent and rinsed with ethanol. Subsequently, the surface is heated to 560 °C to expose OH groups. The surface is alternately or combinatorially subjected to plasma treatment. The surface is silanized with 5 mg / mL Silane-5kPEG-NHS (99.9% ethanol / 0.01% acetic acid), heated to 65 °C, and tested with KOH / detergent / heat to clean the surface. Alternatively or combinatorially, the surface is silanized with 10 mg / mL Silane-5kPEG-NHS (90% DMF / 10% 100 mM MES pH 5.5), heated to 25 °C, and tested with KOH / detergent / heat to clean the surface or plasma treat the surface. Multiple dyes are compatible with these surfaces, such as Cy3-C, R11-U, Cy3.5C, 647N-A, Cy5-G, 660-U, Cy5.5-C (note; dyes only at 200 nM). Exemplary dye mixtures include Cy3-A, Cy3.5-C, Cy5-U, AHO690-G. At low concentrations (5.0×10 4 primers / um 2 ), at high concentrations (1.0×10 7 primers / um 2 ), and at concentrations of values defined within or outside of this range by these endpoints of this range, the surfaces are tunably loaded with primers. Concentration is measured as follows as appropriate. Cy3-dCTP solutions of different concentrations are made and the FL intensity is measured using a GE Typhoon (GE Healthcare Lifesciences, Pittsburgh, PA) or a suitable instrument in a capillary with a fixed size (0.5 mm × 5 mm or other area). When the area is known and the number of molecules is known, this yields primer loading. This pathway has been used to measure at concentrations of 80,000, 160,000, 320,000, 640,000, 1,300,000, 2,600,000, and 5,100,000 primers / um 2 , and other concentrations of values defined within or outside of this range by these endpoints of this range can be readily achieved. These densities are facilitated by the presence of multilayer PEG or other surfaces as disclosed herein. Visible surfaces did not exhibit a significant decrease in stability during a one-week storage period. Densities were measured for multiple surface variants and the results appear as follows. Three-layer multi-arm PEG (8,16,8) on two layers of PEG amine-APTES pre-loaded with 7 uM primers exhibited concentrations on the surface of 2,000,000 to 10,000,000. Similar concentrations were observed using star PEG-amine to replace dumbbell-shaped 16-mers and 64-mers for three-layer multi-arm PEG (8,16,8) and (8,64,8) and three-layer multi-arm PEG (8,8,8) on PEG amine-APTES exposed to 8 uM primers. Using these pathways, it was observed that increased primer density resulted in higher foreground intensity, higher colony density, and higher CNR. For example, 10 pM input at primer density < 1.0×10 4 引子个数 / um 2 generate 10 CNR on the surface, while similar inputs generate 40 - 60 CNR at an initiator density > 1.0×10 6 引子个数 / um 2 under. Example 11 - High CNR The surface generates high-quality data. Test current state-of-the-art surfaces, as well as low and high CNR surfaces such as those disclosed herein, with respect to the fluorescence detected at the first and second channels corresponding to the first and second dyes. We observed that as the CNR increases, we see a clearer resolution of individual detection events. These detection events are aligned along different axes corresponding to the emission spectra of the dyes, rather than with the higher error 'clouds' seen in the top three files of FIG. 27. Turning to the bottom three files of FIG. 27, this more accurate data collection itself manifests as a narrower, taller peak at a specific expected wavelength and fewer data points at intermediate positions. This more clearly resolved data set translates into a more accurate fluorescence-based base identification generated by the analysis performed on the high CNR surface. Example 12 - Cloned and amplified poly-target oligonucleotide molecules Figure 28 provides a schematic diagram of cloned and amplified poly-target oligonucleotide sequences hybridized to a surface (left) containing a high surface density of oligonucleotide adaptor or initiator molecules and hybridized to a surface (right) containing a lower surface density of oligonucleotide adaptor or initiator molecules, and illustrates the resulting improvement in CNR that can be achieved. Prepare several surfaces to have an oligonucleotide density higher than 2000 molecules / uM 2 and the fluorescence image of the surface has a contrast noise ratio greater than 20. Example 13 - The input nucleic acid needs to be reduced Figure 29 provides a comparison of the experimental results of performing conventional hybridization reactions on the low-binding substrate surface of the present invention and optimizing the hybridization reactions on the low-binding substrate surface of the present invention. After oligonucleotide primers are attached to the low-binding substrate surface, efficient hybridization of the target nucleic acid to the oligonucleotide primers may suffer from a decreased collision frequency on the low-binding surface. At least in part, due to the reduced coupling of oligonucleotides captured on prior art surfaces, conventional hybridization methods for adding target DNA to primers bound to the surface require input DNA concentrations of up to 10 nM (see Figure 29 left, showing the binding of labeled target oligonucleotides to a conventional surface). Even at these high concentrations, the coupling of target oligonucleotides is limited. By comparison, using the newly developed hybridization reaction conditions, the target nucleic acid sequence hybridizes to surface-linked oligonucleotides at an input concentration of the target nucleic acid as low as 50 pM. The decrease in the target nucleic acid input concentration indicates an approximately 200-fold increase in hybridization efficiency (see Figure 29 right, showing the binding of labeled target oligonucleotides to the disclosed low-binding surface), which provides a significant advantage for the disclosed low-binding substrate surface for sequencing techniques where the input library DNA may be scarce. The efficient primer coupling method and hybridization conditions allow for the preparation of a surface with low non-specific binding and high surface density of oligonucleotide primers that would not be achieved using conventional primer coupling chemistries or hybridization conditions described in this art. The right side of Figure 28 provides an example of improved hybridization achieved on a low-binding surface using the disclosed hybridization method with a reduced amount of input DNA and a shortened hybridization time compared to the results achieved using the conventional hybridization scheme shown on the left side. At a concentration reported at 90 degrees, use a slow cooling method (2 hours) until reaching 37 degrees and test traditional or standard conditions with 2 - 5 times saline - sodium citrate (SSC) buffer (standard) using a hybridization reporter probe (complementary oligonucleotide sequence labeled with a Cy™3 fluorophore at the 5' end). The surface for both test conditions is an ultra - low non - specific binding surface with a level of non - specific Cy3 dye absorption of less than about 0.25 molecules / μm2. Wash the wells with 50 mM Tris pH 8.0; 50 mM NaCl. When the sample is immersed in the buffer (25 mM ACES, pH 7.4 buffer), maintain for 1 s under non - signal saturation conditions (laser quanta, Gem 532 under this sample, < 1 W / cm2). Use an inverted microscope (Olympus IX83) equipped with a 100 × TIRF objective, NA = 1.4 (Olympus), a dichroic mirror optimized for 532 nm light (Semrock, Di03 - R532 - t1 - 25×36), a band - pass filter optimized for Cy3 emission (Semrock, FF01 - 562 / 40 - 25), and a camera (sCMOS, Andor Zyla) to obtain images. Select conditions 50% ACN + MES with a 1 μm oligonucleotide graft concentration and 25% ACN + MES + 20% PEG + 10% formaldehyde with a 5.1 uM oligonucleotide graft concentration to test the feasibility of these conditions to improve the existing standard surface hybridization protocol for low - binding substrates. Add the oligonucleotide probe at the specified concentration and perform hybridization at 50°C for 2 minutes. Collect images as described above and present the results in the figure. Example 14 - Comparison of traditional oligonucleotide coupling chemistries, hybridization reaction conditions, and amplification techniques on low - binding substrate surfaces Figure 30 - Figure 32 provides a comparison of experimental results for traditional hybridization reactions or improved hybridization reactions on the low - binding substrates of the present invention, where oligonucleotides are linked using conventional coupling chemistries or improved coupling chemistries, and where RCA or bridge amplification is performed after hybridization. Amplification of target DNA on low - binding substrates with an oligonucleotide primer surface density less than different surface density threshold values by PCR - based ( "bridge") or by rolling - circle amplification results in elongated or diffused target molecules, which pose two problems: 1) reduced packing density, and 2) weakened signal. Therefore, scaling up systems for high - throughput sequencing applications based on such surfaces is severely impaired. Figure Figure 30 shows the results of PCR amplification (“bridging”; right) and rolling circle amplification (left) using traditional oligonucleotide-conjugated chemicals on a low-binding surface. In these images, the labeled amplified target DNA can be seen, and it is assumed that the extended conformation will pose serious difficulties for imaging in sequencing applications. In contrast, Figure 31 shows the results of PCR / “bridging” amplification, and Figure 32 shows the results of rolling circle amplification (RCA) on surfaces with an oligonucleotide primer surface density of at least 1,000 molecules / μm 2 . These surfaces support the compaction of the amplified target DNA into highly localized regions, which produce high fluorescence intensities from the self-linking labels and small pixel areas in imaging. The high fluorescence intensity particularly causes an increase in signal when calculating the spot intensity for sequencing applications. Importantly, the enhanced contrast-to-noise ratio generated by using the low-binding surfaces disclosed herein is a function of both the extremely high signal generated by this compaction of the amplified target DNA and the extremely low background provided by the hydrophilic coated surfaces. Example 15 - Comparison of amplification reactions on low-binding substrate surfaces Figure 33 provides non-limiting examples of the fluorescent image of the traditional substrate surface and the low-binding substrate surface of the present invention where the target oligonucleotides have hybridized and amplified. To facilitate sequencing accuracy, each amplified target nucleic acid must be clearly separated from other target nucleic acids in the image on the substrate surface. During the sequencing cycle, each target nucleic acid must also present a signal (such as a fluorescent signal) related to the discrimination of each nucleotide in the sequence (a process called "base identification"). In many cases, this base identification signal, which is only the fluorescence intensity provided by the label attached to the target nucleic acid molecule, must be clearly and accurately resolved above the noise (signal variations within the spot or target) and the background (noise non-specifically generated due to the characteristics of the materials forming the experimental environment). The ratio between the contrast and the noise ("CNR") defines the ability to accurately determine which base is present at each position in the target nucleic acid sequence, as well as the read length, reproducibility, and throughput of the sequencing system. The low-binding substrate surface disclosed in the present invention provides reduced non-specific protein and dye binding, thereby generating a lower background signal and a more compact and brighter foreground signal, thereby generating enhanced CNR and promoting excellent base identification for sequencing applications. Figure 32 shows a comparison between a PEG-coated surface prepared generally according to conventional methods and suitable for binding the cloned and amplified target DNA (top, "conventional") and the low-binding substrate surface of the present invention (top, "element current"). It is clear from the image that the surface disclosed in the present invention particularly shows sharper and denser measurement points of the cloned and amplified DNA than the conventional surface. Quantitative measurements of the spot intensity (attributable to the fluorescence of the cloned and amplified target DNA, equivalent to the sequencing signal) and the background intensity show that the CNR of the disclosed low-binding substrate surface (bottom, "optimal element current") far exceeds the CNR achievable using the conventional surface (bottom, "conventional"), even under ideal imaging conditions, such as when using improved hybridization conditions to bind the target DNA (bottom, 'conventional improvement'). This increase in CNR is greater than the increase reasonably expected using conventional substrate surfaces and represents both qualitative and quantitative improvements relative to prior art surfaces. These improvements are achieved by producing a substrate surface that meets certain criteria, such as reduced non-specific protein and dye binding, connection of oligonucleotides to the calibrated density of the surface, and hybridization / binding of the cloned and amplified target nucleic acid to the surface to produce an image with extremely high CNR that can enhance base identification in sequencing applications. Example 16 - Predictive examples of preparing low-binding substrates using other polymers The glass slides are physically or chemically treated (e.g., using plasma treatment, piranha solution cleaning steps, acid washing, alkali washing, high-temperature glass bonding, or any combination thereof) to remove organic contaminants and activate surface hydroxyl groups for silane coupling. Subsequently, the prepared glass surface is reacted with silane to covalently link a first layer of functional groups (e.g., primary amines) and / or a hydrophilic polymer layer. In some cases, for example, silanes such as (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), or (3-acrylylpropyl)trimethoxysilane are reacted with the surface using standard protocols to covalently link primary amine functional groups to the surface. In other cases, a silane-modified polymer (e.g., a hydrophilic hetero-bifunctional polymer containing a silyl group at one end and a second functional group (e.g., primary amine, carboxyl group, etc.) at the other end) can react directly with the surface (e.g., by contacting the cleaned glass surface with the silane-modified polymer in ethanol at a concentration of 0.1%-2% for about 1 to 2 hours, followed by rinsing with ethanol and water). Examples of suitable silane-modified polymers include, but are not limited to, silane-PEG-NH 2 (where the molecular weight of polyethylene glycol (PEG) is, for example, 1000, 2000, 3400, 5000, or 10 kDa), silane-PEG-COOH (PEG molecular weight is, for example, 1000, 2000, 3400, 5000, or 10 kDa), silane-PEG-maleimide (PEG molecular weight is, for example, 1000, 2000, 3400, 5000, or 10 kDa), silane-PEG-biotin (PEG molecular weight is, for example, 1000, 2000, 3400, 5000, or 10 kDa), silane-PEG-acrylate (PEG molecular weight is, for example, 1000, 2000, 3400, 5000, or 10 kDa), silane-PEG-silane (PEG molecular weight is, for example, 1000, 2000, 3400, 5000, or 10 kDa), silane-modified polypropylene glycol (PPG) of various molecular weights containing additional functional groups, silane-modified poly(vinyl alcohol) (PVA) of various molecular weights containing additional reactive functional groups, silane-modified polyethyleneimine (PEI) of various molecular weights containing additional reactive functional groups, silane-modified poly(lysine) of various molecular weights containing additional reactive functional groups, and their analogs or any combination thereof. In some cases, after initially reacting a surface with a silane or a silane-modified polymer, at least one additional hydrophilic polymer layer is coupled or deposited onto the glass surface. Any of a variety of hydrophilic polymers known to those skilled in the art can be used, including but not limited to polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinyl pyridine), poly(vinyl pyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, dextran, or any combination thereof, where in the case of covalent coupling, polymers containing appropriate mono-functional, homobifunctional, and / or heterobifunctional reactive groups are selected to be compatible with the chosen conjugation chemistry. In some cases, derivative polymers such as PEG-amine, PEG-NHS, or PEG-acrylate are used. In some cases, bifunctional PEG derivatives such as acrylate-PEG-NHS are used. In some cases, these additional hydrophilic polymer layers can be coupled or deposited onto the previous layer by contacting the surface with 0.1% - 2% polymer in ethanol or ethanol / buffered aqueous solution at room temperature for about 5 minutes to about 1 hour, followed by rinsing with ethanol or aqueous buffer solution. In some cases, second, third, fourth, fifth, or more additional layers of hydrophilic polymer can be coupled or deposited onto the initial layer of the substrate surface. In some cases, polymer molecules within a layer can be cross-linked to each other using appropriate homofunctional or heterofunctional cross-linking reagents. In some cases, polymer molecules in different layers can be cross-linked to each other. In some cases, one or more of the hydrophilic polymer layers can comprise branched-chain polymers, such as branched-chain PEG, branched-chain poly(vinyl alcohol) (branched-chain PVA), branched-chain poly(vinyl pyridine), branched-chain poly(vinyl pyrrolidone) (branched-chain PVP), branched-chain poly(acrylic acid) (branched-chain PAA), branched-chain polyacrylamide, branched-chain poly(N-isopropylacrylamide) (branched-chain PNIPAM), branched-chain poly(methyl methacrylate) (branched-chain PMA), branched-chain poly(2-hydroxyethyl methacrylate) (branched-chain PHEMA), branched-chain poly(oligo(ethylene glycol) methyl ether methacrylate) (branched-chain POEGMA), branched-chain polyglutamic acid (branched-chain PGA), branched-chain polylysine, branched-chain polyglucoside, branched-chain dextran, or any combination thereof. One or more of the hydrophilic polymer layers may comprise a plurality of covalently linked oligonucleotide linkers or primer molecules, where the oligonucleotide molecules are covalently coupled to the polymer using any of a variety of suitable conjugation chemistries known to those skilled in the art. In some cases, the oligonucleotide linker or primer molecules are covalently coupled to the polymer in solution, i.e., the polymer is subsequently coupled or deposited on a surface. In some cases, the oligonucleotide linker or primer molecules are covalently coupled to the polymer after they have been coupled to or deposited on a surface. In some cases, at least one hydrophilic polymer layer comprises a plurality of covalently linked oligonucleotide linkers or primer molecules. In some cases, at least two, at least three, at least four, or at least five layers of the hydrophilic polymer comprise a plurality of covalently linked linkers or primer molecules. The choice of polymer used, the number of layers, the degree of crosslinking within and between the layers, the number of layers comprising covalently linked oligonucleotide linkers or primer molecules, and the local concentration or surface density of the oligonucleotide linker or primer molecules can be adjusted independently or in combination to "tune" the properties of the surface to achieve the desired surface wettability (e.g., as indicated by a water contact angle of less than 50 degrees), the desired surface stability under prolonged exposure to sequencing / genotyping reagents and repeated thermal cycling, and the desired surface density of oligonucleotide linkers or primer molecules (e.g., at least 1,000 linkers or primer molecules / µm 2 ), which in turn provides extremely low non-specific binding of dye molecules or other labeled sequencing / genotyping reagents, improved hybridization efficiency, improved amplification efficiency, and the optimal density of specificity of the target sequence being amplified (in terms of the number of clonal colonies per unit area, the number of copies of the target sequence per unit area, or the number of amplified target molecules per unit area), a higher contrast-to-noise ratio (CNR) (e.g., CNR > 20) in an image (e.g., a fluorescence image) of the substrate surface, and ultimately improved detection accuracy or base calling accuracy in genotyping and sequencing applications. While the preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in any combination to practice the invention. It is intended that the following claims define the scope of the invention and thereby cover the methods and structures within the scope of these claims and their equivalents. This patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawings will be provided by the Patent Office upon request and payment of the necessary fee. The novel features of the present invention are set forth in detail in the appended claims. A better understanding of the features and advantages of the present invention will be obtained from the following detailed description of illustrative embodiments that explain the principles of the present invention and their accompanying drawings: Figure 1 provides a schematic diagram of an embodiment of a low-binding solid substrate of the present disclosure, wherein the substrate comprises alternating layers of a glass substrate and a hydrophilic coating, the alternating layers of the hydrophilic coating are covalently or non-covalently adhered to the glass, and further comprises a chemically reactive functional group serving as a linkage site for an oligonucleotide primer. Figure 2 provides a schematic diagram of covalently coupling a first polymer to the surface of a substrate (e.g., glass) using a silane reaction to form a first polymer layer. Figure 3 provides a schematic diagram of covalently coupling a branched-chain polymer to a surface as illustrated in Figure 2 to form a second polymer layer on the substrate surface. Figure 4 provides a schematic diagram of a coupling reaction for covalently linking one or more oligonucleotide adaptors or primer sequences (e.g., sequence 1 (dotted line) and and 2 (dashed line)) to a branched-chain polymer. Figure 5 provides a schematic diagram of covalently coupling a branched-chain polymer containing covalently linked oligonucleotide adaptor or primer sequences to a surface as illustrated in Figure 3 to form a third polymer layer on the substrate surface. Figure 6A - B provides an example of simulated fluorescence intensity data that illustrates the difference between using the signal-to-noise ratio (SNR) and the contrast-to-noise ratio (CNR) as measures of data quality in nucleic acid sequencing and base identification applications. Figure 6A: An example of simulated data where SNR = 2 and CNR = 1.25. Figure 6B: An example of simulated data where SNR = 2 and CNR = 12.29. Figure 7 provides an example of how improving the CNR affects the imaging time required for accurate detection and signal classification (base identification) of a nucleic acid community amplified by cloning on a solid substrate. Figure 8 illustrates different images obtained for different cycles of a nucleic acid sequencing reaction performed on a solid substrate due to incorporation of different labeled nucleotides in the complementary strands of the template molecules selected and amplified. The figure also illustrates different background contributions to the total detection signal of the detection platform, which requires iterative point detection by distinguishing nucleotide-specific signals from noise interspace background and in-space background images. Figure 9 provides an example of image data from a study for determining the relative amounts of non-specific binding of a green fluorescent dye to the surface of a glass substrate treated according to different surface modification schemes. Figure 10 provides an example of image data from a study for determining the relative amounts of non-specific binding of a red fluorescent dye to the surface of a glass substrate treated according to different surface modification schemes. Figure 11 provides an example of oligonucleotide primer grafting data for the surface of a substrate treated according to different surface modification schemes. Figure 12 provides an example of replicated images obtained during non-specific binding tests for the surface of a substrate treated according to different surface modification schemes. Figure 13 provides an example of data on non-specific binding of a sequencing dye mixture to the surface of a substrate treated according to different surface modification schemes. For comparison purposes, the fluorescence intensity measured under the same set of experimental conditions for non-specific binding of the sequencing dye mixture to individual beads grafted with Cy3-labeled oligonucleotides was approximately 1,500 counts. Figure 14 provides examples of images and data on non-specific binding of green and red fluorescent dyes to the surface of a substrate treated according to different surface modification schemes. For comparison purposes, the fluorescence intensity of the selected and amplified template population measured under the same set of experimental conditions after coupling of individual Cy3-labeled nucleotide bases was approximately 1,500 counts. Figure 15 provides examples of images and data demonstrating "tunable" nucleic acid amplification on a low-binding solid substrate by varying the oligonucleotide primer density on the substrate. Blue histogram: low primer density. Red histogram: high primer density. A combination of low non-specific binding and tunable nucleic acid amplification efficiency by adjusting the oligonucleotide primer density results in a high CNR and subsequent improvement in nucleic acid sequencing performance. Figure 16 provides an example of a fluorescence image of the low-binding solid substrate of the present invention, on which the oligonucleotides tethered have been amplified using different primer densities, isothermal amplification methods, and amplification buffer additives. Figure Example of a gel image demonstrating the reduction of non-specific nucleic acid amplification by using an amplification buffer additive while maintaining specific amplification of the target sequence. The gel image reveals bands corresponding to specific amplification of the target (arrow) and other gel quantified amplification products. Figure 18 provides an example of fluorescence images demonstrating the effect of formulation variations for improving amplification specificity on a low-binding substrate surface. Figure 19A - B provides non-limiting examples of image data demonstrating the improvement of hybridization stringency, speed, and efficacy that can be achieved by reforming the hybridization buffer for solid-phase nucleic acid amplification as described herein. Figure 19A provides an example of image data of two different hybridization buffer formulations and protocols. Figure 19B provides an example of the corresponding image data obtained using a standard hybridization buffer and protocol. Figure 20 illustrates a non-limiting example of the workflow for nucleic acid sequencing using the disclosed low-binding substrate and the amplification reaction formulation of the present invention and the processing time that can be achieved. Figure 21 provides an example of a fluorescence image and intensity data of the low-binding substrate of the present invention, where solid-phase nucleic acid amplification is performed on the low-binding substrate of the present invention to form clusters of clonally amplified template oligonucleotide sequences. Figure 22 provides a second example of a fluorescence image and intensity data of the low-binding substrate of the present invention, where solid-phase nucleic acid amplification is performed on the low-binding substrate of the present invention to form clusters of clonally amplified template oligonucleotide sequences. Figure 23 provides an example of a fluorescence image and intensity data of the low-binding substrate of the present invention, where solid-phase nucleic acid amplification is performed on the low-binding substrate of the present invention to form clusters of clonally amplified template oligonucleotide sequences. Figure 24 provides an example of a fluorescence calibration curve for estimating the surface density of primer oligonucleotides tethered to the substrate surface. Figure 25A - B provides non-limiting examples of modified glass and polymer surfaces of the present invention having binding amplicons containing fluorescently labeled nucleotides. Figure 25A: Modified glass surface. In the illustration, we see a CNR of 226 on the surface. Figure 25B: Modified plastic surface. In the illustration, we see a CNR of 109 on the surface. Figure 26A - B provides Figure 25A and Figure Analysis of the images on 25A and Figure On 26A, we see the signal intensity (left) and background intensity (right) for each of the glass and plastic surfaces. For each, the signal intensity is substantially greater than the background intensity. In Figure At 26B, we see a graphical depiction of the CNR values for each of the glass and plastic surfaces. In the left figure, the glass produces a CNR of 226, and in the right figure, the plastic produces a CNR of 109, consistent with the illustrations in Figures 25A and 25B. Figure 27 provides an analysis of the surface regarding the accuracy of its data. The data is collected in two channels and plotted as a scatter plot (upper figure), and quantified from left to right for each of the commercially available low CNR and high CNR surfaces (lower figure). Figure 28 provides a schematic diagram of a polymeric target oligonucleotide sequence hybridized to a surface containing a high surface density of oligonucleotide adaptor or primer molecules (left) and hybridized to a surface containing a lower surface density of oligonucleotide adaptor or primer molecules (right). Figure 29 provides a comparison of the experimental results of a conventional hybridization reaction on the low-binding substrate surface of the present invention and an optimized hybridization reaction on the low-binding substrate surface of the present invention. Figure 30 provides an illustration of the experimental results of a conventional hybridization reaction on the low-binding substrate of the present invention, followed by RCA or bridge amplification. Figure 31 provides an illustration of the experimental results of an optimized hybridization reaction on the low-binding substrate of the present invention, followed by RCA or bridge amplification. Figure 32 provides a comparative description of fluorescence images of surfaces for conventional commercial sequencing devices (e.g., polyacrylamide) and surfaces prepared using optimized hybridization reactions on the low-binding substrates of the present invention and prepared using improved coupling chemistries for attaching oligonucleotide adaptors or primer molecules to the surface, followed by performing RCA or bridge amplification. Figure 33 provides non-limiting examples of fluorescence images of the surface of a substrate for a conventional commercial sequencing device (e.g., polyacrylamide), the surface of a low-binding substrate modified using a conventional coupling chemistry; and the surface of a low-binding substrate of the present invention prepared using the improved coupling chemistries, hybridization methods, and amplification methods described herein. The drawings illustrate the contrast-to-noise ratio measured from the images, such as the images provided in examples of conventional substrate surfaces, improved conventional substrate surfaces, and low-binding substrate surfaces of the present invention in combination with improved solid-phase nucleic acid amplification methods.

Claims

1. A method for nucleic acid sequencing, comprising: (a) providing a surface having at least one hydrophilic polymer coating therewith, the hydrophilic polymer coating comprising a hydrophilic polymer coupled to a first oligonucleotide molecule; (b) contacting the first oligonucleotide molecule with a sample nucleic acid molecule to couple the sample nucleic acid molecule to the first oligonucleotide molecule; (c) amplifying the sample nucleic acid molecule after (b); and (d) obtaining a fluorescence image of the surface, wherein when the surface is immersed in a buffer solution and the fluorescence image of the surface is obtained under non-signal saturation conditions using an inverted fluorescence microscope and a camera, the fluorescence image of the surface exhibits a contrast-to-noise ratio greater than or equal to 20 after (b), and wherein the sample nucleic acid molecule comprises a detectable tag as a fluorescent dye, and wherein, prior to the amplification, a background fluorescence intensity is measured in a region of the surface, the region being a region laterally displaced from a discrete region of the surface having the at least one hydrophilic polymer coating, not exceeding twice the intensity measured in the discrete region.

2. The method of claim 1, wherein when the fluorescent image is obtained, the fluorescent image on the surface exhibits a contrast-to-noise ratio greater than or equal to 40 after (b).

3. The method of claim 1, wherein the at least one hydrophilic polymer coating comprises at least one first polymer layer coupled to the surface and a second polymer layer coupled to the first polymer layer.

4. The method of claim 1, wherein the at least one hydrophilic polymer coating comprises a branched polymer having at least eight branches.

5. The method of claim 1, wherein when the sample nucleic acid molecules are amplified and labeled with cyanine dye-3, and the surface is immersed in the buffer solution, and the fluorescence image of the surface is obtained using an inverted fluorescence microscope and the camera under non-signal saturation conditions, the fluorescence image of the surface exhibits a fluorescence intensity ratio of at least 5:1 between the foreground fluorescence intensity and the nonspecific dye adsorption background (Binter).

6. The method of claim 1, wherein the sequence of the nucleic acid molecule of the sample is determined by sequencing the nucleic acid molecule of the sample.

7. The method of claim 1, wherein the at least one hydrophilic polymer coating comprises: (i) a first layer comprising a first monolayer of polymer molecules coupled to the surface; (ii) a second layer comprising a second monolayer of polymer molecules coupled to the first monolayer of polymer molecules; and (iii) a third layer comprising a third monolayer of polymer molecules coupled to the second monolayer of polymer molecules, wherein the polymer molecules of the first layer, the second layer, and the third layer comprise branched polymer molecules.

8. The method of claim 1, wherein the first oligonucleotide molecule is present at a surface density of greater than or equal to 2,000 molecules / µm².

9. The method of claim 1, wherein the first oligonucleotide molecule is present at a surface density of 10,000 molecules / µm² or greater.

10. The method of claim 1, wherein the first oligonucleotide molecule is present at a surface density of less than or equal to 15,000 molecules / µm².

11. The method of claim 1, wherein (b) comprises contacting the surface with a solution containing the sample nucleic acid molecules, wherein the sample nucleic acid molecules are in a concentration of less than 1 nM in the solution.

12. The method of claim 1, wherein (b) comprises contacting the surface with a solution containing the sample nucleic acid molecules, wherein the sample nucleic acid molecules are in the solution at a concentration not greater than or equal to 50 pM.

13. The method of claim 1, wherein the sample nucleic acid molecule is a single-stranded polynucleotide molecule containing regularly occurring monomeric units.

14. The method of claim 13, wherein the length of the single-stranded polynucleotide molecule is at least 10 kb.

15. The method of claim 1, wherein the sample nucleic acid molecule is a double-stranded polynucleotide molecule containing regularly occurring repeating monomeric units.

16. The method of claim 1, wherein the first oligonucleotide molecule is present at a uniform surface density across the surface.

17. The method of claim 1, wherein the first oligonucleotide molecule is present at a first position on the surface with a first local surface density and at a second position on the surface with a second local surface density, wherein the first local surface density is different from the second local surface density.

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